• View in gallery
    Figure 1—

    Bright-field photomicrographs obtained during monolayer expansion of tendon-derived cells with FGF-2 (100 ng of FGF-2/mL; A) and without FGF-2 (B) and bone marrow–derived cells with FGF-2 (C) and without FGF-2 (D). Unstained; bar = 50 μm.

  • View in gallery
    Figure 2—

    Log10 mean ± SE number of tendon- and bone marrow–derived cells after monolayer expansion with FGF-2 (100 ng of FGF-2/mL) or without FGF-2 followed by culture for 7 days in a pulverized acellular tendon matrix with IGF-I (100 ng of IGF-1/mL) or without IGF-1. Treatments were monolayer expansion without FGF-2 followed by culture without IGF-1 (control treatment; white bars), monolayer expansion with FGF-2 followed by culture without IGF-1 (gray bars), monolayer expansion without FGF-2 followed by culture with IGF-1 (black bars), and monolayer expansion with FGF-2 followed by culture with IGF-1 (crosshatched bars).

  • View in gallery
    Figure 3—

    Mean ± SE values for collagen type I mRNA expression (A), collagen type III mRNA expression (B), and COMP mRNA expression (C) for tendon- and bone marrow–derived cells after monolayer expansion with or without FGF-2 and culture for 7 days in a pulverized acellular tendon matrix with or without IGF-1. Values reported represent results for samples obtained from 4 horses. Values were normalized on the basis of expression for elongation factor-1α and represent the fold increase determined from results for bone marrow–derived cells without FGF-2 or IGF-1. *Value differs significantly (P ≤ 0.05) from the values for bone marrow–derived cells cultured without IGF-I. †Values differ significantly (P ≤ 0.05) from the corresponding values for the bone marrow–derived cells. See Figure 2 for remainder of key.

  • View in gallery
    Figure 4—

    Log10 mean ± SE disintegrations per minute (DPM) as a measure of incorporation of [3H] proline into collagen of the matrix formed by the combination of cells and pulverized acellular tendon. Values reported represent results for samples obtained from 6 horses. Cells were expanded with and without FGF-2 and cultured for 7 days in a pulverized acellular tendon matrix with and without IGF-I. *Value differs significantly (P ≤ 0.05) from the value for bone marrow–derived cells in the control treatment. See Figure 2 for remainder of key.

  • View in gallery
    Figure 5—

    Log10 mean ± SE CPM as a measure of incorporation of 35S-labeled sodium sulfate into GAG of the matrix formed by the combination of cells and pulverized acellular tendon. Values reported represent results for samples obtained from 6 horses. Cells were expanded with and without FGF-2 and cultured for 7 days in a pulverized acellular tendon matrix with and without IGF-I. *Within a cell type, value differs significantly (P ≤ 0.05) from the value for cells in the control treatment. †Values differ significantly (P ≤ 0.05) from the corresponding values for the bone marrow–derived cells. ‡Value differs significantly (P ≤ 0.05) from the value for tendon-derived cells in the control treatment. See Figure 2 for remainder of key.

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Responses of equine tendon- and bone marrow–derived cells to monolayer expansion with fibroblast growth factor-2 and sequential culture with pulverized tendon and insulin-like growth factor-I

Sushmitha S. DurgamDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802

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Allison A. StewartDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802

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Holly C. PondenisDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802

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Angela C. YatesDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802

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Richard B. EvansBiostatistical Retriever, S67 W28303 River Rd, Waukesha, WI 53189.

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Matthew C. StewartDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802

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Abstract

Objective—To compare in vitro expansion of equine tendon- and bone marrow–derived cells with fibroblast growth factor-2 (FGF-2) supplementation and sequential matrix synthesis with pulverized tendon and insulin-like growth factor-I (IGF-I).

Sample—Cells from 6 young adult horses.

Procedures—Progenitor cells were expanded in monolayers with FGF-2, followed by culture with autogenous acellular pulverized tendon and IGF-I for 7 days. Initial cell isolation and subsequent monolayer proliferation were assessed. In pulverized tendon cultures, cell viability and expression of collagen types I and III and cartilage oligomeric matrix protein (COMP) mRNAs were assessed. Collagen and glycosaminoglycan syntheses were quantified over a 24-hour period.

Results—Monolayer expansion with FGF-2 significantly increased the mean ± SE number of tendon-derived cells (15.3 ± 2.6 × 106), compared with bone marrow–derived cells (5.8 ± 1.8 × 106). Overall, increases in collagen type III and COMP mRNAs were seen in tendon-derived cells, compared with results for bone marrow–derived cells. After IGF-I supplementation, increases in collagen type I and type III mRNA expression were seen in bone marrow–derived cells, compared with results for unsupplemented control cells. Insulin-like growth factor-I significantly increased collagen synthesis of bone marrow–derived cells. Monolayer expansion with FGF-2 followed by IGF-I supplementation significantly increased glycosaminoglycan synthesis in tendon-derived cells.

Conclusions and Clinical Relevance—Tendon-derived cells had increased cell numbers and matrix synthesis after monolayer expansion with FGF-2, compared with results for bone marrow–derived cells. In vivo experiments with FGF-2-expanded tendon-derived cells are warranted to evaluate effects on tendon healing.

Abstract

Objective—To compare in vitro expansion of equine tendon- and bone marrow–derived cells with fibroblast growth factor-2 (FGF-2) supplementation and sequential matrix synthesis with pulverized tendon and insulin-like growth factor-I (IGF-I).

Sample—Cells from 6 young adult horses.

Procedures—Progenitor cells were expanded in monolayers with FGF-2, followed by culture with autogenous acellular pulverized tendon and IGF-I for 7 days. Initial cell isolation and subsequent monolayer proliferation were assessed. In pulverized tendon cultures, cell viability and expression of collagen types I and III and cartilage oligomeric matrix protein (COMP) mRNAs were assessed. Collagen and glycosaminoglycan syntheses were quantified over a 24-hour period.

Results—Monolayer expansion with FGF-2 significantly increased the mean ± SE number of tendon-derived cells (15.3 ± 2.6 × 106), compared with bone marrow–derived cells (5.8 ± 1.8 × 106). Overall, increases in collagen type III and COMP mRNAs were seen in tendon-derived cells, compared with results for bone marrow–derived cells. After IGF-I supplementation, increases in collagen type I and type III mRNA expression were seen in bone marrow–derived cells, compared with results for unsupplemented control cells. Insulin-like growth factor-I significantly increased collagen synthesis of bone marrow–derived cells. Monolayer expansion with FGF-2 followed by IGF-I supplementation significantly increased glycosaminoglycan synthesis in tendon-derived cells.

Conclusions and Clinical Relevance—Tendon-derived cells had increased cell numbers and matrix synthesis after monolayer expansion with FGF-2, compared with results for bone marrow–derived cells. In vivo experiments with FGF-2-expanded tendon-derived cells are warranted to evaluate effects on tendon healing.

Tendinitis is a major cause of lameness in performance horses, accounting for up to 50% of racing injuries.1–4 Tendinopathies are a consequence of cumulative microdamage to collagen fibers, which results in matrix degenerative disease.3,5–7 A number of medical and surgical strategies have been developed to treat flexor tendinitis in horses; those strategies have focused on minimizing inflammation, stimulating healing, and preventing further trauma.5,7,8 Despite these treatments, reinjury can be as high as 45% as a result of permanent changes in collagen ECM organization and altered resistance to tensile loading.9,10

Mesenchymal stem cells have been used in the treatment of tendinitis.11,12 Most studies12–14 involving MSCs have focused on stromal cells derived from bone marrow aspirates. Alternate sources of progenitor cells have been described for tendon repair in other species,15–17 and adipose-derived progenitor cells have been evaluated for efficacy in horses with experimentally induced tendinitis.18 Evidence suggests the presence of multipotent, clonogenic progenitor cells or stem cells in tendons within an ECM-rich niche.19–22 In vitro, equine tendon- and muscle-derived cells yield greater cell numbers in a shorter culture time and have superior viability and matrix production in comparison with results for bone marrow–derived cells.23

Fibroblast growth factor-2, also known as basic FGF, is a potent mitogen that binds to heparan sulfate proteoglycans in ECM and is released following matrix degeneration in mice.24 Cultures of MSC supplemented with FGF-2 proliferate more rapidly and have an increased capacity for self-renewal and differentiation.25 In another study26 conducted by our research group, we evaluated the effects of FGF-2 at concentrations of 1, 10, and 100 ng/mL on equine bone marrow–derived MSCs, with optimal effects detected at 100 ng/mL. In addition, several in vitro and in vivo studies27–30 of tendon healing in mice and rabbits have revealed enhanced angiogenesis, tendon fibroblast proliferation, and collagen type III expression in response to FGF-2 administration.

The role of IGF-I in tendon healing of horses has generated a considerable body of research.14,31–33 In vitro, IGF-I stimulates mitogenesis, matrix gene expression, and collagen synthesis by tenocytes.34,35,a Furthermore, exogenous IGF-I injections and gene-assisted delivery of IGF-I have resulted in histologic improvement in tendon healing in horses with collagenase-induced tendinitis.14,31 The dose-dependent effects of IGF-I on in vitro synthesis of the tenocyte matrix were evaluated.34 In that study,34 collagen synthesis was significantly increased at doses of 100 and 250 μg of IGF-I/mL.

For clinically viable applications of cell-based treatments, the in vitro expansion of putative progenitor cell populations must be optimized to reduce the time required for generation of adequate cell numbers. Additionally, the biosynthetic activities of reimplanted cells need to be augmented to promote healing through effective tissue repair. Therefore, the study reported here was conducted to address 2 major objectives. First, the mitogenic effects of FGF-2 on tendon- and bone marrow–derived cell populations were assessed during in vitro monolayer expansion. Second, the synthetic and phenotypic responses of monolayer-expanded cells to exogenous IGF-I were assessed via an in vitro powdered matrix. Pulverized tendon derived from an autogenous source was used as substrate material to provide a 3-D substrate for cell adhesion and proliferation and matrix synthesis.

The overall objective of the study reported here was to determine whether sequential administration of FGF-2 during monolayer expansion and IGF-I in culture with pulverized tendon would improve cell expansion and subsequent matrix synthesis. Our hypothesis was that tendon-derived cells expanded with FGF-2 and cultured with pulverized tendon and IGF-I will have increased cell viability and proliferation, matrix gene expression, and matrix synthesis, compared with results for bone marrow–derived cells cultured under similar conditions.

Materials and Methods

Samples—Bone marrow aspirates and tendon specimens were collected aseptically from 6 horses (2 to 4 years old) that were euthanized for reasons unrelated to musculoskeletal disease. Horses were sedated with detomidine (0.01 to 0.03 mg/kg, IV). After collection of bone marrow aspirates, all horses were euthanized via injection of sodium pentobarbital (104 mg/kg, IV). Immediately after the horses were euthanized, the lateral digital extensor tendon from a hind limb and both superficial digital flexor tendons were harvested aseptically from each horse. All samples were obtained in accordance with guidelines reviewed and approved by the Institutional Animal Care and Use Committee of the University of Illinois.

Processing of bone marrow–derived cells—Bone marrow aspirates were collected from the sternum by use of a bone marrow biopsy needle.14,b Approximately 15 to 20 mL of bone marrow was aspirated into syringes that contained 1,000 U of heparin. Each bone marrow aspirate was diluted with 15 mL of PBS solution and centrifuged at 300 × g for 10 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 DMEMc supplemented with 10% FBS,d 300 μg of l-glutaminee/mL, 100 U of sodium penicillinf/mL, and 100 μg of streptomycin sulfatef/mL. Resuspended cells were placed in 75-cm2 flasks and incubated at 37°C in a 5% carbon dioxide atmosphere with 90% humidity. Bone marrow–derived cells were passaged after they reached focal confluence. Monolayer expansion of cells was achieved via culture in medium supplemented with or without FGF-2 supplementation (100 ng of FGF-2g/mL) for 2 passages, which provided a sufficient number of cells for subsequent experiments. Time to confluence and cell counts at the time of trypsinization were recorded.

Processing of tendon-derived cells—A 4 × 1-cm sample of lateral digital extensor tendon was reserved for cell isolation. The specimen for cell isolation was diced into 0.25-cm3 pieces and digested for 16 hours at 37°C in 0.2% collagenaseh high-glucose DMEM supplemented with 1% FBS, 100 U of sodium penicillin/mL, and 100 μg of streptomycin sulfate/mL. After digestion, the isolated cells were passed through a 40-μm filter. Isolated cells were collected by use of centrifugation at 300 × g for 5 minutes. Supernatant was removed, and the cell pellet was resuspended in DMEM supplemented with 1% FBS, 100 U of sodium penicillin/mL, and 100 μg of streptomycin sulfate/mL. Cell viability was determined via trypan blue dye exclusion.36

Monolayer expansion of tendon cells—Progenitor cells were collected from tendons in accordance with a previously described protocol.23,37 Tendon-derived cells were seeded at 13,300 cells/cm2 in culture flasks in high-glucose DMEM supplemented with 20% FBS, 300 μg of l-glutamine/mL, 100 U of sodium penicillin/mL, and 100 μg of streptomycin sulfate/mL. The slowly adherent, tendon-derived cells were preferentially isolated from the rapidly adherent, fibroblast-like cells by differential attachment. Culture medium and unattached cells were serially transferred to fresh culture flasks every 24 hours during the first 6 days of culture.23,37 Tendon-derived cells that adhered on day 6 of the transfer protocol (day 0 was the first day of cell culture) were maintained until confluence and expanded in monolayers with or without supplementation of FGF-2 (100 ng/mL) for 2 passages to generate a sufficient number of cells for subsequent experiments.

Tendon matrix culture—Superficial digital flexor tendons were pulverized in a freezer milli with liquid nitrogen. Pulverized tendon was subjected to 4 rounds of freeze-thaw cycles at −80° and 4°C to kill resident tenocytes. A 1% (wt/vol) acellular tendon matrix suspension was prepared with tenogenic medium (high-glucose DMEM supplemented with 10% FBS, 300 μg of l-glutamine/mL, 100 U of sodium penicillin/mL, 100 μg of streptomycin sulfate/mL, and 37.5 μg of ascorbic acid/mL). The tendon matrix suspension was maintained in culture without additional cells to serve as a negative control product (matrix only) or seeded with 250,000-cell aliquots of the expanded tendon- and bone marrow–derived cells supplemented with or without IFG-1 supplementation (100 ng of IGF-Ig/mL) in 24-well ultralow attachment culture plates.j A concentration of 100 ng of IGF-I/mL was selected for use in the study here on the basis of results for other studies34,35 and the outcomes of a stem cell–focused studya completed by the authors' research group.

The experimental design comprised 9 treatment groups. Each treatment group contained 18 replicates. Twelve replicates were used for RNA isolation, 3 replicates were used for collagen synthesis, and 3 replicates were used for GAG synthesis. The mean of the replicates for each treatment group was calculated to provide a single data point. This was repeated for each of the 6 horses included in the study. Each treatment well was supplemented with 1 mL of tenogenic medium that contained 0 or 100 ng of IGF-I/mL. Exhausted medium was removed from all wells and replaced with fresh medium every 2 days. All culture samples were collected on day 7 by use of centrifugation to separate the medium from the matrix with cells.

Cell number—Three replicates of each treatment group were used to measure cell numbers on day 7 by use of a mitochondrial metabolic assayk in accordance with the manufacturer's instructions. In brief, 50 μL of the assay reagent containing tetrazolium was added to fresh medium in each well, and the cells were then incubated at 37°C for 2.5 hours. One hundred microliters of medium from each well was transferred to a 96-well plate, and absorbance was measured at 492 nm in a microplate readerl to detect concentrations of the metabolic product, formazan. All samples were assayed in duplicate, and a mean value was calculated to provide a single data point. Optical density data were converted to cell numbers via reference to standard curves generated from known numbers of tendon- and bone marrow–derived cell cultures.

RNA isolation—Twelve replicates from each treatment group were pooled, snap-frozen in liquid nitrogen, and stored at −80°C for RNA isolation. Total RNA was extracted by use of a guanidinium thiocyanate–phenolchloroform solution reagentm used in accordance with the manufacturer's suggested protocol and purified in silica columns.n Concentration and purity of RNA were assessed via UV spectrophotometry and agarose gel electrophoresis, respectively. One microgram of RNA in each sample was converted to cDNA with a commercial reverse transcription kito and oligo(dT) primers. Target cDNAs were amplified via real-time PCR assay by use of Taq DNA polymerasep and gene-specific primers designed with a multiple sequence alignment programq from published sequences available in GenBank. Specificity was confirmed by cloning and sequencing the PCR products (Appendix). Real-time quantitative PCR assay was performed in triplicate for collagen I, collagen III, and COMP mRNAs and for the reference gene, elongation factor-1α. A fluorescence detection systemr was used to measure PCR-generated cDNA and generate threshold cycle values. All reactions were conducted individually, and the relative gene expression was quantified by use of the 2−ΔΔCT method.38

Collagen synthesis—Collagen synthesis was determined by incorporation of [3H] proline conducted in accordance with a published protocol.39 On day 6, 3 wells of each treatment group were radiolabeled with 50 μCi of [3H] prolines/mL of tenogenic medium and incubated for 24 hours. Samples were washed 3 times with 0.5 mL of PBS solution containing 1mM proline and stored at −80°C. Radiolabeled samples were frozen-thawed 3 times, digested, and homogenizedt prior to RNase treatment. Total protein was precipitated with tricholoroacetic acid and washed 3 times with l-proline to remove traces of unincorporated [3H] proline. The resulting pellets were digested with purified collagenaseu and centrifuged at 3,220 × g for 10 minutes. Supernatant and pellets were separated and transferred to scintillation liquid. Radioactivity was measured in a scintillation counter.v Newly synthesized collagen was detected on the basis of radioactivity in the sample supernatants obtained after collagenase digestion. Values were expressed as the number of disintegrations/min/250,000 cells.

GAG synthesis—Glycosaminoglycan synthesis was determined by measuring incorporation of 35SO4 into each sample. Three wells of each treatment group were radiolabeled with 10 μCi of 35S-labeled sodium sulfatew/mL during the last 24 hours of the experiments.40 Samples were washed 3 times with PBS solution and then digested in 1 mL of buffer containing 0.5 mg of papainx at 65°C for 16 hours. Aliquots (25 μL) of 35S-labeled, papain-digested samples were placed in multi-well punch plates,y precipitated with Alcian blue dye, and counted by use of a scintillation counter.23,40 All CPM values were adjusted for the decay of 35S-radioisotope from the time of radiolabeling until assay. Values were expressed as the CPM/250,000 cells.

Statistical analysis—Mean ± SE values were calculated for each cell type and for supplementation with FGF-2, IGF-1, and the combination of FGF-2 and IGF-1. Background values detected in the matrix-only group were subtracted from the values of the other groups for quantification of collagen and GAG synthesis. Data for cell numbers, collagen synthesis, and GAG synthesis were logarithmically transformed to provide a normal distribution. The effect of cell type was analyzed by use of a mixed-effects model, with horse as a random effect. For the cell types (tendon- and bone marrow–derived cells), the effect of supplementation with growth factors was evaluated by use of a 2-way repeated-measures ANOVA to control for differences among horses. When group differences for growth factor supplementation were detected, pairwise multiple comparisons were made by use of the Holm-Sidak nonparametric test. A commercially available statistical programz was used to perform statistical analyses. Values of P ≤ 0.05 were considered significant.

Results

Monolayer cell expansion—Overall, mean cell numbers following monolayer expansion were significantly (P = 0.011) higher in tendon-derived cells, compared with results for the bone marrow–derived cells (Table 1). Monolayer expansion of tendon-derived cells with FGF-2 significantly (P = 0.027) increased cell numbers, compared with results for the unsupplemented tendon-derived cultures (Figure 1). In contrast, monolayer expansion with FGF-2 did not significantly (P = 0.311) affect proliferation of bone marrow–derived cells.

Figure 1—
Figure 1—

Bright-field photomicrographs obtained during monolayer expansion of tendon-derived cells with FGF-2 (100 ng of FGF-2/mL; A) and without FGF-2 (B) and bone marrow–derived cells with FGF-2 (C) and without FGF-2 (D). Unstained; bar = 50 μm.

Citation: American Journal of Veterinary Research 73, 1; 10.2460/ajvr.73.1.162

Table 1—

Mean ± SE number of tendon- and bone marrow–derived cells (n = 6 horses) after monolayer expansion with FGF-2 (100 ng of FGF-2/mL) or without FGF-2.

Cell typeWith FGF-2Without FGF-2
Tendon-derived cells15.34 × 106 ± 2.60 × 106*†9.14 × 106 ± 1.03 × 106*
Bone marrow–derived cells5.87 × 106 ± 1.79 × 1063.06 × 106 ± 0.85 × 106

Within a column, value differs significantly (P ≤ 0.05) from the value for the bone marrow–derived cells.

Within a row, value differs significantly (P = 0.027) from the value without FGF-2 supplementation.

Cell numbers—After 7 days in culture with pulverized tendon matrix and IGF-I, there was no significant effect of cell type on mean cell numbers, as determined by use of the mitochondrial metabolic assay (Figure 2). Monolayer expansion with FGF-2 did not significantly change the number of tendon- (P = 0.052) or bone marrow–derived (P = 0.096) cells cultured with tendon matrix. There was no significant (P = 0.178) effect of IGF-I supplementation on cell numbers for either cell type. The cell number of the matrix-only control group was zero, which confirmed the absence of viable cells.

Figure 2—
Figure 2—

Log10 mean ± SE number of tendon- and bone marrow–derived cells after monolayer expansion with FGF-2 (100 ng of FGF-2/mL) or without FGF-2 followed by culture for 7 days in a pulverized acellular tendon matrix with IGF-I (100 ng of IGF-1/mL) or without IGF-1. Treatments were monolayer expansion without FGF-2 followed by culture without IGF-1 (control treatment; white bars), monolayer expansion with FGF-2 followed by culture without IGF-1 (gray bars), monolayer expansion without FGF-2 followed by culture with IGF-1 (black bars), and monolayer expansion with FGF-2 followed by culture with IGF-1 (crosshatched bars).

Citation: American Journal of Veterinary Research 73, 1; 10.2460/ajvr.73.1.162

ECM gene expression—No mRNA was isolated from the acellular matrix samples, which verified that no viable endogenous tenocytes remained. Therefore, gene expression data derived from bone marrow–derived cells cultured with tendon matrix without growth factor supplementation (bone marrow–derived cells only) were used as reference values for comparative analyses.

Collagen type I—Collagen type I mRNA expression was not significantly (P = 0.087) different for tendon-derived cell groups and bone marrow–derived cell groups (Figure 3). Among the tendon-derived cell groups, supplementation with IGF-I did not significantly (P = 0.095) increase collagen type I mRNA expression. In the bone marrow–derived cell groups, IGF-I significantly (P = 0.028) increased collagen type I mRNA expression. Monolayer expansion with FGF-2 had no effect on subsequent collagen type I mRNA expression in tendon- or bone marrow–derived cell types cultured with tendon matrix.

Figure 3—
Figure 3—

Mean ± SE values for collagen type I mRNA expression (A), collagen type III mRNA expression (B), and COMP mRNA expression (C) for tendon- and bone marrow–derived cells after monolayer expansion with or without FGF-2 and culture for 7 days in a pulverized acellular tendon matrix with or without IGF-1. Values reported represent results for samples obtained from 4 horses. Values were normalized on the basis of expression for elongation factor-1α and represent the fold increase determined from results for bone marrow–derived cells without FGF-2 or IGF-1. *Value differs significantly (P ≤ 0.05) from the values for bone marrow–derived cells cultured without IGF-I. †Values differ significantly (P ≤ 0.05) from the corresponding values for the bone marrow–derived cells. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 73, 1; 10.2460/ajvr.73.1.162

Collagen type III mRNA expression—Overall, tendon-derived cell groups cultured with tendon matrix expressed significantly (P = 0.003) more collagen type III mRNA than did bone marrow–derived cell groups (Figure 3). Within the tendon-derived cell groups, there was no significant effect of FGF-2 (P = 0.623) or IGF-I (P = 0.119) on collagen type III mRNA expression. In the bone marrow–derived cell groups, IGF-I supplementation significantly (P = 0.048) increased collagen type III mRNA expression. Supplementation with FGF-2 during monolayer expansion of bone marrow–derived cells did not significantly (P = 0.523) affect collagen type III expression.

COMP mRNA expression—Overall, tendon-derived cell groups cultured with tendon matrix expressed significantly (P = 0.001) more COMP mRNA than did bone marrow–derived cell groups (Figure 3). However, there was no significant effect of FGF-2 or IGF-I on COMP mRNA expression in tendon- or bone marrow–derived cells.

Collagen synthesis—The mean collagen synthesis rate of tendon-derived cell groups was less, but not significantly (P = 0.055) different, than that of bone marrow–derived cell groups. Monolayer expansion of tendon-derived cells with FGF-2 did not significantly (P = 0.367) affect subsequent collagen synthesis. Furthermore, IGF-I did not significantly (P = 0.055) affect collagen synthesis by tendon-derived cells. There was no significant (P = 0.532) effect of FGF-2 during monolayer expansion on collagen synthesis by bone marrow–derived cells. Treatment with IGF-I significantly (P = 0.028) increased collagen synthesis by bone marrow–derived cells, compared with results for the unsupplemented control cells (Figure 4). However, collagen synthesis in the bone marrow–derived cell groups supplemented with IGF-I remained lower than collagen synthesis in the tendon-derived groups.

Figure 4—
Figure 4—

Log10 mean ± SE disintegrations per minute (DPM) as a measure of incorporation of [3H] proline into collagen of the matrix formed by the combination of cells and pulverized acellular tendon. Values reported represent results for samples obtained from 6 horses. Cells were expanded with and without FGF-2 and cultured for 7 days in a pulverized acellular tendon matrix with and without IGF-I. *Value differs significantly (P ≤ 0.05) from the value for bone marrow–derived cells in the control treatment. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 73, 1; 10.2460/ajvr.73.1.162

GAG synthesis—Glycosaminoglycan synthesis was significantly (P = 0.006) higher in the tendon-derived cell groups than in the bone marrow–derived cells (Figure 5). Within the tendon-derived cell groups, monolayer expansion with FGF-2 significantly (P = 0.030) increased GAG synthesis. Furthermore, IGF-I supplementation of the tendon-derived cell-matrix cultures also significantly (P = 0.016) increased GAG synthesis. In the bone marrow–derived cell groups, monolayer expansion with FGF-2 did not significantly (P = 0.305) affect GAG synthesis. However, IGF-I significantly (P = 0.022) increased GAG synthesis by bone marrow–derived cells, compared with GAG synthesis by unsupplemented cultures.

Figure 5—
Figure 5—

Log10 mean ± SE CPM as a measure of incorporation of 35S-labeled sodium sulfate into GAG of the matrix formed by the combination of cells and pulverized acellular tendon. Values reported represent results for samples obtained from 6 horses. Cells were expanded with and without FGF-2 and cultured for 7 days in a pulverized acellular tendon matrix with and without IGF-I. *Within a cell type, value differs significantly (P ≤ 0.05) from the value for cells in the control treatment. †Values differ significantly (P ≤ 0.05) from the corresponding values for the bone marrow–derived cells. ‡Value differs significantly (P ≤ 0.05) from the value for tendon-derived cells in the control treatment. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 73, 1; 10.2460/ajvr.73.1.162

Discussion

The objective of the study reported here was to evaluate the effect of sequential growth factors, FGF-2 and IGF-I, on tendon-derived cells that may be used as an option for cell-based treatments of tendinitis. Bone marrow–derived cells were used as the criterion-referenced standard for comparison. Overall, FGF-2 increased cell proliferation during monolayer expansion and IGF-I increased subsequent matrix synthesis in the tendon-derived cells. Tendon-derived cells were more proliferative in culture in vitro and required a shorter amount of time to generate clinically relevant numbers than did bone marrow–derived cells. In addition, less interhorse variability was detected in the data derived for tendon-derived cells, compared with that for bone marrow–derived cells. Results of this study were derived from samples obtained from 6 young adult horses, and the statistical power for significance ranged from 0.6 to 0.9.

Analysis of results of the present study suggested that tendon-derived cells can be obtained in sufficient numbers from an autogenous specimen for use in cell-based treatment of tendinitis in horses.12,14,18 Studies19–22 have been conducted to evaluate the presence of a stem cell population within a tendon ECM niche. Moreover, differentiation toward adipogenic, osteogenic, and chondrogenic pathways has been reported,20,21,aa which confirms the multipotential capacity of the tendon stem cell populations in mice, rats, rabbits, horses, and humans. In 1 study,41 investigators found an increase in chondrogenic capacity of caprine tendon–derived cells, compared with results for control bone marrow–derived MSCs, which suggests that tendon-derived progenitor cells are feasible for use in cell-based treatments directed toward tissues other than tendon. In the present study, tendon-derived cells had an overall increase in expression of collagen type III mRNA, compared with results for bone marrow–derived cells. An increase in the expression of collagen type III can alter the collagen type I-to-type III ratio and eventually affect the quality of the resulting structure. Determinations of collagen type I and III protein production or consequent ECM organization were not performed in the present study. These analyses will require an in vivo study to clarify the clinical benefits of sequential FGF-2 and IGF-I supplementation in culture media of cells used for cell-based repair of tendon injuries. In addition, following monolayer expansion with FGF-2, both tendon- and bone marrow–derived cells were cultured in tenogenic medium with pulverized tendon and IGF-I. It is possible that this medium may have provided optimum conditions for matrix synthesis of tendon-derived cells versus bone marrow–derived cells.

In the present study, FGF-2 significantly increased proliferation of tendon-derived cells during monolayer expansion; however, FGF-2 did not significantly affect proliferation of bone marrow–derived cells. This contrast may have been attributable to the considerable variation in bone marrow rates of proliferation and formation of ECM among donors. Fibroblast growth factor-2 had minimal effects on cell viability and adherence of both tendon- and bone marrow–derived cells on acellular pulverized tendon. In addition, FGF-2 did not influence ECM gene expression or matrix synthesis in either cell type. To our knowledge, there have been no reports in which the effects of FGF-2 during mono-layer expansion on subsequent activities of tendon-derived cells have been described. In vitro studies26,42 conducted to evaluate the effect of FGF-2 on bone marrow–derived MSCs have revealed mitogenic effects and a protective effect on subsequent multilineage potential during proliferation in vitro. Furthermore, in vivo studies27–30 that involved intratendinous injections of FGF-2 in mice and dogs indicated angiogenic stimulation in the early stages of tendon healing. On the basis of results of the present study, researchers should consider supplementation of culture medium with FGF-2 to increase the proliferation rate of tendon-derived cells in vitro, without detrimental effects on subsequent matrix synthesis.

In an in vitro study,43 investigators described IGF-I–enhanced tenocyte proliferation and matrix synthesis in equine tendon explants. In the present study, IGF-I increased collagen and proteoglycan synthesis in both tendon and bone marrow–derived cells, although this effect was only seen at a transcriptional level in the bone marrow–derived cells. In addition, sequential administration of IGF-I to FGF-2–expanded tendon-derived cells significantly increased GAG synthesis, compared with GAG synthesis for tendon-derived cells without growth factor supplementation. However, it should be mentioned that results of matrix synthesis in the study reported here were expressed in relation to the total number of cells seeded at the start of the experiment. Therefore, caution should be used for interpretation of the results because this does not account for cell death or proliferation during the course of the experiment. These variables may have altered per-cell biosynthetic rates among the treatment groups. These increases in matrix synthesis suggest that IGF-I supplementation is justified for tissue regeneration applications of both cell types, as supported by results of 2 in vivo studies14,31 in which investigators used collagenase to experimentally induce tendinitis in horses. Both of those in vivo studies14,31 indicated improvement in biomechanical properties, which is a critical outcome for successful tendon repair. In the present study, the improvement in gene expression and matrix synthesis was apparent in bone marrow–derived cells cultured in medium supplemented with IGF-I and was similar to that reported in other in vitro studies.43,44 However, the increases in gene expression and matrix synthesis by bone marrow–derived cells cultured in medium supplemented with IGF-I remained lower than the corresponding activities of tendon-derived cells cultured in medium supplemented with IGF-I.

In the present study, acellular pulverized tendon was used to provide a 3-D substrate for cell adherence with the goal of simulating the in vivo microenvironment of a damaged tendon, in contrast to acellular tendon matrix explants, which provide an intact surface for attachment. In other studies,23,a tendon-derived cells had superior viability and adherence to acellular tendon matrix explant, compared with results for bone marrow–derived cells. In contrast, the results from the present study indicated no difference in viability between tendon- and bone marrow–derived cells cocultured with pulverized matrix suspension. This suggests that the powdered tendon used in the present study has cell adhesion and survival properties that differ from those of acellular tendon matrix explants used previously. Furthermore, acellular tendon explants may provide topographic cues for cell attachment as a result of the organized fiber pattern, which is similar to in vivo conditions. Additional studies need to be conducted to assess the differential effects of specific substrate characteristics on progenitor cell colonization and survival time.

Tenectomies of the lateral digital extensor tendon of the hind limb were performed to obtain tendon-derived cells and tendon matrix. In this technique, a 4 × 1-cm tendon specimen provided sufficient numbers of tendon-derived cells for potential use in clinical applications. This procedure is used clinically for the treatment of refractory stringhalt; however, the long-term safety and morbidity associated with lateral digital extensor tenectomies need further evaluation. In addition, investigators in 1 study45 detected metabolic and homeostatic differences between equine flexor and extensor tenocytes. Common digital extensor tenocytes were less proliferative and had reduced matrix synthetic capacity in vitro, compared with results for superficial and deep digital flexor tenocytes.45 It is possible that cells derived from the lateral digital extensor tendon, although an expedient source of tendon-derived progenitors, are not biosynthetically ideal for the repair or regeneration of injuries to digital flexor tendons in horses.

Analysis of results of the present study revealed that in vitro tendon-derived cells have increased matrix gene expression and matrix synthetic capacity, compared with results for bone marrow–derived cells. In general, growth factor supplementation of culture medium had more pronounced effects on bone marrow–derived cells. However, tendon-derived cells proliferated more rapidly than did bone marrow–derived cells in monolayer culture with FGF-2 supplementation. Also, GAG synthesis of tendon-derived cells was increased after FGF-2 during monolayer expansion and IGF-I supplementation during cell-matrix suspension culture. These beneficial results were obtained via an in vitro method and require careful interpretation and in vivo assessment before these techniques can be used in clinical treatments. On the basis of the results obtained from this study, further in vivo research on the use of tendon-derived cells and growth factor enhancement of these cells for applications in the treatment of tendinitis is warranted.

ABBREVIATIONS

COMP

Cartilage oligomeric matrix protein

CPM

Counts per minute

DMEM

Dulbecco modified Eagle medium

ECM

Extracellular matrix

FBS

Fetal bovine serum

GAG

Glycosaminoglycan

IGF

Insulin-like growth factor

MSC

Mesenchymal stem cell

a.

Durgam SS, Stewart AA, Stewart MC, et al. In vitro comparison of IGF-I enhanced tendon- and bone marrow–derived progenitor cells cultured on tendon matrix (abstr), in Proceedings. 43rd Am Coll Vet Surg Symp 2008;10.

b.

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

c.

Mediumtech Inc, Herndon, Va.

d.

Gemini Bioproducts Inc, Woodland, Calif.

e.

Invitrogen Corp, Carlsbad, Calif.

f.

Penicillin-streptomycin, BioWhittaker, Cambrex BioScience Inc, Walkersville, Md.

g.

R & D Systems Inc, Minneapolis, Minn.

h.

Collagenase type II, Worthington Biochemical Corp, Lakewood, NJ.

i.

SPEX CertiPrep, Matuchen, NJ.

j.

24-well ultra-low attachment plates, Fisher Scientific Inc, Pittsburgh, Pa.

k.

Cell Titer 96 Aqueous One Solution Cell Proliferation Assay, Promega Corp, Madison, Wis.

l.

FLUOstar Optima, BMG Laboratories, Durham, NC.

m.

Trizol, Invitrogen Corp, Carlsbad, Calif.

n.

RNeasy, Qiagen Inc, Valencia, Calif.

o.

Superscript II, Invitrogen Corp, Carlsbad, Calif.

p.

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

q.

Clustal W, multiple-sequence alignment, European Bioinformatics Institute, Cambridge, Cambridgeshire, England.

r.

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

s.

Sigma Chemical Co, St Louis, Mo.

t.

Handheld or postmounted homogenizer, PRO Scientific Inc, Oxford, Conn.

u.

Worthington Biochemical Corp, Lakewood, NJ.

v.

LS6500 multipurpose scintillation counter, Beckman Coulter Inc, Fullerton, Calif.

w.

MP Biochemicals LLC, Irvine, Calif.

x.

Sigma-Aldrich Corp, St Louis, Mo.

y.

PDVF plate, Millipore, Bedford, Mass.

z.

SigmaStat, version 3.0, Systat Software Inc, San Jose, Calif.

aa.

Barrett JG, Stewart AA, Yates AC, et al. Tendon-derived progenitor cells can differentiate toward multiple lineages (abstr), in Proceedings. 34th Annu Conf Vet Orthop Soc 2007;31.

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Appendix

Primers used for real-time PCR amplification of genes of equine bone marrow- and tendon-derived cells.

GeneSequenceAmplicon (bp)
Equine Col ISense: GAA AAC ATC CCA GCC AAG AA 
 Antisense: GAT TGC CAG TCT CCT CAT CC231
Equine Col IIISense: AGG GGA CCT GGT TAC TGC TT 
 Antisense: TCT CTG GGT TGG GAC AGT CT215
Equine COMPSense: TCA TGT GGA AGC AGA TGG AG 
 Antisense: TAG GAA CCA GCG GTA GGA TG223
Equine EF-1αSense: CCC GGA CAC AGA GAC TTC AT 
 Antisense: AGC ATG TTG TCA CCA TTC CA328

COL = Collagen. EF-1α = Elongation factor-1α.

Contributor Notes

Supported by the American Quarter Horse Foundation.

Presented in part as a poster at the 43rd American College of Veterinary Surgeons Symposium, San Diego, October 2008.

Address correspondence to Dr. Allison Stewart (aaw@illinois.edu).