• View in gallery
    Figure 1—

    Log10 mean ± SE cell numbers for tendon- and bone marrow–derived cells cultured on tendon matrix for 7 days with IGF-I (100 ng/mL [white bars]) or without IGF-I (gray bars). Values represent results for samples obtained from 7 horses. *Value differs significantly (P ≤ 0.05) from the corresponding value for the bone marrow–derived cells. †Within a cell type, value differs significantly (P ≤ 0.05) from the value for cells cultured without IGF-I.

  • View in gallery
    Figure 2—

    Log10 mean ± SE disintegrations per minute (DPM) as a measure of incorporation of [3H] proline into collagen of the tendon matrix after seeding with tendon- and bone marrow–derived cells and culture for 7 days. Values represent results for samples obtained from 7 horses. See Figure 1 for key.

  • View in gallery
    Figure 3—

    Log10 mean ± SE CPM as a measure of incorporation of 35S-labeled sodium sulfate into GAG of the tendon matrix after seeding with tendon- and bone marrow–derived cells and culture for 7 days. Values represent results for samples obtained from 7 horses. See Figure 1 for key.

  • View in gallery
    Figure 4—

    Photomicrographs of sections of tendon matrix seeded with equine tendon-derived cells and cultured with (A) and without (B) IGF-I for 7 days or with bone marrow–derived cells and cultured with (C) and without (D) IGF-1 for 7 days. All cells are adhered to the surface of the acellular matrix. Subjectively, tendon-derived cells had a higher number of cells arranged in a linear fashion, compared with results for bone marrow–derived cells. Tendon-derived cells cultured with IGF-I had the highest number of cells, compared with results for the other treatment groups. H&E stain; bar = 50 μm.

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Comparison of equine tendon- and bone marrow–derived cells cultured on tendon matrix with or without insulin-like growth factor-I supplementation

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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Santiago M. Gutierrez-NibeyroDepartment 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, explant colonization, and matrix synthesis of equine tendon- and bone marrow–derived cells in response to insulin-like growth factor-I (IGF-I) supplementation.

Sample—Cells isolated from 7 young adult horses.

Procedures—Tendon- and bone marrow–derived progenitor cells were isolated, evaluated for yield, and cultured on autogenous cell-free tendon matrix for 7 days. Samples were analyzed for cell viability and expression of collagen type I, collagen type III, and cartilage oligomeric matrix protein mRNAs. Collagen and glycosaminoglycan syntheses were quantified over a 24-hour period.

Results—Tendon- and bone marrow–derived cells required 17 to 19 days of monolayer culture to reach 2 passages. Mean ± SE number of monolayer cells isolated was higher for tendon-derived cells (7.9 ± 0.9 × 106) than for bone marrow–derived cells (1.2 ± 0.1 × 106). Cell numbers after culture for 7 days on acellular tendon matrix were 1.6- to 2.8-fold higher for tendon-derived cells than for bone marrow–derived cells and 0.8- to 1.7-fold higher for IGF-I supplementation than for untreated cells. New collagen and glycosaminoglycan syntheses were significantly greater in tendon-derived cell groups and in IGF-I–supplemented groups. The mRNA concentrations of collagen type I, collagen type III, and cartilage oligomeric matrix protein were not significantly different between tendon- and bone marrow–derived groups.

Conclusions and Clinical Relevance—In vitro results of this study suggested that tendon-derived cells supplemented with IGF-I may offer a useful resource for cell-based strategies in tendon healing.

Abstract

Objective—To compare in vitro expansion, explant colonization, and matrix synthesis of equine tendon- and bone marrow–derived cells in response to insulin-like growth factor-I (IGF-I) supplementation.

Sample—Cells isolated from 7 young adult horses.

Procedures—Tendon- and bone marrow–derived progenitor cells were isolated, evaluated for yield, and cultured on autogenous cell-free tendon matrix for 7 days. Samples were analyzed for cell viability and expression of collagen type I, collagen type III, and cartilage oligomeric matrix protein mRNAs. Collagen and glycosaminoglycan syntheses were quantified over a 24-hour period.

Results—Tendon- and bone marrow–derived cells required 17 to 19 days of monolayer culture to reach 2 passages. Mean ± SE number of monolayer cells isolated was higher for tendon-derived cells (7.9 ± 0.9 × 106) than for bone marrow–derived cells (1.2 ± 0.1 × 106). Cell numbers after culture for 7 days on acellular tendon matrix were 1.6- to 2.8-fold higher for tendon-derived cells than for bone marrow–derived cells and 0.8- to 1.7-fold higher for IGF-I supplementation than for untreated cells. New collagen and glycosaminoglycan syntheses were significantly greater in tendon-derived cell groups and in IGF-I–supplemented groups. The mRNA concentrations of collagen type I, collagen type III, and cartilage oligomeric matrix protein were not significantly different between tendon- and bone marrow–derived groups.

Conclusions and Clinical Relevance—In vitro results of this study suggested that tendon-derived cells supplemented with IGF-I may offer a useful resource for cell-based strategies in tendon healing.

Tendinitis is a common cause of breakdown injury in equine athletes and accounts for 30% to 50% of all racing injuries.1–3 Tendon injuries are often degenerative injuries, and the prevalence of tendinitis increases with age.3 Despite improvements in early detection and monitoring, the tissue that is deposited during the process of tendon repair does not restore the original matrix organization and biomechanical properties.4–8 As a consequence, reinjury is common (up to 43% of tendon strain injuries).1

Mesenchymal stem cells are characterized by their ability to maintain considerable proliferative activity and differentiate along several tissue lineages (ie, multi-potency). These characteristics have generated research interest for use in addressing the potential of MSCs in tissue regeneration.9–13 Most of these studies11,12,14 have focused on MSCs derived from bone marrow aspirates. Both bone marrow- and adipose-derived cells have been used empirically for the treatment of tendinitis in horses. Only a few controlled studies15–19 have been reported. It has been indicated in several studies19–22 that alternative sources of progenitor cells might also be beneficial for specific therapeutic applications, which include treatment of tendon injuries. In another study22 conducted by our research group, it was found that tendon- and muscle-derived cells proliferated more rapidly in vitro and had better viability and greater tenogenic matrix production, compared with results for bone marrow–derived cells.

Tendons are composed of longitudinally oriented fibrillar collagen, predominantly collagen type I, that accounts for 75% of the tissue.8,23 Tendons also contain collagen type III, which comprises approximately 14% of total collagen in physiologically normal tendons.24 The COMP is a glycoprotein secreted by tenocytes that helps regulate the diameter of collagen fibrils.25 Synthesis of both collagen and proteoglycan is increased in healing tendons.25,26 Specifically, collagen type III and COMP concentrations are increased in the acute stage of healing, and both participate in collagen type I fibrillogenesis.25,27–30 Independent of the matrix synthesis responses, restoration of the normal architecture of the ECM is critical for effective tendon repair because a high degree of alignment of collagen fibrils is critical for the ability of flexor tendons to withstand high recurrent tensile loads.

Based on in vitro and in vivo studies,31–34 investigators have reported benefits for the administration of growth factor to tenocytes. Exogenous injections and transfection of bone marrow–derived MSCs with IGF-I stimulated tendon healing in horses with tendinitis experimentally induced by the injection of collagenase.15,34 In several studies,31–33 IGF-I stimulated matrix synthesis and ECM production in tendons and ligaments. In early stages of tendon repair, IGF-I protein concentrations decrease by 40%, compared with protein concentrations in physiologically normal tendon; however, by 4 weeks after injury, tissue concentrations of IGF-I peak, although they remain elevated through 8 weeks after injury.26 Therefore, supplementation with exogenous IGF-I during the early phases of tendon repair may provide a therapeutic advantage.15,26,33,34 The plasma concentration of IGF-I ranges from 25 to 82 ng/mL in clinically normal horses.35 In the study reported here, we used an IGF-I concentration of 100 ng/mL, which was selected on the basis of the results of an in vitro flexor tendon explant study33 conducted by other investigators and a stem cell studya conducted by our research group. This IGF-I concentration significantly increases in vitro cell number and matrix synthesis.31,33

The objective of the study reported here was to compare in vitro growth characteristics of tendon- and bone marrow–derived cells during monolayer expansion. We also assessed matrix production and matrix gene expression of monolayer-expanded cells cultured on acellular tendon matrix. The effects of IGF-I supplementation on gene expression and matrix production were assessed in both cell populations. The hypothesis tested was that tendon-derived cells supplemented with IGF-I would grow and persist on matrix better and produce more tendon ECM than would cells derived from bone marrow.

Materials and Methods

Samples—Samples of bone marrow and tendon were collected aseptically from 7 young horses (2 to 4 years of age) euthanized for reasons unrelated to musculoskeletal disease. Horses were sedated with xylazine hydrochloride (0.5 to 1.0 mg/kg, IV), and anesthesia was induced by IV administration of ketamine hydrochloride (2.2 mg/kg) and diazepam (0.1 mg/kg). Anesthesia was maintained by IV infusion of a solution of 5% guaifenesin, 1 mg of ketamine/mL, and 1 mg of xylazine/mL. Bone marrow aspirates were collected as described elsewhere.36 Then, all horses were euthanized by IV administration of sodium pentobarbital (104 mg/kg, IV). Immediately after the horses were euthanized, 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.

Collection and processing of bone marrow–derived cells—The right tuber coxae was clipped of hair and aseptically prepared. A bone marrow biopsy needleb was used to aspirate 15 to 20 mL of bone marrow into syringes that contained 1,000 U of heparin. The bone marrow aspirate was transferred to a centrifuge tube, 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, 100 μg of streptomycin sulfatef/mL, and 1mM sodium pyruvateg/mL. Resuspended cells were placed in a 75-cm2 flask and incubated at 37°C in a 5% carbon dioxide atmosphere with 90% humidity. To obtain adequate cell numbers for subsequent experiments, bone marrow–derived cells were passaged twice. Time to confluence and cell counts at the time of trypsinization were recorded for all cell types at each passage.

Collection and processing of tendon-derived cells—A 6 × 1-cm sample of each superficial digital flexor tendon was reserved for cell isolation. A 2-cm3 sample was snap-frozen in liquid nitrogen for RNA isolation, and the remainder was cryopreserved for cell-free tendon matrix production. The specimen for cell isolation was diced into small 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.i The isolated cells were collected by centrifugation at 300 × g for 5 minutes. Supernatant was removed, and the cell pellet was resuspended in culture medium containing high-glucose DMEM supplemented with 20% FBS, 300 mg of l-glutamine/mL, 100 U of sodium penicillin/mL, and 100 μg of streptomycin sulfate/mL. Cell yields were determined by use of a hemacytometer, and viability was determined via trypan blue dye exclusion.37,g

Culture of tendon cells—Progenitor cells were collected from tendon samples by use of previously described preplating procedures.23,32,j Tendon-derived cells were seeded at 13,300 cells/cm2 in culture flasks in high-glucose DMEM supplemented with 20% FBS, 300 mg of l-glutamine/mL, 100 U of sodium penicillin/mL, and 100 μg of streptomycin sulfate/mL. Rapidly adherent fibroblast-like cells were excluded by differential attachment whereby the culture medium and unattached cells were serially transferred to fresh culture flasks every 24 hours during the first 72 hours of culture.22,38,j Tendon-derived cells that adhered after the transfer at 72 hours were expanded in monolayer for subsequent experiments.

Tendon matrix culture—Samples of superficial digital flexor tendons were cut longitudinally with a dermatome to produce uniformly flat 0.5-mm-thick tendon sheets. These sheets were cut into 1 × 1-cm square explants. The explants were subjected to 4 rounds of freeze-thaw cycles at −80° and 4°C to kill endogenous cells. These cell-free tendon explants (matrix only) were maintained in culture without the addition of cells to serve as negative control samples or seeded with aliquots (125,000 cells) of the monolayer-expanded bone marrow- or tendon-derived cells.

Experiments comprised 5 treatment groups (matrix only, matrix and bone marrow–derived cells, matrix and bone marrow–derived cells with IGF-I,k matrix and tendon-derived cells, and matrix and tendon-derived cells with IGF-I). Each treatment group had 14 replicates. The 14 replicates were distributed into 3 samples for cell numbers, 3 samples for collagen synthesis, 5 samples for GAG synthesis, and 2 samples for histologic evaluation. The mean of all replicates was calculated to yield a single data point for each horse. Total RNA was isolated from 3 of the 7 horses. Treatment groups established for RNA isolation had an additional 12 replicates. There were 70 to 130 samples for each of the 7 horses.

Cell-seeded tendon matrices were supplemented with 1 mL of tenogenic media that contained 0 or 100 ng of IGF-I. Tenogenic media consisted of high-glucose DMEM supplemented with 10% FBS, 37.5 μg of ascorbic acidl/mL, 300 μg of l-glutamine/mL, 100 U of sodium penicillin/mL, and 100 μg of streptomycin sulfate/mL. Media were changed every 2 days. All culture samples were collected on day 7 (day 0 was the first day of cell culture).

Cell numbers on matrices—Three cell matrices from each treatment group were used to measure cell numbers on day 7 via a mitochondrial metabolic assay,m which was used in accordance with the manufacturer's instructions. In brief, 50 μL of the assay reagent containing tetrazolium was added to fresh media in each well, and wells were incubated at 37°C for 2.5 hours. One hundred microliters of media from each well was transferred to a 96-well plate, and absorbance was measured at 492 nm in a microplate readern 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. These optical density values were converted to cell numbers via reference to standard curves determined from plated tendon- and bone marrow–derived cells from each horse.

Collagen synthesis—Collagen synthesis was determined by measuring [3H] prolineo incorporation by use of a published protocol.39 On day 6, 3 cell matrices of each treatment group were radiolabeled with 50 μCi of [3H] proline/mL of tenogenic medium and incubated for 24 hours. The samples were washed 3 times with 0.5 mL of PBS solution containing 1mM proline and stored at −80°C. Radiolabeled samples were frozenthawed 3 times, digested, and homogenized to disrupt cells and matrix 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 collagenaseg and centrifuged at 3,220 × g for 10 minutes. Supernatant and the pelleted material were separated and added to scintillation liquid, and radioactivity was counted by use of a scintillation counter.p Newly synthesized collagen was detected on the basis of radioactivity in the sample supernatants following collagenase digestion.39 Collagen synthesis was normalized for background amounts by subtracting the number of disintegrations per minute of the explant-only control samples. Values were expressed as the number of disintegrations per minute per explant seeded with 125,000 cells.

GAG synthesis—Synthesis of GAG was determined by measuring 35SO4 incorporation into each sample. Five cell matrices of each treatment group were radiolabeled with 10 μCi of 35S-labeled sodium sulfateq/mL during the last 24 hours of the experiment. Samples were washed 3 times with PBS solution and then digested in 1 mL of buffer that contained 0.5 mg of papaing at 65°C for 16 hours.40 Aliquots (25 mL) of 35S-labeled papain-digested tendon matrices were placed in multi-well punch plates,r precipitated with Alcian blue dye, and counted by use of a scintillation counter. All CPM values were adjusted for radioisotope decay from the time of radiolabeling to assay. Proteoglycan synthesis was normalized for background amounts by subtracting the CPM of the explant-only control samples. Values were expressed as the number of CPM per explant seeded with 125,000 cells.

RNA isolation—Sufficient numbers of cells were generated from 3 horses to support transcriptional analyses. Twelve cell matrices from each treatment group were pooled, snap-frozen in liquid nitrogen, and stored at −80°C for RNA isolation. The RNA was isolated by use of a protocol adapted from a technique for cartilage RNA isolation.41 Briefly, tissues were pulverized under liquid nitrogen, homogenized in guanidinium isothiocyanate lysis buffer, extracted with phenol-chloroform, precipitated with isopropanol, and purified by use of a commercially available column-based procedure.s This procedure included an on-column DNase treatment to exclude contamination of the genomic template.

Real-time PCR gene expression—One microgram of RNA from each sample was converted to cDNA by use of a commercial reverse transcription kitt and oligo(dT) primers. Target cDNAs were amplified via real-time PCR assay by use of Taq DNA polymeraseu and gene-specific primers designed by use of a multiple sequence alignment programv from published sequences available in GenBank. Primer specificity was confirmed by cloning and sequencing the PCR products (Appendix 1). Real-time quantitative PCR assay was performed in triplicate for the equine-specific primers, including collagen type I, collagen type III, and COMP mRNAs, and the reference gene, elongation factor-1α (Appendix 2). The generated amplicons were quantified by incorporation of a fluorescent dye by use of a commercial fluorescent PCR detection system.w To relate in vitro expression to in vivo expression, RNA from freshly collected, snap-frozen tendon samples was used as a reference for gene expression analyses. Relative gene expression was quantified by use of the 2−ΔΔCT method.42

Gene expression on northern blots—Northern blot analyses of collagen type I, collagen type III, and COMP mRNAs were performed on samples obtained from 1 horse. Gel electrophoresis and northern blot analyses of RNA samples were performed in accordance with standard protocols.43 Radiolabeled probes were synthesized from gel-purified partial cDNA templates by use of 32P-dCTP and random 9-mer primersx and were purified through spin columns.y Consistency of RNA sample loading was assessed via electrophoresis followed by ethidium bromide staining of the gels. The RNAs were transferred to nylon hybridization membranesz via capillary transfer by use of a high-salt buffer, as described elsewhere.43 Elongation factor-1α was used for normalization of gene expression. Prehybridization, hybridization, and wash conditions were those described in protocols recommended by the manufacturer of the nylon membranes. Northern blot data were quantified by use of a computer workstationaa and commercially available software.bb

Histologic examination—Two tendon samples from each treatment group were fixed in 4% paraformaldehyde and embedded in paraffin in accordance with routine protocols. Sections (thickness of 6 μm) were stained with H&E for evaluation of the cell layers colonizing the matrix surfaces.

Statistical analysis—Mean ± SE value for each variable was calculated for each cell type and IGF-I supplementation status for tendon samples obtained from each of the 7 horses in the study. Background values detected in the matrix-only group were subtracted from values for the other groups. Data for cell number, GAG synthesis, and collagen synthesis were logarithmically transformed to accommodate between-horse variability. In addition, collagen type I, collagen type III, and COMP mRNA expression were normalized on the basis of expression of the reference gene, elongation factor-1α. The effects of cell type and IGF-I supplementation were evaluated by use of generalized estimating equations, which is a method robust to violation of assumptions required for a repeated-measures ANOVA.44 When group differences were detected, pairwise multiple comparisons were conducted by use of nonparametric tests. A statistical programcc was used to perform statistical analyses. Values of P ≤ 0.05 were considered significant. Mean ± SE values were determined for days to confluence and cell number following monolayer expansion of bone marrow- and tendon-derived cells.

Results

Cell isolation and expansion—After the completion of preplating procedures and cell attachment, the cells from bone marrow and tendon proliferated in focal clones of tightly packed cells with fusiform morphological characteristics. During the first passage, bone marrow–derived cells reached focal confluence significantly (P = 0.006) more rapidly than did tendon-derived cells (Table 1). However, during the second passage, tendon-derived cells reached confluence significantly (P = 0.018) more rapidly than did bone marrow–derived cells. After 2 passages, there was no significant (P = 0.127) difference in the number of days to confluence between bone marrow- and tendon-derived cells. Both cell types required 17 to 19 days to complete 2 passages. Cell numbers at the first and second passage were significantly (P = 0.012 and 0.004, respectively) higher for tendon-derived cells than for bone marrow–derived cells. Tendon-derived cell expansion yielded significantly (P = 0.004) more cells (mean of approx 6.7 × 106 more cells), compared with the number of cells in bone marrow–derived cell cultures.

Table 1—

Mean ± SE values for number of days to passage 1 and 2 and number of cells at the time of passage during monolayer culture of bone marrow- and tendon-derived cells.

VariableBone marrowTendon
No. of days to passage 112.8 ± 0.517.3 ± 0.3*
No. of days from passage 1 to passage 24.5 ± 1.02.3 ± 0.3*
Total No. of days to passage 217.3 ± 0.919.5 ± 0.5
No. of cells at passage 1 (× 106)0.28 ± 0.751.18 ± 0.22*
No. of cells at passage 2 (× 106)0.94 ± 0.066.30 ± 0.99*
Total No. of cells for passage 1 and 2 (× 106)1.21 ± 0.117.95 ± 0.86*

Values represent results for samples obtained from 7 horses.

Value differs significantly (P ≤ 0.05) from the value for bone marrow–derived cells.

Cell numbers on matrices—After culture for 7 days, the mean log10 numbers of cells adherent to the acellular tendon matrices were significantly (P < 0.001) increased in the tendon-derived cell groups, compared with the numbers in the bone marrow–derived cell groups (Figure 1). In addition, cell numbers in the IGF-I–treated groups were significantly (P = 0.002) increased, compared with the cell numbers in cell groups cultured without IGF-I. Evaluation of cell numbers for the cell-free matrix-only group revealed that there were no viable cells in these control cultures. Results of statistical analysis for transformed and untransformed data were summarized (Table 2).

Figure 1—
Figure 1—

Log10 mean ± SE cell numbers for tendon- and bone marrow–derived cells cultured on tendon matrix for 7 days with IGF-I (100 ng/mL [white bars]) or without IGF-I (gray bars). Values represent results for samples obtained from 7 horses. *Value differs significantly (P ≤ 0.05) from the corresponding value for the bone marrow–derived cells. †Within a cell type, value differs significantly (P ≤ 0.05) from the value for cells cultured without IGF-I.

Citation: American Journal of Veterinary Research 73, 1; 10.2460/ajvr.73.1.153

Table 2—

Mean ± SE values for number of cells, collagen synthesis, and GAG synthesis for equine bone marrow- and tendon-derived cells cultured with and without IGF-I on a tendon matrix for 7 days.

 Bone marrowTendon
VariableWithout IGF-IWith IGF-1Without IGF-1With IGF-I
No. of cells/explant85,649 ± 43,619190,924 ± 87,173215,430 ± 100,923472,970 ± 105,612
Log10 No. of cells/explant4.81 ± 2.60*8.21 ± 2.36*†10.03 ± 1.8512.93 ± 0.21
Collagen synthesis (DPM/explant)52,361 ± 21,87042,545 ± 11,57461,418 ± 11,95699,987 ± 20,172
Log10 collagen synthesis (Log10 DPM/explant)10.37 ± 0.52*10.40 ± 0.37*10.87 ± 0.2911.29 ± 0.37
GAG synthesis (CPM/explant)6,232 ± 1,57412,776 ± 5,19125,605 ± 8,76449,133 ± 16,520
Log10 GAG synthesis (Log10 CPM/explant)8.41 ± 0.39*8.98 ± 0.42*†9.86 ± 0.3010.53 ± 0.30

Values represent results for samples obtained from 7 horses.

Within a row, value differs significantly (P ≤ 0.05) from the value for the corresponding tendon-derived cells.

Within a cell type within a row, value differs significantly (P ≤ 0.05) from the value for cells cultured without IGF-I.

DPM = Disintegrations per minute.

Collagen synthesis—Mean log10 collagen synthesis was significantly (P < 0.001) increased in the tendon-derived cell groups, compared with synthesis in the bone marrow–derived cell groups (Figure 2). Treatment with IGF-I significantly (P = 0.012) increased the mean log10 collagen synthesis of tendon-derived cells. There was no significant effect of IGF-I supplementation on collagen synthesis by bone marrow–derived cells. Results of statistical analysis for transformed and untransformed data were summarized (Table 2).

Figure 2—
Figure 2—

Log10 mean ± SE disintegrations per minute (DPM) as a measure of incorporation of [3H] proline into collagen of the tendon matrix after seeding with tendon- and bone marrow–derived cells and culture for 7 days. Values represent results for samples obtained from 7 horses. See Figure 1 for key.

Citation: American Journal of Veterinary Research 73, 1; 10.2460/ajvr.73.1.153

GAG synthesis—Mean log10 GAG synthesis was significantly (P = 0.001) increased in the tendon-derived cell groups, compared with GAG synthesis in the bone marrow–derived cell groups (Figure 3). Treatment with IGF-I significantly (P < 0.001) increased the mean log10 GAG synthesis in both the tendon- and bone marrow–derived cell groups. Results of statistical analysis for transformed and untransformed data were summarized (Table 2).

Figure 3—
Figure 3—

Log10 mean ± SE CPM as a measure of incorporation of 35S-labeled sodium sulfate into GAG of the tendon matrix after seeding with tendon- and bone marrow–derived cells and culture for 7 days. Values represent results for samples obtained from 7 horses. See Figure 1 for key.

Citation: American Journal of Veterinary Research 73, 1; 10.2460/ajvr.73.1.153

mRNA expression in the ECM—No mRNA was isolated from the acellular control tendon matrices that were subjected to 4 freeze-thaw cycles. Sufficient quantities of RNA for further analyses were obtained from only 3 of 7 horses in the study because of inherent difficulty in isolating RNA from a relatively small number of viable cells within a large volume of dense, acellular matrix. Quantitative PCR analyses of collagen type I mRNA expression in the cell-matrix groups revealed no significant differences between bone marrow- and tendon-derived cell groups (P = 0.082) or in response to IGF-I administration (P = 0.083; Table 3). Similarly, there was no significant difference in collagen type III expression between tendon-derived cell groups (P = 0.951) or in response to IGF-I (P = 0.322). Although tendon-derived cells expressed 3- to 5-fold more COMP mRNA than did bone marrow–derived cells, the expressions did not differ significantly (P = 0.163). Supplementation with IGF-I did not significantly (P = 0.800) affect COMP mRNA expression.

Table 3—

Results for real-time PCR evaluation of mRNA expression of bone marrow- and tendon-derived cells after culture with and without IGF-I on a tendon matrix for 7 days.

 TendonBone marrow
VariableWithout IGF-IWith IGF-IWithout IGF-IWith IGF-I
Collagen type I1.00 ± 01.76 ± 0.222.12 ± 0.362.09 ± 0.56
Collagen type III1.00 ± 01.27 ± 0.620.96 ± 0.541.07 ± 0.48
COMP1.00 ± 01.15 ± 0.500.37 ± 0.280.21 ± 0.10

Values reported are mean ± SE; values for mRNA expression of collagen type I, collagen type III, and COMP were normalized on the basis of expression for elongation factor-1α. Values represent results for samples obtained from 3 horses. A physiologically normal tendon was used as a positive control sample.

Northern blot analyses of collagen types I and III and COMP mRNAs from total RNA isolated from cells collected from 1 horse had expression profiles that were consistent with the real-time PCR results (Table 4).

Table 4—

Results for northern blot evaluation of mRNA expression for bone marrow- and tendon-derived cells after culture with and without IGF-I on a tendon matrix for 7 days.

  TendonBone marrow
VariableControl tendon*Without IGF-IWith IGF-IWithout IGF-IWith IGF-I
Collagen type I1.061.641.571.492.11
Collagen type III0.393.664.324.323.54
COMP0.810.830.700.820.89

Values reported are the mean mRNA expression of collagen type I, collagen type III, and COMP after normalization on the basis of expression for elongation factor-1α. Values represent results for samples obtained from 3 horses.

Physiologically normal tendon (positive control sample).

Histologic examination—In all cell-treated groups, the cells were predominantly adherent to the surface of the autogenous matrices (Figure 4). Tendon-derived cells were present in higher numbers than were bone marrow–derived cells, and IGF-I treatment increased matrix-associated cell numbers in both cell types, which was consistent with the cell count data. In addition, tendon-derived cells appeared to have a more elongate tenocyte-like appearance, compared with the appearance of the bone marrow–derived cells.

Figure 4—
Figure 4—

Photomicrographs of sections of tendon matrix seeded with equine tendon-derived cells and cultured with (A) and without (B) IGF-I for 7 days or with bone marrow–derived cells and cultured with (C) and without (D) IGF-1 for 7 days. All cells are adhered to the surface of the acellular matrix. Subjectively, tendon-derived cells had a higher number of cells arranged in a linear fashion, compared with results for bone marrow–derived cells. Tendon-derived cells cultured with IGF-I had the highest number of cells, compared with results for the other treatment groups. H&E stain; bar = 50 μm.

Citation: American Journal of Veterinary Research 73, 1; 10.2460/ajvr.73.1.153

Discussion

In the study reported here, initial yields of tendon-derived cells were significantly higher than were yields from bone marrow aspirates, and tendon-derived cells were easier to culture during the first passage. Tendon- and bone marrow–derived cell cultures both required approximately 17 to 19 days to reach second-passage confluence. However, after 2 passages, sufficient numbers of tendon-derived cells were generated for most cell-based treatments that have been used in the treatment of tendinitis.15,19 These results are similar to those of another study22 in which more cells were obtained at confluence from tendon-derived cell cultures than from bone marrow–derived cell cultures. It is possible that bone marrow–derived cells could have provided higher cell yields with different isolation techniques, such as density gradient centrifugation and initial RBC lysis.45,46 Although greater numbers of tendon-derived cells can be obtained in a shorter period, the use of tendon as a source of cells remains a concern for donor site morbidity. These concerns were addressed in a recent studya in which our research group evaluated tendon-derived cells obtained from the lateral digital extensor tendon as a clinically relevant sample. To our knowledge, there were no adverse effects for at least 4 months after tenectomy for collection of tissue from the lateral digital extensor tendon.dd Longer follow-up evaluation of the lateral digital extensor tenectomy site in clinically normal horses is warranted prior to use in clinically affected horses.

In the present study, tendon-derived cells persisted on the acellular matrix and continued to proliferate during the 7-day culture period. In contrast, bone marrow–derived cell numbers did not change from the original seeding density. Supplementation with IGF-I increased (2-fold increase) tendon- and bone marrow–derived cell numbers, compared with cell numbers for unsupplemented cultures. The IGF-I–mediated increase in cell numbers could have resulted from an increase in cell survival rate or cell proliferation. Investigators in several other in vitro studies31–33 have detected similar increases in tenocyte cell numbers with IGF-I supplementation. In a recent study47 it was found that IGF-I supplementation increases cell numbers by reversing cell cycle arrest, which suggests that cell proliferation was a factor in the increase in cell numbers in the study reported here.

Considerable increases in collagen and GAG synthesis were detected in tendon-derived cells, compared with results for bone marrow–derived cells, which was consistent with results of another study.22 Supplementation with IGF-I further increased matrix synthesis by tendon-derived cells, compared with synthesis in unsupplemented control samples. These results are similar to those of several in vivo and in vitro studies15,31–33 in which investigators detected IGF-I enhancement of tendon matrix synthesis. Some of these in vitro anabolic effects of IGF-I are mediated through increases in mitogenesis, with consequent increases in population-wide matrix synthesis. However, it should be mentioned that the matrix synthesis results in the present study were expressed in terms of cell numbers at the start of the experiments and did not account for possible changes in cell numbers as a consequence of differential persistence or proliferation during the course of the experiments. Further evaluation is required before specific conclusions can be drawn regarding the biosynthetic capacities of these cell types and their responses to IGF-I supplementation.

In the present study, there were no significant differences in expression of collagen type I, collagen type III, and COMP mRNAs by cell type or in response to IGF-I supplementation. However, values of P > 0.05 but < 0.10 were obtained for collagen type I (with and without IGF-I supplementation) and collagen type III (without IGF-I supplementation). The mRNA data in the present study were derived from samples of only 3 horses, and this may have been an insufficient sample size to adequately assess differences in mRNA concentrations because of low statistical power. In addition, results from another study22 conducted by our research group revealed significantly greater collagen type III expression by tendon-derived cells than by bone marrow–derived cells (data derived from samples of 4 horses). In that study,22 there was no difference in collagen type I or COMP mRNA expression in these cell types. Furthermore, the results of the present study are similar to those of other studies33,48 in which IGF-I supplementation had minimal effects on ECM gene expression despite overall increases in tendon matrix synthesis. Regardless, increases in matrix synthesis per explant are a more important outcome variable than are changes in mRNA expression.

Results from the present study support the hypothesis that tendon-derived cells supplemented with IGF-I grow and adhere to acellular matrix better and produce more ECM than do cells derived from bone marrow. These results suggest that tendon-derived cells may be a better source for cells used in the repair of tendon injuries. Supplementation with IGF-I enhances cell persistence and proliferation and matrix synthesis. Results of this study further support in vivo evaluation of tendon-derived cells and IGF-I for use in tendon repair.

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, Pondenis H, et al. In-vitro comparison of FGF-2/IGF-I enhanced tendon- and bone marrow–derived progenitor cells cultured on tendon matrix (abstr), in Proceedings. 44th Am Coll Vet Surg Symp 2009;32.

b.

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

c.

Mediatech Inc, Herndon, Va.

d.

Gemini Bioproducts Inc, Woodland, Calif.

e.

l-glutamine, 200mM, Invitogen Corp, Carlsbad, Calif.

f.

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

g.

Sigma-Aldrich Corp, St Louis, Mo.

h.

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

i.

BD Biosciences, Bedford, Mass.

j.

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

k.

R & D Systems Inc, Minneapolis, Minn.

l.

WAKO Chemicals USA Inc, Richmond, Va.

m.

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

n.

FLUOstar Optima, BMG Laboratories, Durham, NC.

o.

Collagenase purified, Worthington Biochemical Corp, Lakewood, NJ.

p.

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

q.

MP Biochemicals LLC, Irvine, Calif.

r.

PDVF plate, Millipore, Bedford, Mass.

s.

RNeasy mini kit, Qiagen Inc, Valencia, Calif.

t.

Superscript II, Invitrogen Corp, Carlsbad, Calif.

u.

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

v.

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

w.

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

x.

Prime-It II random primer labeling kit, Stratagene Corp, La Jolla, Calif.

y.

G-50 Sephadex spin columns, Boehringer-Mannheim, Indianapolis, Ind.

z.

MSI, Westboro, Mass.

aa.

Model 445, Molecular Dynamics Phosphorimager, Amersham Biosciences, Piscataway, NJ.

bb.

ImageQuant software, Molecular Dynamics Phosphorimager, Amersham Biosciences, Piscataway, NJ.

cc.

R, version 2.9.1, R Foundation for Statistical Computing, Vienna, Austria. Available at: www.r-project.org/. Accessed Jun 12, 2009.

dd.

Durgam SS, Stewart AA, Caporali E, et al. Effect of tendon-derived progenitor cells on a collagenase-induced model of tendinitis in horses (abstr), in Proceedings. 36th Vet Orthop Soc Conf 2009;44.

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Appendix 1

Primers used for real-time PCR amplification of genes of equine bone marrow- and tendon-derived cells cultured with and without IGF-I supplementation.

GeneSequenceAmplicon (bp)
Eq Col ISense: GAA AAC ATC CCA GCC AAG AA 
 Antisense: GAT TGC CAG TCT CCT CAT CC231
Eq Col IIISense: AGG GGA CCT GGT TAC TGC TT 
 Antisense: TCT CTG GGT TGG GAC AGT CT215
Eq COMPSense: TCA TGT GGA AGC AGA TGG AG 
 Antisense: TAG GAACCAGCG GTAGGATG223
Eq EF-1αSense: CCC GGA CAC AGA GAC TTC AT 
 Antisense: AGC ATG TTG TCA CCA TTC CA329

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

Appendix 2

The cDNA probes used for northern blot analysis of genes in a study of equine bone marrow- and tendon-derived cells cultured with and without IGF-I supplementation.

GeneSequenceAmplicon (bp)
Eq Col ISense: AGC CAG CAG ATC GAG AAC AT 
 Antisense: CGC CAT ACT CGA ACT GGA AT303
Eq Col IIISense: AAG GGT GAA ACT GGT GAA CG 
 Antisense: AAC TGA AAG CCA CCA TCC AC734
Eq COMPSense: GAC TCA GAC AGC GAT GGT CA 
 Antisense: TAG GAA CCA GCG GTA GGA TG687
Eq EF-1αSense: CCC GGA CAC AGA GAC TTC AT 
 Antisense: AGC ATG TTG TCA CCA TTC CA329

See Appendix 1 for key.

Contributor Notes

Dr. Yates' present address is Franklin Animal Clinic, 2990 N Morton St, Franklin, IN 46131.

Supported by the Grayson-Jockey Club Research Foundation.

Presented in part as an oral presentation at the 44th American College of Veterinary Surgeons Symposium, Washington, DC, October 2009.

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