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.
Objective—To evaluate the effects of glucosamine on equine articular chondrocytes and synoviocytes at concentrations clinically relevant to serum and synovial fluid concentrations.
Sample Population—Articular cartilage and synovium with normal gross appearance from metacarpophalangeal and metatarsophalangeal joints of 8 horses (1 to 10 years of age).
Procedures—In vitro chondrocyte and synoviocyte cell cultures from 8 horses were treated with glucosamine (0.1 to 20 μg/mL) with or without interleukin-1 (IL-1; 10 ng/mL) for 48 hours. Negative control cultures received no glucosamine or IL-1, and positive control cultures received only IL-1. Cultures were assayed for production of proteoglycan (via media containing sulfur 35 (35S)-labeled sodium sulfate and Alcian blue precipitation), prostaglandin E2 (PGE2; via a colorimetric assay), cyclooxygenase-2 (via real-time reverse-transcriptase PCR assay), microsomal PGE2 synthase (mPGEs; via real-time reverse-transcriptase PCR assay), and matrix metalloproteinase (MMP)-13 (via a colorimetric assay).
Results—Glucosamine had no impact on proteoglycan production or MMP-13 production under noninflammatory (no IL-1) or inflammatory (with IL-1) conditions. Glucosamine at 0.1 and 0.5 μg/mL significantly decreased IL-1–stimulated production of mPGEs by chondrocytes, compared with that of positive control chondrocytes. Glucosamine at 0.1 and 5 μg/mL significantly decreased IL-1–stimulated production of mPGEs and PGE2, respectively, compared with that of positive control synoviocytes.
Conclusions and Clinical Relevance—Glucosamine had limited effects on chondrocyte and synoviocyte metabolism at clinically relevant concentrations, although it did have some anti-inflammatory activity on IL-1–stimulated articular cells. Glucosamine may have use at clinically relevant concentrations in the treatment of inflammatory joint disease.
Objective—To evaluate the effects of methylprednisolone acetate (MPA) on proteoglycan production by equine chondrocytes and to investigate whether glucosamine hydrochloride modulates these effects at clinically relevant concentrations.
Sample Population—Articular cartilage with normal gross appearance from metacarpophalangeal and metatarsophalangeal joints of 8 horses (1 to 10 years of age).
Procedures—In vitro chondrocyte pellets were pretreated with glucosamine (0, 1, 10, and 100 μg/mL) for 48 hours and exposed to MPA (0, 0.05, and 0.5 mg/mL) for 24 hours. Pellets and media were assayed for proteoglycan production (Alcian blue precipitation) and proteoglycan content (dimethylmethylene blue assay), and pellets were assayed for DNA content.
Results—Methylprednisolone decreased production of proteoglycan by equine chondrocytes at both concentrations studied. Glucosamine protected proteoglycan production at all 3 concentrations studied.
Conclusions and Clinical Relevance—Methylprednisolone, under noninflammatory conditions present in this study, decreased production of proteoglycan by equine chondrocytes. Glucosamine had a protective effect against inhibition of proteoglycan production at all 3 concentrations studied. This suggested that glucosamine may be useful as an adjunct treatment when an intra-articular injection of a corticosteroid is indicated and that it may be efficacious at concentrations relevant to clinical use.
Objective—To determine whether fibroblast growth factor-2 (FGF-2) treatment of equine mesenchymal stem cells (MSCs) during monolayer expansion enhances subsequent chondrogenesis in a 3-dimensional culture system.
Animals—6 healthy horses, 6 months to 5 years of age.
Procedures—Bone marrow–derived MSCs were obtained from 6 horses. First-passage MSCs were seeded as monolayers at 10,000 cells/cm2 and in medium containing 0, 1, 10, or 100 ng of FGF-2/mL. After 6 days, MSCs were transferred to pellet cultures (200,000 cells/pellet) and maintained in chondrogenic medium. Pellets were collected after 15 days. Pellets were analyzed for collagen type II content by use of an ELISA, total glycosaminoglycan content by use of the dimethylmethylene blue dye–binding assay, and DNA content by use of fluorometric quantification. Semiquantitative PCR assay was performed to assess relative concentrations of collagen type II and aggrecan mRNAs.
Results—Use of 100 ng of FGF-2/mL significantly increased pellet DNA and glycosaminoglycan content. Collagen type II content of the pellet was also increased by use of 10 and 100 ng of FGF-2/mL. Collagen type II and aggrecan mRNA transcripts were increased by treatment with FGF-2. Some control samples had minimal evidence of collagen type II and aggrecan transcripts after 35 cycles of amplification.
Conclusions and Clinical Relevance—FGF-2 treatment of bone marrow–derived MSC monolayers enhanced subsequent chondrogenic differentiation in a 3-dimensional culture. This result is important for tissue engineering strategies dependent on MSC expansion for cartilage repair.
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.
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.
Objective—To determine the effects of sodium hyaluronate (HA) in combination with methylprednisolone acetate (MPA) on interleukin-1 (IL-1)–induced inflammation in equine articular cartilage pellets.
Sample Population—Chondrocytes collected from 7 horses euthanatized for problems unrelated to the musculoskeletal system.
Procedures—Chondrocyte pellets were treated with medium (negative control); medium containing IL-1 (positive control); or medium containing IL-1 with MPA only (0.05 or 0.5 mg/mL), HA only (0.2 or 2 mg/mL), or MPA (0.05 or 0.5 mg/mL) and HA (0.2 or 2 mg/mL) in combination. Proteoglycan (PG) synthesis was determined by incorporation of sulfur 35–labeled sodium sulfate into PGs. Glycosaminoglycan (GAG) content of the media and the pellets and total pellet DNA content were determined.
Results—Methylprednisolone acetate at 0.5 mg/mL caused an increase in PG synthesis, whereas HA had no effect alone. The combination of MPA, both 0.05 mg/mL and 0.5 mg/mL, with HA at 2 mg/mL increased PG synthesis, compared with IL-1–treated control. All treatment groups containing the high concentration of MPA (0.5 mg/mL) and the high concentration of HA (2.0 mg/mL) had pellets with increased GAG content. The addition of HA caused an increase in total GAG content in the media, regardless of MPA treatment. Cyclooxygenase-2 mRNA and aggrecan mRNA expression was significantly reduced with MPA treatment. Total pellet DNA content was unchanged by any treatment.
Conclusions and Clinical Relevance—Our results indicate that MPA in combination with HA has beneficial effects on PG metabolism of IL-1–treated equine chondrocytes.
Objective—To determine concentrations of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in equine chondrocytes and synoviocytes and to quantify changes in the OPG:RANKL ratio in response to exogenous factors.
Sample Population—Samples of articular cartilage and synovium with grossly normal appearance obtained from metacarpophalangeal and metatarsophalangeal joints of 5 adult (1- to 8-year-old) horses.
Procedures—Cell cultures of chondrocytes and synoviocytes were incubated with human recombinant interleukin-1B (hrIL-1β; 10 ng/mL), lipopolysaccharide (LPS; 10 μg/mL), or dexamethasone (100nM) for 48 hours. Negative control cultures received no treatment. Cells and spent media were assayed for RANKL and OPG concentrations by use of western blot and immunocytochemical analyses. Spent media were also assayed for OPG concentration by use of an ELISA.
Results—RANKL and OPG were expressed in equine chondrocytes and synoviocytes in vitro. Cell-associated RANKL and OPG concentrations were not impacted by exogenous factors. Soluble RANKL release into media was significantly increased by hrIL-1β in chondrocyte but not in synoviocyte cultures. Soluble OPG release into media was significantly increased by hrIL-1β and LPS in chondrocyte but not in synoviocyte cultures. The soluble OPG:RANKL ratio was significantly increased by LPS in chondrocyte cultures. Dexamethasone decreased OPG expression in synoviocytes.
Conclusions and Clinical Relevance—RANKL and OPG proteins were expressed in equine articular cells. Release of these proteins may affect osteoclastogenesis within adjacent subchondral bone. Thus, RANKL and OPG may have use as biomarkers and treatment targets in horses with joint disease.
Objective—To compare the effects of autologous equine serum (AES) and autologous conditioned serum (ACS) on equine articular chondrocyte metabolism when stimulated with recombinant human (rh) interleukin (IL)-1β.
Sample—Articular cartilage and nonconditioned and conditioned serum from 6 young adult horses.
Procedures—Cartilage samples were digested, and chondrocytes were isolated and formed into pellets. Chondrocyte pellets were treated with each of the following: 10% AES, 10% AES and rhIL-1β, 20% AES and rhIL-1β, 10% ACS and rhIL-1β, and 20% ACS and rhIL-1β, and various effects of these treatments were measured.
Results—Recombinant human IL-1β treatment led to a decrease in chondrocyte glycosaminoglycan synthesis and collagen II mRNA expression and an increase in medium matrix metalloproteinase-3 activity and cyclooxygenase-2 mRNA expression. When results of ACS and rhIL-1β treatment were compared with those of AES and rhIL-1β treatment, no difference was evident in glycosaminoglycan release, total glycosaminoglycan concentration, total DNA content, or matrix metalloproteinase-3 activity. A significant increase was found in chondrocyte glycosaminoglycan synthesis with 20% AES and rhIL-1β versus 10% ACS and rhIL-1β. The medium from ACS and rhIL-1β treatment had a higher concentration of IL-1β receptor antagonist, compared with medium from AES and rhIL-1β treatment. Treatment with 20% ACS and rhIL-1β resulted in a higher medium insulin-like growth factor-I concentration than did treatment with 10% AES and rhIL-1β. No difference in mRNA expression was found between ACS and rhIL-1β treatment and AES and rhIL-1β treatment.
Conclusions and Clinical Relevance—Minimal beneficial effects of ACS treatment on proteoglycan matrix metabolism in equine chonrocytes were evident, compared with the effects of AES treatment.