Macrophage phenotype impacts in vitro equine intrasynovial deep digital flexor tenocyte matrix metalloproteinase gene expression and secretion.

OBJECTIVE: To investigate matrix metalloproteinase (MMP) and their inhibitors tissue inhibitor matrix metalloproteinase (TIMP) gene expression and secretion during equine deep digital flexor tendon (DDFT) tenocyte and macrophage (undifferentiated, proinflammatory, and regulatory) co-culture. SAMPLE: Third passage DDF tenocytes and donor-matched macrophages differentiated from peripheral blood CD14+ monocytes from 5 healthy horses ages 9-11 years, euthanized for reasons unrelated to musculoskeletal conditions. METHODS: Passage 3 DDT tenocyte aggregate cultures were co-cultured with undifferentiated (control), proinflammatory (granulocyte-macrophage colony-stimulating factor; GM-CSF pretreated and lipopolysaccharide + interferon gamma-primed; LPS+IFN-γ) or regulatory (interleukin-4 and interleukin-10-primed; IL-4 + IL-10) macrophages in direct and transwell co-cultures for 72 hours. MMP-1, -2, -3, -9, -13, and TIMP -1, -2 mRNA were measured via real-time Polymerase Chain Reaction (rtPCR). Co-culture media MMP -3, -9, and TIMP -1, -2 concentrations were quantified via ELISA. RESULTS: Direct co-culture of DDF tenocytes with proinflammatory macrophages for 72 hours increased MMP-1, -3, and -13 mRNA levels whereas, MMP-9 mRNA levels decreased. Direct and transwell co-culture with proinflammatory and regulatory macrophages resulted in increased MMP-3 and decreased MMP-9 media concentrations. While direct co-culture with regulatory macrophages significantly increased TIMP-1 mRNA, overall, TIMP mRNA and culture media concentrations were largely unchanged. CLINICAL RELEVANCE: Cell-to-cell contact between DDF tenocytes and macrophages is not essential to induce MMP gene expression and secretion. Co-culture systems offer a viable in vitro platform to screen and evaluate immunomodulatory properties of therapies aimed at improving equine intrasynovial tendon healing.

Basal and inducible Osterix expression reflect equine mesenchymal progenitor cell osteogenic capacity.

Introduction: Mesenchymal stem cells are characterized by their capacities for extensive proliferation through multiple passages and, classically, tri-lineage differentiation along osteogenic, chondrogenic and adipogenic lineages. This study was carried out to compare osteogenesis in equine bone marrow-, synovium- and adipose-derived cells, and to determine whether osteogenic capacity is reflected in the basal expression of the critical osteogenic transcription factors Runx2 and Osterix. Methods: Bone marrow, synovium and adipose tissue was collected from six healthy 2-year-old horses. Cells were isolated from these sources and expanded through two passages. Basal expression of Runx2 and Osterix was assessed in undifferentiated third passage cells, along with their response to osteogenic culture conditions. Results: Bone marrow-derived cells had significantly higher basal expression of Osterix, but not Runx2. In osteogenic medium, bone-marrow cells rapidly developed dense, multicellular aggregates that stained strongly for mineral and alkaline phosphatase activity. Synovial and adipose cell cultures showed far less matrix mineralization. Bone marrow cells significantly up-regulated alkaline phosphatase mRNA expression and enzymatic activity at 7 and 14 days. Alkaline phosphatase expression and activity were increased in adipose cultures after 14 days, although these values were less than in bone marrow cultures. There was no change in alkaline phosphatase in synovial cultures. In osteogenic medium, bone marrow cultures increased both Runx2 and Osterix mRNA expression significantly at 7 and 14 days. Expression of both transcription factors did not change in synovial or adipose cultures. Discussion: These results demonstrate that basal Osterix expression differs significantly in progenitor cells derived from different tissue sources and reflects the osteogenic potential of the cell populations.

Interleukin-1ß and methylprednisolone acetate demonstrate differential effects on equine deep digital flexor tendon and navicular bone fibrocartilage cells in vitro.

OBJECTIVE: To investigate the effects of interleukin-1ß (IL-1ß) and methylprednisolone acetate (MPA) on equine intrabursal deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) cells in vitro. SAMPLE: Third passage DDFT and NBF cells from 5 healthy donor horses ages 11-17 years euthanized for reasons unrelated to musculoskeletal conditions. PROCEDURES: Aggregate cultures were incubated with culture medium alone (control), 10 ng/mL IL-1ß, 10 ng/mL IL-1ß + 0.05 mg/mL MPA, or 10 ng/mL IL-1ß + 0.5 mg/mL MPA for 24 hours. Extracellular matrix (ECM) gene expressions were assessed via real-time polymerase chain reaction (rtPCR). Culture media matrix metalloproteinase (MMP) -3 and -13 concentrations were quantified via ELISA. Total glycosaminoglycan (GAG) content in the cell pellets and culture media was also assessed. RESULTS: IL-1ß and IL-1ß combined with MPA significantly downregulated ECM gene expression to a greater extent in NBF cells compared with DDFT cells. IL-1ß and IL-1ß combined with MPA significantly upregulated MMP-3 culture media concentrations in DDFT cells only, and MMP-13 culture media concentrations to a greater extent in NBF cells compared with DDFT cells. CLINICAL RELEVANCE: NBF cells were more susceptible to IL-1ß and MPA-mediated ECM gene expression downregulation in vitro. These results serve as a first step for future work to determine intrabursal corticosteroid regimens that limits or resolve the inflammation as well as take into consideration NBF cell biosynthesis in horses with navicular disease, for which currently no information exists.

Equine peripheral blood CD14+ monocyte-derived macrophage in-vitro characteristics after GM-CSF pretreatment and LPS+IFN-γ or IL-4+IL-10 differentiation.

Macrophages are a heterogeneous population of immune cells that exhibit dynamic plasticity, polarize into inflammatory or regulatory/pro-resolving macrophages, and influence the healing tissue microenvironment. This study evaluated the in-vitro morphological, proliferative, cell surface marker expression and cytokine/soluble factor secretion characteristics of control, GM-CSF pretreated and inflammatory (LPS+IFN-γ) and regulatory (IL-4 + IL-10) differentiated equine CD14+ monocyte-derived macrophages. Phase contrast microscopy demonstrated that LPS+IFN-γ-primed macrophages exhibited a rounded, granular morphology, whereas IL-4 +IL-10-primed macrophages were elongated with a spindle-shaped morphology. GM-CSF enhanced the proliferation rate of monocytes/macrophages during adherent in-vitro culture. Flow cytometry analysis showed that GM-CSF alone and GM-CSF pretreatment with LPS+IFN-γ or IL-4 +IL-10 priming increased CD86 immunopositivity by 2-fold (p = 0.6); and CD206 immunopositivity remained unchanged. GM-CSF pretreatment and subsequent priming with LPS and IFN-γ yielded inflammatory macrophages that secrete significantly increased quantities of IL-1ß compared to control (p = 0.012) and IL-4 +IL-10-primed (p = 0.0047) macrophages. GM-CSF pretreatment followed by both LPS + IFN-γ and IL-4 + IL-10 priming significantly increased IL-1Ra secretion by 6-fold (p < 0.05). There were no differences in TGFß-1 secretion among control, LPS+IFN-γ or IL-4 + IL-10 primed macrophages (p = 0.85). All groups contained an average of 643 ± 51.5 pg/mL of TGFß1. Among the culture conditions evaluated, IL-4 +IL-10 priming for 24 h after 6 days of adherent culture yielded macrophages that were the least inflammatory compared to GM-CSF pretreated and LPS+IFN-γ or IL-4 +IL-10-primed macrophages. These results provide a basis for subsequent in-vitro and in-vivo studies that investigate macrophage-tissue cell interactions and related biological mechanisms relevant to the field of immunomodulatory approaches for enhancing tissue healing.

Autologous platelet-rich plasma effects on Staphylococcus aureus-induced chondrocyte death in an in vitro bovine septic arthritis model.

OBJECTIVE: To investigate the chondroprotective effects of autologous platelet-rich plasma (PRP), ampicillin-sulbactam (AmpS), or PRP combined with AmpS (PRP+AmpS) in an in vitro chondrocyte explant model of bovine Staphylococcus aureus-induced septic arthritis. SAMPLE: Autologous PRP and cartilage explants obtained from 6 healthy, adult, nonlactating Jersey-crossbred cows. PROCEDURES: Autologous PRP was prepared prior to euthanasia using an optimized double centrifugation protocol. Cartilage explants collected from grossly normal stifle joints were incubated in synovial fluid (SF) alone, S aureus-inoculated SF (SA), or SA supplemented with PRP (25% culture medium volume), AmpS (2 mg/mL), or both PRP (25% culture medium volume) and AmpS (2 mg/mL; PRP+AmpS) for 24 hours. The metabolic activity, percentage of dead cells, and glycosaminoglycan content of cartilage explants were measured with a resazurin-based assay, live-dead cell staining, and dimethylmethylene blue assay, respectively. Treatment effects were assessed relative to the findings for cartilage explants incubated in SF alone. RESULTS: Application of PRP, AmpS, and PRP+AmpS treatments significantly reduced S aureus-induced chondrocyte death (ie, increased metabolic activity and cell viability staining) in cartilage explants, compared with untreated controls. There were no significant differences in chondrocyte death among explants treated with PRP, AmpS, or PRP+AmpS. CLINICAL RELEVANCE: In this in vitro explant model of S aureus-induced septic arthritis, PRP, AmpS, and PRP+AmpS treatments mitigated chondrocyte death. Additional work to confirm the efficacy of PRP with bacteria commonly associated with clinical septic arthritis in cattle as well as in vivo evaluation is warranted.

Zonal characterization and differential trilineage potentials of equine intrasynovial deep digital flexor tendon-derived cells.

BACKGROUND: Intrasynovial deep digital flexor tendon (DDFT) injuries occur frequently and are often implicated in cases of navicular disease with poor outcomes and reinjuries. Cell-based approaches to tendon healing are gaining traction in veterinary medicine and ultimately may contribute to improved DDFT healing in horses. However, a better understanding of the innate cellular characteristics of equine DDFT is necessary for developing improved therapeutic strategies. Additionally, fibrocartilaginous, intrasynovial tendons like the DDFT are common sites of injury and share a poor prognosis across species, offering translational applications of this research. The objective of this study is to isolate and characterize tendon-derived cells (TDC) from intrasynovial DDFT harvested from within the equine forelimb podotrochlear bursa. TDC from the fibrocartilaginous and tendinous zones are separately isolated and assessed. Flow cytometry is performed for mesenchymal stem cell (MSC) surface markers (CD 29, CD 44, CD 90). Basal tenogenic, osteogenic and chondrogenic markers are assessed via quantitative real time-PCR, and standard trilineage differentiation is performed with third passage TDC from the fibrocartilaginous (fTDC) and tendinous (tTDC) zones of DDFT. RESULTS: Low-density plating isolated homogenous TDC populations from both zones. During monolayer passage, both TDC subpopulations exhibited clonogenicity, high in vitro proliferation rate, and fibroblast-like morphology. fTDC and tTDC were positive for MSC surface markers CD90 and CD29 and negative for CD44. There were no significant differences in basal tenogenic, osteogenic or chondrogenic marker expression between zones. While fTDC were largely restricted to chondrogenic differentiation, tTDC underwent osteogenic and chondrogenic differentiation. Both TDC subpopulations displayed weak adipogenic differentiation potentials. CONCLUSIONS: TDC at the level of the podotrochlear bursa, that potentially could be targeted for enhancing DDFT injury healing in horses were identified and characterized. Pending further investigation, promoting chondrogenic properties in cells administered exogenously into the intrasynovial space may be beneficial for intrasynovial tendon regeneration.

In vitro Effects of Methylprednisolone Acetate on Equine Deep Digital Flexor Tendon-Derived Cells.

Primary deep digital flexor tendon (DDFT) pathologies and those accompanying degenerative changes of navicular bone fibrocartilage are major causes of lameness associated with navicular disease. Intrasynovial corticosteroids are mainstay in the treatment due to the anti-inflammatory effects, but their effect on DDFT cell biosynthesis are unknown. The objective of this in-vitro study was to investigate the effects of methylprednisolone acetate (MPA) on cells isolated from the dorsal fibrocartilaginous region of forelimb DDFTs (DDFT-derived cells) of 5 horses (aged 11-17 years). Non-adherent aggregate cultures were established from third passage cells over a 72 to 96-h duration prior to treating with medium containing 0 (control), 0.05 and 0.5 mg/mL MPA for 24 h. Tendon and cartilage extracellular matrix (ECM) related gene expression, cell aggregate and culture medium GAG contents, culture medium collagen and MMP-3 and-13 concentrations were measured. After 24 h of treatment, only the higher MPA concentration (0.5 mg/mL) significantly down-regulated tendon ECM related genes; whereas, both MPA doses significantly down-regulated cartilage ECM related genes. MPA treatment did not affect the total GAG content of DDFT-derived cells or total GAG, soluble collagen and MMP-3 and-13 contents in culture medium compared to untreated controls. Future studies to determine the response of DDFT-derived cells with longer exposure times to corticosteroids and in the presence of inflammatory cytokines are necessary. These results are a first step in assessing the effects of intrasynovial medications on equine DDFT, for which currently no information exists.

Percutaneous ultrasonic tenotomy effectively debrides tendons of the extensor mechanism of the knee: A technical note.

BACKGROUND: Percutaneous ultrasonic tenotomy (PUT) is a minimally-invasive method of treating patellar tendinosis, but its immediate effect on tendon structure has never been studied. Given the crucial nature of the extensor mechanism of the knee, it is important to understand the procedure's effect on tendon structure prior to clinical implementation. The aim of this study was to analyze the tendon structure of the extensor mechanism of the knee after PUT in a cadaveric model. METHODS: Four fresh-frozen cadaveric specimens (two patellar and two quadriceps tendons) underwent PUT. The tendons were then sectioned and stained with hematoxilin & eosin (H&E). The sections were analyzed for a clear area of debridement. The area of debridement was calculated as an average of three measurements. RESULTS: All four tendons demonstrated a clear area of debridement limited to the treatment area without damaging any surrounding tissue. The area of debridement for the patellar and quadriceps tendons treated was 2.89 mm2, 1.5 mm2, 2.98 mm2 and 7.29 mm2, respectively. CONCLUSIONS: Percutaneous ultrasonic tenotomy effectively debrided the treatment area in all tendons without damaging surrounding tissue. Further work is needed to report clinical outcomes, assess the risk of post-procedure tendon rupture and define return-to-sport progression.

Investigation of a novel prosthesis technique for extracapsular stabilization of cranial cruciate ligament-deficient stifle joints in adult cattle.

OBJECTIVE: To evaluate a novel prosthesis technique for extracapsular stabilization of cranial cruciate ligament (CCL)-deficient stifle joints in adult cattle. SAMPLE: 13 cadaveric bovine stifle joint specimens. PROCEDURES: In the first of 3 study phases, the most isometric points on the distal aspect of the femur (distal femur) and proximal aspect of the tibia (proximal tibia) were determined from measurements obtained from lateromedial radiographs of a stifle joint specimen maintained at angles of 135°, 90°, 65°, and 35°. During phase 2, 800-lb-test monofilament nylon leader line was cut into 73-cm-long segments. Each segment was secured in a loop by use of 2, 3, or 4 crimping sleeves such that there were 12 replicates for each construct. Each loop was distracted to failure at a constant rate of 1 mm/s. Mean force at failure and elongation and mode of failure were compared among the 3 constructs. During phase 3, bone tunnels were created in the distal femur and proximal tibia at the isometric points identified during phase 1 in each of 12 CCL-deficient stifle joint specimens. The 3-sleeve construct was applied to each specimen. Specimens were distracted to failure at a constant rate of 1 mm/s. RESULTS: Among the 3 constructs evaluated, the 3-sleeve construct was considered optimal in terms of strength and amount of foreign material. In phase 3, all replicates failed because of suture slippage. CONCLUSIONS AND CLINICAL RELEVANCE: Use of 800-lb-test monofilament nylon leader line as a prosthesis might be a viable alternative for extracapsular stabilization of CCL-deficient stifle joints in adult cattle. Further in vivo studies are necessary.

Cellular Components and Growth Factor Content of Platelet-Rich Plasma With a Customizable Commercial System.

BACKGROUND: Platelet-rich plasma (PRP) is an autologous orthobiologic treatment option for musculoskeletal conditions with favorable results in a limited number of high-quality clinical trials. Because different blood-processing methods result in PRP with varying cellular and growth factor content, it is critical that clinicians understand the content of the specific PRP being used in clinical practice. One adjustable system, the Angel System, has few independent laboratory reports on the specific composition of its PRP. The goal of this study was to quantify the cellular and growth factor composition of PRP produced by this system at its lowest hematocrit settings. HYPOTHESIS: The authors hypothesized that the system would significantly concentrate platelets over baseline and, at the lowest hematocrit settings, would reduce leukocytes to produce leukocyte-poor PRP. STUDY DESIGN: Descriptive laboratory study. METHODS: Ten healthy male volunteers donated 150 mL of whole blood for processing. Three separate processing cycles were performed for each sample at the 0%, 1%, and 2% hematocrit settings. The resultant PRP from each cycle was sent for complete blood counts and enzyme-linked immunosorbent assay to quantify the following growth factors: platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF). RESULTS: The system consistently concentrated platelets 5-fold over baseline, with no significant differences among settings. Leukocytes were concentrated at all settings, between 2 and 5 times over baseline. The 0% and 1% settings had significantly lower leukocyte concentrations than the 2% setting. Lymphocytes made up >89% of the leukocyte differential, while neutrophils represented <11% of the differential at each setting. There was a significant increase in PDGF and bFGF, a significant decrease in IGF-1, and no change in VEGF, with no difference among settings. CONCLUSION: The system consistently concentrated platelets 5 times but was unable to reduce leukocytes, therefore resulting in leukocyte-rich PRP at each setting tested. Leukocytes had a differential composition of >89% lymphocytes and <11% neutrophils. For all settings, PDGF and bFGF were concentrated; IGF-1 was reduced; and VEGF was not significantly different from baseline. CLINICAL RELEVANCE: These data can serve to guide clinicians considering using this particular PRP system. It consistently yielded leukocyte-rich PRP with a lymphocyte-predominant/neutrophil-reduced profile. Further research is needed to better understand how to apply this specific PRP in clinical practice.

Insulin Enhances the In Vitro Osteogenic Capacity of Flexor Tendon-Derived Progenitor Cells.

There is increased incidence of tendon disorders and decreased tendon healing capacity in people with diabetes mellitus (DM). Recent studies have also suggested pathological ossification in repair tendon of people with DM. Therefore, the objective of this study is to investigate the effects of insulin supplementation, an important pathophysiologic stimulus of DM, on tendon progenitor cell (TPC) proliferation and in vitro osteogenic capacity. Passage 3 TPCs were isolated from collagenase-digested, equine superficial digital flexor tendons. TPC proliferation was measured via MTT assay after 3 days of monolayer culture in medium supplemented with 0, 0.007, 0.07, and 0.7 nM insulin. In vitro osteogenic capacity of TPCs (Alizarin Red staining, osteogenic mRNA expression, and alkaline phosphatase bioactivity) was assessed with 0, 0.07, and 0.7 nM insulin-supplemented osteogenic induction medium. Insulin (0.7 nM) significantly increased TPC proliferation after 3 days of monolayer culture. Alizarin Red staining of insulin-treated TPC osteogenic cultures demonstrated robust cell aggregation and mineralized matrix secretion, in a dose-dependent manner. Runx2, alkaline phosphatase, and Osteonectin mRNA and alkaline phosphatase bioactivity of insulin-treated TPC cultures were significantly higher at day 14 of osteogenesis compared to untreated controls. Addition of picropodophyllin (PPP), a selective IGF-I receptor inhibitor, mitigated the increased osteogenic capacity of TPCs, indicating that IGF-I signaling plays an important role. Our findings indicate that hyperinsulinemia may alter TPC phenotype and subsequently impact the quality of repair tendon tissue.

Tendon-derived progenitor cells improve healing of collagenase-induced flexor tendinitis.

Tendinitis is a common and a performance-limiting injury in athletes. This study describes the value of intralesional tendon-derived progenitor cell (TDPC) injections in equine flexor tendinitis. Collagenase-induced tendinitis was created in both front superficial digital flexor (SDF) tendons. Four weeks later, the forelimb tendon lesions were treated with 1 × 107 autogenous TDPCs or saline. Tendinitis was also induced by collagenase in one hind SDF tendon, to study the survival and distribution of DiI-labeled TDPCs 1, 2, 4, and 6 weeks after injection. The remaining normal tendon was used as a "control." Twelve weeks after forelimb TDPC injections, tendons were harvested for assessment of matrix gene expression, biochemical, biomechanical, and histological characteristics. DiI-labeled TDPCs were abundant 1 week after injection but gradually declined over time and were undetectable after 6 weeks. Twelve weeks after TDPC injection, collagens I and III, COMP and tenomodulin mRNA levels were similar (p = 0.3) in both TDPC and saline groups and higher (p < 0.05) than normal tendon. Yield and maximal stresses of the TDPC group were significantly greater (p = 0.005) than the saline group's and similar (p = 0.6) to normal tendon. However, the elastic modulus of the TDPC and saline groups were not significantly different (p = 0.32). Histological assessment of the repair tissues with Fourier transform-second harmonic generation imaging demonstrated that collagen alignment was significantly better (p = 0.02) in TDPC group than in the saline controls. In summary, treating collagenase-induced flexor tendon lesions with TDPCs improved the tensile strength and collagen fiber alignment of the repair tissue. Study Design © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2162-2171, 2016.

Effects of serum and autologous conditioned serum on equine articular chondrocytes treated with interleukin-1ß.

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)-1ß. 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-1ß, 20% AES and rhIL-1ß, 10% ACS and rhIL-1ß, and 20% ACS and rhIL-1ß, and various effects of these treatments were measured. RESULTS: Recombinant human IL-1ß 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-1ß treatment were compared with those of AES and rhIL-1ß 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-1ß versus 10% ACS and rhIL-1ß. The medium from ACS and rhIL-1ß treatment had a higher concentration of IL-1ß receptor antagonist, compared with medium from AES and rhIL-1ß treatment. Treatment with 20% ACS and rhIL-1ß resulted in a higher medium insulin-like growth factor-I concentration than did treatment with 10% AES and rhIL-1ß. No difference in mRNA expression was found between ACS and rhIL-1ß treatment and AES and rhIL-1ß 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.

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

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 × 10(6)) than for bone marrow-derived cells (1.2 ± 0.1 × 10(6)). 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.

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.

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 × 10(6)), compared with bone marrow-derived cells (5.8 ± 1.8 × 10(6)). 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.

Evaluation of experimentally induced injury to the superficial digital flexor tendon in horses by use of low-field magnetic resonance imaging and ultrasonography.

OBJECTIVE: To evaluate tendon injuries in horses over a 16-week period by use of ultrasonography and low-field magnetic resonance imaging (MRI). SAMPLE: Tendons of 8 young adult horses. PROCEDURES: The percentage of experimentally induced tendon injury was evaluated in cross section at the maximal area of injury by use of ultrasonography and MRI at 3, 4, 6, 8, and 16 weeks after collagenase injection. The MRI signal intensities and histologic characteristics of each tendon were determined at the same time points. RESULTS: At 4 weeks after collagenase injection, the area of maximal injury assessed on cross section was similar between ultrasonography and MRI. In lesions of > 4 weeks' duration, ultrasonography underestimated the area of maximal cross-sectional injury by approximately 18%, compared with results for MRI. Signal intensity of lesions on T1-weighted images was the most hyperintense of all the sequences, lesions on short tau inversion recovery images were slightly less hyperintense, and T2-weighted images were the most hypointense. Signal intensity of tendon lesions was significantly higher than the signal intensity for the unaltered deep digital flexor tendon. Histologically, there was a decrease in proteoglycan content, an increase in collagen content, and minimal change in fiber alignment during the 16 weeks of the study. CONCLUSIONS AND CLINICAL RELEVANCE: Ultrasonography may underestimate the extent of tendon damage in tendons with long-term injury. Low-field MRI provided a more sensitive technique for evaluation of tendon injury and should be considered in horses with tendinitis of > 4 weeks' duration.

Effects of sodium hyaluronate and triamcinolone acetonide on glucosaminoglycan metabolism in equine articular chondrocytes treated with interleukin-1.

OBJECTIVE-To determine whether the effects of a high-molecular-weight sodium hyaluronate alone or in combination with triamcinolone acetonide can mitigate chondrocyte glyocosaminoglycan (GAG) catabolism caused by interleukin (IL)-1 administration. SAMPLE POPULATION-Chondrocytes collected from metacarpophalangeal joints of 10 horses euthanized for reasons unrelated to joint disease. PROCEDURES-Chondrocyte pellets were treated with medium (negative control), medium containing IL-1 only (positive control), or medium containing IL-1 with hyaluronic acid only (0.5 or 2.0 mg/mL), triamcinolone acetonide only (0.06 or 0.6 mg/mL), or hyaluronic acid (0.5 or 2.0 mg/mL) and triamcinolone acetonide (0.06 or 0.6 mg/mL) in combination. Chondrocyte pellets were assayed for newly synthesized GAG, total GAG content, total DNA content, and mRNA for collagen type II, aggrecan, and cyclooxygenase (COX)-2. RESULTS-High-concentration hyaluronic acid increased GAG synthesis, whereas high-concentration triamcinolone acetonide decreased loss of GAG into the medium. High concentrations of hyaluronic acid and triamcinolone acetonide increased total GAG content. There was no change in DNA content with either treatment. Triamcinolone acetonide reduced COX-2 mRNA as well as aggrecan and collagen type II expression. Treatment with hyaluronic acid had no effect on mRNA for COX-2, aggrecan, or collagen type II. CONCLUSIONS AND CLINICAL RELEVANCE-Results indicated that high concentrations of hyaluronic acid or triamcinolone acetonide alone or in combination mitigated effects of IL-1 administration on GAG catabolism of equine chondrocytes.

Comparison of equine tendon-, muscle-, and bone marrow-derived cells cultured on tendon matrix.

OBJECTIVE: To compare viability and biosynthetic capacities of cells isolated from equine tendon, muscle, and bone marrow grown on autogenous tendon matrix. SAMPLE POPULATION: Cells from 4 young adult horses. PROCEDURES: Cells were isolated, expanded, and cultured on autogenous cell-free tendon matrix for 7 days. Samples were analyzed for cell viability, proteoglycan synthesis, collagen synthesis, and mRNA expression of collagen type I, collagen type III, and cartilage oligomeric matrix protein (COMP). RESULTS: Tendon- and muscle-derived cells required less time to reach confluence (approx 2 weeks) than did bone marrow-derived cells (approx 3 to 4 weeks); there were fewer bone marrow-derived cells at confluence than the other 2 cell types. More tendon- and muscle-derived cells were attached to matrices after 7 days than were bone marrow-derived cells. Collagen and proteoglycan synthesis by tendon- and muscle-derived cells was significantly greater than synthesis by bone marrow-derived cells. On a per-cell basis, tendon-derived cells had more collagen synthesis, although this was not significant. Collagen type I mRNA expression was similar among groups. Tendon-derived cells expressed the highest amounts of collagen type III and COMP mRNAs, although the difference for COMP was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: Tendon- and muscle-derived cells yielded greater cell culture numbers in shorter time and, on a per-cell basis, had comparable biosynthetic assays to bone marrow-derived cells. More in vitro experiments with higher numbers may determine whether tendon-derived cells are a useful resource for tendon healing.