Interleukin-6 upregulates extracellular matrix gene expression and transforming growth factor ß1 activity of tendon progenitor cells.

BACKGROUND: Prolonged inflammation during tendon healing and poor intrinsic healing capacity of tendon are causal factors associated with tendon structural and functional degeneration. Tendon cells, consisting of mature tenocytes and tendon progenitor cells (TPC) function to maintain tendon structure via extracellular matrix (ECM) synthesis. Tendon cells can succumb to tissue cytokine/chemokine alterations during healing and consequently contribute to tendon degeneration. Interleukin-(IL-)1ß, IL-6 and TNFα are key cytokines upregulated in injured tendons; the specific effects of IL-6 on flexor tendon-derived TPC have not been discerned. METHODS: Passage 3 equine superficial digital flexor tendon (SDFT)-derived TPC were isolated from 6 horses. IL-6 impact on the viability (MMT assay with 0, 1, 5 and 10 ng/mL concentrations), migration (scratch motility assay at 0, 10ng/mL concentration) of TPC in monolayer culture were assessed. IL-6 effect on tendon ECM and chondrogenic gene expression (qRT-PCR), TGFß1 gene expression and activity (ELISA), and MMP-1, -3 and - 13 gene expression of TPC was evaluated. RESULTS: IL-6 decreased TPC viability and migration. IL-6 treatment at 10 ng/mL significantly up-regulated TGFß1 gene expression (6.3-fold; p = 0.01) in TPC, and significantly increased the TGFß1 concentration in cell culture supernates. IL-6 (at 10 ng/mL) significantly up-regulated both tendon ECM (COL1A1:5.3-fold, COL3A1:5.4-fold, COMP 5.5-fold) and chondrogenic (COL2A1:3.9-fold, ACAN:6.2-fold, SOX9:4.8-fold) mRNA expression in TPC. Addition of SB431542, a TGFß1 receptor inhibitor, to TPC in the presence of IL-6, attenuated the up-regulated tendon ECM and chondrogenic genes. CONCLUSION: IL-6 alters TPC phenotype during in vitro monolayer culture. Pro- and anti-inflammatory roles of IL-6 have been implicated on tendon healing. Our findings demonstrate that IL-6 induces TGFß1 activity in TPC and affects the basal TPC phenotype (as evidenced via increased tendon ECM and chondrogenic gene expressions). Further investigation of this biological link may serve as a foundation for therapeutic strategies that modulate IL-6 to enhance tendon healing.

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.

Effect of Cortical Screw Diameter on Reduction and Stabilization of Type III Distal Phalanx Fractures: An Equine Cadaveric Study.

OBJECTIVE: To compare reduction of type III distal phalangeal fractures using 4.5 and 5.5 mm cortical screws placed in lag fashion and an intact hoof capsule model. STUDY DESIGN: Cadaveric experimental study. SAMPLE POPULATION: Hooves from 12 adult horses (n=24). METHODS: Sagittal fractures were created in pairs of distal phalanges after distal interphalangeal joint disarticulation and were reduced with either 4.5 or 5.5 mm cortical screws placed in lag fashion. Contralateral phalanges served as non-reduced controls. Fracture reduction following screw placement was assessed by comparing pre-reduction and post-reduction fracture gap measurements from radiographs using paired t-tests. Effects of incremental loading (0, 135, 270, 540, 800, 1070, and 1335 kg) on fracture gaps in 6 phalanges reduced with 4.5 mm screws and 5 phalanges reduced with 5.5 mm screws were measured from fluoroscopic images and assessed by 2-way ANOVA. Significance was set at P<.05. RESULTS: Type III distal phalanx fractures were reliably created. Only 5.5 mm cortical screws, not 4.5 mm screws, significantly reduced fracture gaps and constrained fracture gap expansion 3 cm distal to the articular surface. Compressive loading closed the fracture gaps at the articular surface in both non-reduced control groups and those reduced with either 5.5 or 4.5 mm screws. CONCLUSION: The 5.5 mm cortical screws were more effective than 4.5 mm screws in reducing type III distal phalanx fractures and restricting distal fracture gap expansion under load.

Imaging horse tendons using multimodal 2-photon microscopy.

Injuries and damage to tendons plague both human and equine athletes. At the site of injuries, various cells congregate to repair and re-structure the collagen. Treatments for collagen injury range from simple procedures such as icing and pharmaceutical treatments to more complex surgeries and the implantation of stem cells. Regardless of the treatment, the level of mechanical stimulation incurred by the recovering tendon is crucial. However, for a given tendon injury, it is not known precisely how much of a load should be applied for an effective recovery. Both too much and too little loading of the tendon could be detrimental during recovery. A mapping of the complex local environment imparted to any cell present at the site of a tendon injury may however, convey fundamental insights related to their decision making as a function of applied load. Therefore, fundamentally knowing how cells translate mechanical cues from their external environment into signals regulating their functions during repair is crucial to more effectively treat these types of injuries. In this paper, we studied systems of tendons with a variety of 2-photon-based imaging techniques to examine the local mechanical environment of cells in both normal and injured tendons. These tendons were chemically treated to instigate various extents of injury and in some cases, were injected with stem cells. The results related by each imaging technique distinguish with high contrast and resolution multiple morphologies of the cells' nuclei and the alignment of the collagen during injury. The incorporation of 2-photon FLIM into this study probed new features in the local environment of the nuclei that were not apparent with steady-state imaging. Overall, this paper focuses on horse tendon injury pattern and analysis with different 2-photon confocal modalities useful for wide variety of application in damaged tissues.

Equine bone marrow MSC-derived extracellular vesicles mitigate the inflammatory effects of interleukin-1ß on navicular tissues in vitro.

BACKGROUND: Safe, efficacious therapy for treating degenerate deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) in navicular horses is critically necessary. While archetypal orthobiologic therapies for navicular disease are used empirically, their safety and efficacy are unknown. Mesenchymal stem cell-derived extracellular vesicles (EV) may overcome several limitations of current orthobiologic therapies. OBJECTIVES: To (1) characterise cytokine and growth factor profiles of equine bone marrow mesenchymal stem cell (BM-MSC)-derived extracellular vesicles (BM-EV) and (2) evaluate the in vitro anti-inflammatory and extracellular matrix (ECM) protective potentials of BM-EV on DDFT and NBF explant co-cultures in an IL-1ß inflammatory environment. STUDY DESIGN: In vitro experimental study. METHODS: Cytokines (IL-1ß, IL-6, IL-10, IL-1ra and TNF-α) and growth factors (TGFß1, VEGF, IGF1 and PDGF) in equine BM-EV isolated via ultracentrifugation and precipitation methods were profiled. Forelimb DDFT and NBF explant co-cultures from seven horses were exposed to media alone, or media containing 2 × 109 ± 0.1 × 109 particles/mL or 10 µg/mL BM-EV (BM-EV), 10 ng/mL interleukin-1ß (IL-1ß), or IL-1ß + BM-EV for 48 h. Co-culture media IL-6, TNF-α, MMP-3, MMP-13 concentrations and explant sulphated glycosaminoglycan (sGAG) content were quantified. RESULTS: IL-6, IGF1 and VEGF concentrations were 102.1 (37.61-256.2) and 182.3 (163.1-226.3), 72.3 (8-175.6) and 2.4 (0.1-2.6), 108.3 (38.3-709.1) and 211.4 (189.1-318.2) pg/mL per 2 × 109 ± 0.1 × 109 particles/mL or 10 µg/mL 10 µg of BM-EV isolated via ultracentrifugation and precipitation methods, respectively. Co-culture media MMP-3 in BM-EV- (p = 0.03) and BM-EV + IL-1ß-treated (p = 0.01) groups were significantly lower than the respective media and IL-1ß groups. DDFT explant sGAG content of BM-EV (p = 0.003) and BM-EV + IL-1ß groups were significantly higher compared with IL-1ß group. MAIN LIMITATIONS: Specimen numbers are limited, in vitro model may not replicate clinical case conditions, lack of non-MSC-derived EV control group. CONCLUSIONS: Equine BM-EV contains IL-6 and growth factors, IGF1 and VEGF. The anti-inflammatory and ECM protective potentials of BM-EV were evident as increased IL-6 and decreased MMP-3 concentrations in the DDFT-NBF explant co-culture media. These results support further evaluation of BM-EV as an acellular and 'off-the-shelf' intra-bursal/intrasynovial therapy for navicular pathologies.

Microfragmented Adipose Tissue Is Equivalent to Platelet-Rich Plasma for Knee Osteoarthritis at 12 Months Posttreatment: A Randomized Controlled Trial.

Background: Platelet-rich plasma (PRP) is an effective treatment for knee osteoarthritis (OA). Microfragmented adipose tissue (MFAT) is another orthobiologic that holds promise, but data supporting its use are limited. Previous studies showed that MFAT created using the Lipogems device was equivalent to PRP created via noncommercial laboratory-based processes. Purpose: To perform a comparison of commercially available MFAT and PRP systems for treatment of knee OA. Study Design: Randomized controlled trial; Level of evidence, 2. Methods: A total of 71 patients with symptomatic knee OA (Kellgren-Lawrence grades 1-4) were randomized to receive a single injection of either leukocyte-rich PRP (Angel; Arthrex) or MFAT (Lipogems) under ultrasound guidance. Patient-reported outcomes (Knee injury and Osteoarthritis Outcome Score [KOOS], visual analog scale for pain with activities of daily living [VAS pain], and Tegner activity level) were recorded at baseline and at 1, 3, 6, and 12 months after injection. The primary outcome was the KOOS-Pain subscale score at 12 months after injection. Results: Overall, 49 patients completed their 12-month follow-up (PRP group, n = 23; MFAT group, n = 26). All demographic features were similar between groups, except that more men were randomized to the PRP group and more women to the MFAT group. At 12 months posttreatment, KOOS-Pain scores improved in both groups, with no significant group difference (PRP, 78 ± 17.9 vs MFAT, 77.8 ± 19.3; P = .69). Similarly, other KOOS subscales, VAS pain scores, and Tegner scores improved at 12 months, with no differences between treatment groups. Conclusion: Both PRP and MFAT injections for knee OA resulted in improved patient-reported outcomes at 12 months posttreatment, with no differences found between treatments. Registration: NCT04351087 (ClinicalTrials.gov identifier).

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.

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.

Platelet-Rich Plasma Versus Microfragmented Adipose Tissue for Knee Osteoarthritis: A Randomized Controlled Trial.

Background: Platelet-rich plasma (PRP) has been established as safe and effective for knee osteoarthritis (OA). Another orthobiologic therapy, microfragmented adipose tissue (MFAT), has gained attention because of its heterogeneous cell population (including mesenchymal stem cells). However, prospective comparative data on MFAT are lacking. Because of the safety, efficacy, and simplicity of PRP, new therapeutics such as MFAT should be compared directly with PRP. Purpose: To compare patient-reported outcomes of a single injection of PRP versus MFAT for knee OA. Study Design: Randomized controlled trial; Level of evidence, 2. Methods: A total of 58 patients with symptomatic knee OA (Kellgren-Lawrence grades 1-4) were randomized to receive a single injection of either leukocyte-rich PRP or MFAT under ultrasound guidance. PRP was created by processing 156 mL of whole blood. MFAT was created by harvesting 30 mL of adipose tissue via standard lipoaspiration. Scores for the Knee injury and Osteoarthritis Outcome Score (KOOS) subscales and visual analog scale for pain with Activities of Daily Living (VAS-ADL) were recorded at baseline and at 1, 3, and 6 months after the injection. The primary outcome was the KOOS-Pain subscore at 6 months after the injection. Results: The PRP group (n = 30) had a mean volume of 5.12 ± 1.12 mL injected. This consisted of a mean platelet count of 2673.72 ± 1139.04 × 103/µL and mean leukocyte count of 25.36 ± 13.27 × 103/µL (67.81% lymphocytes, 18.66% monocytes, and 12.33% neutrophils). The MFAT group (n = 28) had a mean volume of 7.92 ± 3.87 mL injected. The mean total nucleated cell count was 3.56 ± 4.62 million/mL. In both groups, KOOS subscale and VAS-ADL scores improved from baseline, and there was no significant difference between the PRP and MFAT groups in the final KOOS-Pain subscore (80.38 ± 16.07 vs 81.61 ± 16.37, respectively; P = .67) or any other outcome score. Conclusion: A single injection of either PRP or MFAT resulted in a clinically meaningful improvement for patients with knee OA at 6 months, with no difference between treatment groups. Registration: NCT04351087 (ClinicalTrials.gov identifier).

Adipose-Derived Stem Cells, Obesity, and Inflammation: A Systematic Review and Implications for Osteoarthritis Treatment.

ABSTRACT: Adipose is a known source of mesenchymal stem cells that can be used to treat musculoskeletal disorders, such as osteoarthritis. Because obesity often coexists with osteoarthritis, excess adiposity may be a useful source of mesenchymal stem cells. However, obesity is associated with systemic inflammation, which may influence the quality of adipose-derived stem cells. We performed a systematic review of the literature examining adipose-derived stem cell behavior, cytokine, and growth factor profiles from obese and nonobese patients. Two independent reviewers applied the inclusion/exclusion criteria and independently extracted data including mesenchymal stem cell count/viability/behavior, growth factor, and/or cytokine expression. Twenty-two articles met criteria for inclusion. Samples from obese patients had increased mesenchymal stem cell content (n = 6), but decreased proliferative ability (n = 3), and increased expression of interleukin 1 (n = 3), interleukin 6 (n = 3), and tumor necrosis factor α (n = 6). There was also greater macrophage content (n = 4). Weight loss normalized cellular function. In vitro behavior and quality of adipose-derived stem cell are significantly different between obese and nonobese patients. Samples from obese patients had greater adipose-derived stem cell content, lower proliferative ability, increased senescence, and increased proinflammatory cytokine expression. Differences in cellular function should be considered when using adipose to treat musculoskeletal pathology in obese and nonobese patients.

Ex vivo effects of corticosteroids on equine deep digital flexor and navicular fibrocartilage explant cell viability.

OBJECTIVE: To investigate the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on the viability of resident cells within the fibrocartilage on the dorsal surface of the deep digital flexor tendon (FC-DDFT) and fibrocartilage on the flexor surface of the navicular bone (FC-NB) of horses. SAMPLE: 12 to 14 explants of FC-DDFT and of FC-NB from grossly normal forelimbs of 5 cadavers of horses aged 9 to 15 years without evidence of musculoskeletal disease. PROCEDURES: Explants were incubated with culture medium (control) or TA-supplemented (0.6 or 6 mg/mL) or MPA-supplemented (0.5 or 5 mg/mL) medium for 6 or 24 hours. Explant metabolic activity and percentage of dead cells were assessed with a resazurin-based assay and live-dead cell staining, respectively, at each time point. Drug effects were assessed relative to findings for the respective control group. RESULTS: Application of TA (at both concentrations) did not significantly change the cell viability of FC-DDFT explants. For FC-NB explants, TA at 6 mg/mL significantly reduced the metabolic activity and increased the percentage of dead cells at both time points. With either MPA concentration, FC-DDFT and FC-NB explants had reduced metabolic activity and an increased percentage of dead cells at 24 hours, whereas only MPA at 5 mg/mL was cytotoxic at the 6-hour time point. CONCLUSIONS AND CLINICAL RELEVANCE: In ex vivo explants, TA was less cytotoxic to equine FC-DDFT and FC-NB cells, compared with MPA. Further work is warranted to characterize the drugs' transcriptional and translational effects as well as investigate their cytotoxicity at lower concentrations.

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.

Platelet-Rich Plasma Content of Active Spinal Cord Injured Patients: A Controlled Laboratory Study.

OBJECTIVE: Platelet-rich plasma has potential uses for patients with spinal cord injuries. However, no study has quantified the cellular and growth factor content of platelet-rich plasma in this population. This study aimed to analyze (1) platelet-rich plasma content of spinal cord injury subjects and (2) the effect of high-intensity interval exercise on their platelet-rich plasma. DESIGN: Ten spinal cord injury patients and 10 controls were enrolled. At rest, platelet-rich plasma was created from both groups. The spinal cord injury group then performed high-intensity interval exercise and underwent a second blood draw to create post-high-intensity interval exercise platelet-rich plasma. Complete blood counts and growth factor analysis (via enzyme-linked immunosorbent assay) was performed on all platelet-rich plasma. RESULTS: The spinal cord injury group had lower platelets (305,700 ± 85,697/µl vs 380,800 ± 57,301/µl, P = 0.015) and transforming growth factor ß (12.84 ± 1.58 vs 14.33 ± 0.63 ng/ml, P = 0.023). Four minutes of high-intensity interval exercise increased the platelets (305,700 ± 85,697/µl to 399,200 ± 96,251/µl, P = 0.004), leukocytes (906 ± 930 vs 2504 ± 3765/µl, P = 0.002) and transforming growth factor ß (12.84 ± 1.58 to 14.28 ± 1.21 ng/ml, P = 0.020). CONCLUSIONS: Spinal cord injury patients have fewer platelets and transforming growth factor ß in their platelet-rich plasma at baseline compared with controls. Exercise increased platelet, leukocyte, and transforming growth factor ß yield, compensating for the baseline deficits.

Quantitative analysis of collagen fiber organization in injured tendons using Fourier transform-second harmonic generation imaging.

Fourier transform-second harmonic generation (FT-SHG) imaging is used as a technique for evaluating collagenase-induced injury in horse tendons. The differences in collagen fiber organization between normal and injured tendon are quantified. Results indicate that the organization of collagen fibers is regularly oriented in normal tendons and randomly organized in injured tendons. This is further supported through the use of additional metrics, in particular, the number of dark (no/minimal signal) and isotropic (no preferred fiber orientation) regions in the images, and the ratio of forward-to-backward second-harmonic intensity. FT-SHG microscopy is also compared with the conventional polarized light microscopy and is shown to be more sensitive to assessing injured tendons than the latter. Moreover, sample preparation artifacts that affect the quantitative evaluation of collagen fiber organization can be circumvented by using FT-SHG microscopy. The technique has potential as an assessment tool for evaluating the impact of various injuries that affect collagen fiber organization.

Quantitative Assessment of Tendon Hierarchical Structure by Combined Second Harmonic Generation and Immunofluorescence Microscopy.

Histological evaluation of healing tendons is primarily focused on monitoring restoration of longitudinal collagen alignment, although the elastic property of energy-storing flexor tendons is largely attributed to interfascicular sliding facilitated by the interfascicular matrix (IFM). The objectives of this study were to explore the utility of second harmonic generation (SHG) imaging to objectively assess cross-sectional tendon fascicle architecture, to combine SHG microscopy with elastin immunofluorescence to assess the ultrastructure of collagen and elastin in longitudinal and transverse sections, and lastly, to quantify changes in IFM elastin and fascicle collagen alignment of normal and collagenase-injured flexor tendons. Paraffin-embedded transverse and longitudinal histological sections (10-µm thickness) derived from normal and collagenase-injured (6- and 16-week time-points) equine superficial digital flexor tendons were de-paraffinized, treated with Tris EDTA at 80°C for epitope retrieval, and incubated with mouse monoclonal anti-elastin antibody (1:100 dilution) overnight. Anti-mouse IgG Alexa Flour 546 secondary antibody was applied, and sections were mounted with ProLong Gold reagent with 4',6-diamidino-2-phenylindole (DAPI). Nuclei (DAPI) and elastin (Alexa Fluor 546) signals were captured by using standard confocal imaging with 405 and 543 nm excitation wavelengths, respectively. The SHG signal was captured by using a tunable Ti:Sapphire laser tuned to 950 nm to visualize type I collagen. Quantitative measurements of fascicle cross-sectional area (CSA), IFM thickness in transverse SHG-DAPI merged z-stacks, fascicle/IFM elastin area fraction (%), and elastin-collagen alignment in longitudinal SHG-elastin merged z-stacks were conducted by using ImageJ software. Using this methodology, fascicle CSA, IFM thickness, and IFM elastin area fraction (%) at 6 weeks (∼2.25-fold; ∼2.8-fold; 60% decrease; p < 0.001) and 16 weeks (∼2-fold; ∼1.5-fold; 70% decrease; p < 0.001) after collagenase injection, respectively, were found to be significantly different from normal tendon. IFM elastin and fascicle collagen alignment characterized via fast Fourier transform (FFT) frequency plots at 16 weeks demonstrated that collagen re-alignment was more advanced than that of elastin. The integration of SHG-derived quantitative measurements in transverse and longitudinal tendon sections supports comprehensive assessment of tendon structure. Our findings demonstrate the importance of including IFM and non-collagenous proteins in tendon histological evaluations, tasks that can be effectively carried out by using SHG and immunofluorescence microscopy. Impact statement This work demonstrated that second harmonic generation microscopy in conjunction with elastin immunofluorescence provided a comprehensive assessment of multiscale structural re-organization in healing tendon than when restricted to longitudinal collagen fiber alignment alone. Utilizing this approach for tendon histomorphometry is ideal not only to improve our understanding of hierarchical structural changes that occur after tendon injury and during remodeling but also to monitor the efficacy of therapeutic approaches.

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.

High Intensity Interval Exercise Increases Platelet and Transforming Growth Factor-ß Yield in Platelet-Rich Plasma.

BACKGROUND: Platelet-rich plasma (PRP) is an emerging orthobiologic treatment for musculoskeletal conditions like osteoarthritis. Two studies have demonstrated the influence of longer duration exercise on PRP composition, but no study has ever explored the impact of high intensity interval exercise (HIIE) on PRP content. OBJECTIVE: To quantify cellular and growth factor content changes in PRP after 4 minutes of HIIE. DESIGN: Controlled laboratory pilot study. SETTING: Academic sports medicine center. PARTICIPANTS: Ten healthy volunteers (5 male, 5 female). INTERVENTION: Volunteers had PRP prepared from 15 mL of whole blood using a single spin, plasma-based system (autologous conditioned plasma [ACP]) immediately before and after 4 minutes of HIIE on a stationary exercise bike (Tabata protocol). MAIN OUTCOME MEASURE: The PRP was sent for complete blood counts and enzyme-linked immunosorbent assay (ELISA) to quantify transforming growth factor (TGF)-ß, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF)-1, and vascular endothelial growth factor (VEGF). RESULTS: Mean platelet count in PRP increased from 367.4 ± 57.5 k/µL to 497.7 ± 93.3 k/µL after 4 minutes of HIIE (P < .001). TGF-ß also increased from 8237.2 ± 7676.5 pg/mL to 21 535.7 ± 4062.6 pg/mL postexercise (P = .004). The other cellular components (leukocytes, red blood cells, and mean platelet volume) and growth factors (PDGF, IGF-1, and VEGF) were not significantly changed. CONCLUSIONS: A short 4-minute bout of HIIE significantly increased the total platelet count and TGF-ß concentration in PRP.