Intra-articular Recombinant Human Proteoglycan 4 Mitigates Cartilage Damage After Destabilization of the Medial Meniscus in the Yucatan Minipig

Background: Lubricin, or proteoglycan 4 (PRG4), is a glycoprotein responsible for joint boundary lubrication. PRG4 has been shown previously to be down-regulated after traumatic joint injury such as a meniscal tear. Preliminary evidence suggests that intra-articular injection of PRG4 after injury will reduce cartilage damage in rat models of surgically induced posttraumatic osteoarthritis. Objective: To determine the efficacy of intra-articular injection of full-length recombinant human lubricin (rhPRG4) for reducing cartilage damage after medial meniscal destabilization (DMM) in a preclinical large animal model. Study Design: Controlled laboratory study. Methods: Unilateral DMM was performed in 29 Yucatan minipigs. One week after DMM, animals received 3 weekly intra-articular injections (3 mL per injection): (1) rhPRG4 (1.3 mg/mL; n = 10); (2) rhPRG4+hyaluronan (1.3 mg/mL rhPRG4 and 3 mg/mL hyaluronan [~950 kDA]; n = 10); and (3) phosphate-buffered saline (PBS; n = 9). Hindlimbs were harvested 26 weeks after surgery. Cartilage integrity was evaluated by use of macroscopic (India ink) and microscopic (safranin O–fast green and hematoxylin and eosin) scoring systems. Secondary outcomes evaluated via enzyme-linked immunosorbent assay (ELISA) included PRG4 levels in synovial fluid, carboxy-terminal telepeptide of type II collagen (CTX-II) concentrations in urine and serum, and interleukin 1β (IL-1β) levels in synovial fluid and serum. Results: The rhPRG4 group had significantly less macroscopic cartilage damage in the medial tibial plateau compared with the PBS group ( P = .002). No difference was found between the rhPRG4+hyaluronan and PBS groups ( P = .23). However, no differences in microscopic damage scores were observed between the 3 groups ( P = .70). PRG4 production was elevated in the rhPRG4 group synovial fluid compared with the PBS group ( P = .033). The rhPRG4 group presented significantly lower urinary CTX-II levels, but not serum levels, when compared with the PBS ( P = .013) and rhPRG4+hyaluronan ( P = .011) groups. In serum and synovial fluid, both rhPRG4 ( P = .006; P = .017) and rhPRG4+hyaluronan groups ( P = .009; P = .03) presented decreased IL-1β levels. Conclusion: All groups exhibited significant cartilage degeneration after DMM surgery. However, animals treated with rhPRG4 had the least amount of cartilage damage and less inflammation, providing evidence that intra-articular injections of rhPRG4 may slow the progression of posttraumatic osteoarthritis. Clinical Relevance: Patients with meniscal trauma are at high risk for posttraumatic osteoarthritis. This study demonstrates that an intra-articular injection regimen of rhPRG4 may attenuate cartilage damage after meniscal injury.

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CTRP9 protects against MIA-induced inflammation and knee cartilage damage by deactivating the MAPK/NF-κB pathway in rats with osteoarthritis

Open Life Sciences ◽

10.1515/biol-2020-0105 ◽

2020 ◽

Vol 15 (1) ◽

pp. 971-980

Author(s):

Shicheng Zheng ◽

Jing Ren ◽

Sihai Gong ◽

Feng Qiao ◽

Jinlong He

Keyword(s):

Synovial Fluid ◽

Nuclear Factor Kappa B ◽

Cartilage Damage ◽

Cartilage Tissue ◽

Enzyme Linked Immunosorbent Assay ◽

Nuclear Factor ◽

Knee Cartilage ◽

Monosodium Iodoacetate ◽

Dose Dependent ◽

Different Doses

AbstractC1q/TNF-related protein 9 (CTRP9), the closest paralog of adiponectin, has been reported to protect against inflammation-related diseases. However, its role in regulating osteoarthritis (OA) has not been fully elucidated. First, a rat model of OA was generated. Furthermore, rats with OA were injected with different doses of recombinant CTRP9 protein (rCTRP9), and the knee cartilage damage was evaluated. Finally, the phosphorylation of p38 and the secretion of matrix metalloproteinases (MMPs) were detected by Western blotting and enzyme-linked immunosorbent assay. Results revealed that CTRP9 was highly expressed in adipose tissue, followed by skeletal muscle and cartilage tissue, and less expressed in liver, kidney and lung. Moreover, the expression of CTRP9 significantly decreased in the monosodium iodoacetate (MIA) group in the knee cartilage and knee synovial fluid, and the contents of interleukin-1β (IL-1β) and IL-6 significantly increased in knee synovial fluid. In addition, rCTRP9 alleviated MIA-induced inflammation, oxidative stress and knee cartilage damage in a dose-dependent way. In addition, rCTRP9 could attenuate the expression of p38MAPK and p-p38 and suppress the expression of nuclear factor-kappa B (NF-κB), p65 and MMPs. Collectively, the results of the present study suggested that CTRP9 alleviates the inflammation of MIA-induced OA through deactivating p38MAPK and NF-κB signaling pathways in rats.

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A Translational Porcine Model for Human Cell–Based Therapies in the Treatment of Posttraumatic Osteoarthritis After Anterior Cruciate Ligament Injury

The American Journal of Sports Medicine ◽

10.1177/0363546520952353 ◽

2020 ◽

Vol 48 (12) ◽

pp. 3002-3012

Author(s):

Thomas J. Kremen ◽

Tina Stefanovic ◽

Wafa Tawackoli ◽

Khosrowdad Salehi ◽

Pablo Avalos ◽

...

Keyword(s):

Animal Model ◽

Anterior Cruciate Ligament ◽

Synovial Fluid ◽

Human Cell ◽

Bioluminescence Imaging ◽

Cruciate Ligament ◽

Acl Injury ◽

Large Animal Model ◽

Large Animal ◽

Anterior Cruciate

Background: There is a high incidence of posttraumatic osteoarthritis (PTOA) after anterior cruciate ligament (ACL) injury, and these injuries represent an enormous health care economic burden. In an effort to address this unmet clinical need, there has been increasing interest in cell-based therapies. Purpose: To establish a translational large animal model of PTOA and demonstrate the feasibility of intra-articular human cell–based interventions. Study Design: Descriptive laboratory study. Methods: Nine Yucatan mini-pigs underwent unilateral ACL transection and were monitored for up to 12 weeks after injury. Interleukin 1 beta (IL-1β) levels and collagen breakdown were evaluated longitudinally using enzyme-linked immunosorbent assays of synovial fluid, serum, and urine. Animals were euthanized at 4 weeks (n = 3) or 12 weeks (n = 3) after injury, and injured and uninjured limbs underwent magnetic resonance imaging (MRI) and histologic analysis. At 2 days after ACL injury, an additional 3 animals received an intra-articular injection of 107 human bone marrow–derived mesenchymal stem cells (hBM-MSCs) combined with a fibrin carrier. These cells were labeled with the luciferase reporter gene (hBM-MSCs-Luc) as well as fluorescent markers and intracellular iron nanoparticles. These animals were euthanized on day 0 (n = 1) or day 14 (n = 2) after injection. hBM-MSC-Luc viability and localization were assessed using ex vivo bioluminescence imaging, fluorescence imaging, and MRI. Results: PTOA was detected as early as 4 weeks after injury. At 12 weeks after injury, osteoarthritis could be detected grossly as well as on histologic analysis. Synovial fluid analysis showed elevation of IL-1β shortly after ACL injury, with subsequent resolution by 2 weeks after injury. Collagen type II protein fragments were elevated in the synovial fluid and serum after injury. hBM-MSCs-Luc were detected immediately after injection and at 2 weeks after injection using fluorescence imaging, MRI, and bioluminescence imaging. Conclusion: This study demonstrates the feasibility of reproducing the chondral changes, intra-articular cytokine alterations, and body fluid biomarker findings consistent with PTOA after ACL injury in a large animal model. Furthermore, we have demonstrated the ability of hBM-MSCs to survive and express transgene within the knee joint of porcine hosts without immunosuppression for at least 2 weeks. Clinical Relevance: This model holds great potential to significantly contribute to investigations focused on the development of cell-based therapies for human ACL injury–associated PTOA in the future (see Appendix Figure A1 , available online).

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Integrin α10β1-Selected Mesenchymal Stem Cells Mitigate the Progression of Osteoarthritis in an Equine Talar Impact Model

The American Journal of Sports Medicine ◽

10.1177/0363546519899087 ◽

2020 ◽

Vol 48 (3) ◽

pp. 612-623

Author(s):

Michelle L. Delco ◽

Margaret Goodale ◽

Jan F. Talts ◽

Sarah L. Pownder ◽

Matthew F. Koff ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Synovial Fluid ◽

Prostaglandin E2 ◽

Collagen Type ◽

Type Ii ◽

Collagen Type Ii ◽

Posttraumatic Osteoarthritis ◽

India Ink ◽

Joint Trauma

Background: Early intervention with mesenchymal stem cells (MSCs) after articular trauma has the potential to limit progression of focal lesions and prevent ongoing cartilage degeneration by modulating the joint environment and/or contributing to repair. Integrin α10β1 is the main collagen type II binding receptor on chondrocytes, and MSCs that are selected for high expression of the α10 subunit have improved chondrogenic potential. The ability of α10β1-selected (integrin α10high) MSCs to protect cartilage after injury has not been investigated. Purpose: To investigate integrin α10high MSCs to prevent posttraumatic osteoarthritis in an equine model of impact-induced talar injury. Study Design: Controlled laboratory study. Methods: Focal cartilage injuries were created on the tali of horses (2-5 years, n = 8) by using an impacting device equipped to measure impact stress. Joints were treated with 20 × 106 allogenic adipose-derived α10high MSCs or saline vehicle (control) 4 days after injury. Synovial fluid was collected serially and analyzed for protein content, cell counts, markers of inflammation (prostaglandin E2, tumor necrosis factor α) and collagen homeostasis (procollagen II C-propeptide, collagen type II cleavage product), and glycosaminoglycan content. Second-look arthroscopy was performed at 6 weeks, and horses were euthanized at 6 months. Joints were imaged with radiographs and quantitative 3-T magnetic resonance imaging. Postmortem examinations were performed, and India ink was applied to the talar articular surface to identify areas of cartilage fibrillation. Synovial membrane and osteochondral histology was performed, and immunohistochemistry was used to assess type I and II collagen and lubricin. A mixed effect model with Tukey post hoc and linear contrasts or paired t tests were used, as appropriate. Results: Integrin α10high MSC-treated joints had less subchondral bone sclerosis on radiographs ( P = .04) and histology ( P = .006) and less cartilage fibrillation ( P = .04) as compared with control joints. On gross pathology, less India ink adhered to impact sites in treated joints than in controls, which may be explained by the finding of more prominent lubricin immunostaining in treated joints. Prostaglandin E2 concentration in synovial fluid and mononuclear cell synovial infiltrate were increased in treated joints, suggesting possible immunomodulation by integrin α10high MSCs. Conclusion: Intra-articular administration of integrin α10high MSCs is safe, and evidence suggests that the cells mitigate the effects of joint trauma. Clinical Relevance: This preclinical study indicates that intra-articular therapy with integrin α10high MSCs after joint trauma may be protective against posttraumatic osteoarthritis.

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T1ρ Mri Assessment of Cartilage in a Large Animal Model of Traumatic Joint Injury

Osteoarthritis and Cartilage ◽

10.1016/j.joca.2017.02.441 ◽

2017 ◽

Vol 25 ◽

pp. S262-S263

Author(s):

D.R. Thedens ◽

D.J. Heckelsmiller ◽

B.J. Laughlin ◽

M. Saad Eldine ◽

D.R. Pedersen ◽

...

Keyword(s):

Animal Model ◽

Large Animal Model ◽

Large Animal ◽

Joint Injury

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Synovial Fluid Mitochondrial DNA Concentration Reflects the Degree of Cartilage Damage After Naturally Occurring Articular Injury

10.1101/2021.08.26.457571 ◽

2021 ◽

Author(s):

Lindsay A Seewald ◽

Isabella G Sabino ◽

Kaylee L Montney ◽

Michelle L Delco

Keyword(s):

Mitochondrial Dna ◽

Synovial Fluid ◽

Cartilage Damage ◽

Joint Surface ◽

Therapeutic Success ◽

Joint Injury ◽

Naturally Occurring ◽

Molecular Pattern ◽

Extracellular Release ◽

Sensitive Marker

Posttraumatic osteoarthritis (PTOA) is a debilitating sequela to joint injury with no current therapeutics that can slow its progression. Early intervention, prior to the development of degenerative joint changes, has the potential for greater therapeutic success but requires early detection of joint injury. In other tissue types, trauma is associated with the extracellular release of mitochondrial DNA (mtDNA), which serves as a mitochondria-specific Damage Associated Molecular Pattern (mDAMP) to perpetuate inflammation. We demonstrated that chondrocytes release mtDNA following cellular stress and that mtDNA is increased in equine synovial fluid following experimental and naturally occurring mechanical injury to the joint surface. Moreover, we found a strong correlation between the degree of cartilage damage and mtDNA concentration. Finally, impact-induced mtDNA release was mitigated by mitoprotective treatment. These data suggest synovial fluid mtDNA may represent a sensitive marker of early articular injury, prior to the onset of changes on standard diagnostic imaging modalities.

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Synovial fluid levels and serum pharmacokinetics in a large animal model following treatment with oral glucosamine at clinically relevant doses

Arthritis & Rheumatism ◽

10.1002/art.20762 ◽

2005 ◽

Vol 52 (1) ◽

pp. 181-191 ◽

Cited By ~ 92

Author(s):

Sheila Laverty ◽

John D. Sandy ◽

Christophe Celeste ◽

Pascal Vachon ◽

Jean-Francois Marier ◽

...

Keyword(s):

Animal Model ◽

Synovial Fluid ◽

Large Animal Model ◽

Large Animal ◽

Model Following ◽

Fluid Levels

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Traumatic joint injury induces acute catabolic bone turnover concurrent with articular cartilage damage in a rat model of posttraumatic osteoarthritis

Journal of Orthopaedic Research® ◽

10.1002/jor.24903 ◽

2020 ◽

Author(s):

Tristan Maerz ◽

Michael D. Newton ◽

Mackenzie Fleischer ◽

Samantha E. Hartner ◽

Karissa Gawronski ◽

...

Keyword(s):

Articular Cartilage ◽

Bone Turnover ◽

Rat Model ◽

Cartilage Damage ◽

Joint Injury ◽

Posttraumatic Osteoarthritis ◽

Articular Cartilage Damage

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The Effect of Intense Exercise on Equine Serum Proteoglycan-4/Lubricin

Frontiers in Veterinary Science ◽

10.3389/fvets.2020.599287 ◽

2020 ◽

Vol 7 ◽

Author(s):

Austyn Matheson ◽

Suresh C. Regmi ◽

Gregory D. Jay ◽

Tannin A. Schmidt ◽

W. Michael Scott

Keyword(s):

Serum Levels ◽

Enzyme Linked Immunosorbent Assay ◽

Intense Exercise ◽

Joint Injury ◽

Thoroughbred Horses ◽

Proteoglycan 4 ◽

Joint Health ◽

Biomechanical Stimuli ◽

And Function

Objective: Local biological and biomechanical-stimuli modulate proteoglycan-4 secretion within synovial joints. For the horse, changes to proteoglycan-4 concentration and function are notable in acute joint injury and osteoarthritis. Proteoglycan-4 (also known as Lubricin) is present in the blood, however the effect of exercise on equine serum levels is unknown. The overall objective of this study was, therefore, to investigate the effect of intense exercise on serum proteoglycan-4 in thoroughbred horses.Methods: Samples of blood were taken from thoroughbreds (n = 12) during a chuckwagon racing event (Alberta, Canada). The chuckwagon race is a sprint racing event where teams of horses pull a combined 1,325 lbs (601 kg) of wagon and driver around a 5/8th mile (1 km) of dirt track, racing at full gallop to the finish. Blood samples were collected 30-min before the race start, and several timepoints post-race: 5-min, 90-min, 3-h, 12-h, and 23-h. Proteoglycan-4 concentrations in serum were quantified by enzyme-linked-immunosorbent-assay using recombinant-human proteoglycan-4 standards and anti-proteoglycan-4 mAb 9G3. The molecular weight of immunoreactive proteoglycan-4 in serum was assessed by western blot.Results: Proteoglyan-4 in serum demonstrated the expected high MW immunoreactivity to mAb 9G3, consistent with that of full length PRG4. Serum proteoglycan-4 decreased five-minutes post-race from baseline concentration (0.815 ± 0.175 to 0.466 ± 0.090 μg/mL, μ ± SEM, p < 0.01).Conclusions: The concentration of serum proteoglycan-4 in horses decreased significantly five min post-exercise. A potential explanation for this finding could be increased proteoglycan-4 clearance from the circulation. Further investigations could extend to complete the detailed characterization of proteoglycan-4 structure and its potential function within the blood as it relates to joint health and exercise.

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