A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit

Post-traumatic osteoarthritis (PTOA) is responsible for 12% of all osteoarthritis cases in the United States. PTOA can be initiated by a single traumatic ...

Indiana University School of Medicine

Indiana University-Purdue University Indianapolis

Collagen crosslinking enhances many beneficial properties of articular cartilage, including resistance to chemical degradation and mechanical wear, but many crosslinking agents are cytotoxic. The purpose of this study was to evaluate the effectiveness of genipin, a crosslinking agent with favorable biocompatibility and cytotoxicity, as a potential treatment to prevent the degradation and wear of articular cartilage. First, the impact of genipin concentration and treatment duration on the viscoelastic properties of bovine articular cartilage was quantified. Next, two short-term (15 min) genipin crosslinking treatments were chosen, and the change in collagenase digestion, cartilage wear, and the friction coefficient of the tissue with these treatments was measured. Finally, chondrocyte viability after exposure to these genipin treatments was assessed. Genipin treatment increased the stiffness of healthy, intact cartilage in a dose-dependent manner. The 15-min crosslinking treatments improved cartilage's resistance to both chemical degradation, particularly at the articular surface, and to damage due to mechanical wear. These enhancements were achieved without sacrificing the low coefficient of friction of the tissue and at a genipin dose that preserved chondrocyte viability. The results of this study suggest that collagen crosslinking via genipin may be a promising preventative treatment to slow the degradation of cartilage.

Genipin crosslinking of cartilage enhances resistance to biochemical degradation and mechanical wear.

High energy trauma to cartilage causes surface fissures and microstructural damage, but the degree to which this damage renders the tissue more susceptible to wear and contributes to the progression of post-traumatic osteoarthritis (PTOA) is unknown. Additionally, no treatments are currently available to strengthen cartilage after joint trauma and to protect the tissue from subsequent degradation and wear. The purposes of this study were to investigate the role of mechanical damage in the degradation and wear of cartilage, to evaluate the effects of impact and subsequent genipin crosslinking on the changes in the viscoelastic parameters of articular cartilage, and to test the hypothesis that genipin crosslinking is an effective treatment to enhance the resistance to biochemical degradation and mechanical wear. Results demonstrate that cartilage stiffness decreases after impact loading, likely due to the formation of fissures and microarchitectural damage, and is partially or fully restored by crosslinking. The wear resistance of impacted articular cartilage was diminished compared to undamaged cartilage, suggesting that mechanical damage that is directly induced by the impact may contribute to the progression of PTOA. However, the decrease in wear resistance was completely reversed by the crosslinking treatments. Additionally, the crosslinking treatments improved the resistance to collagenase digestion at the impact-damaged articular surface. These results highlight the potential therapeutic value of collagen crosslinking via genipin in the prevention of cartilage degeneration after traumatic injury. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:558-565, 2017.

Genipin crosslinking decreases the mechanical wear and biochemical degradation of impacted cartilage in vitro.

Successful fracture healing requires the simultaneous regeneration of both the bone and vasculature; mesenchymal stem cells (MSCs) are directed to replace the bone tissue, while endothelial progenitor cells (EPCs) form the new vasculature that supplies blood to the fracture site. In the elderly, the healing process is slowed, partly due to decreased regenerative function of these stem and progenitor cells. MSCs from older individuals are impaired with regard to cell number, proliferative capacity, ability to migrate, and osteochondrogenic differentiation potential. The proliferation, migration and function of EPCs are also compromised with advanced age. Although the reasons for cellular dysfunction with age are complex and multidimensional, reduced expression of growth factors, accumulation of oxidative damage from reactive oxygen species, and altered signaling of the Sirtuin-1 pathway are contributing factors to aging at the cellular level of both MSCs and EPCs. Because of these geriatric-specific issues, effective treatment for fracture repair may require new therapeutic techniques to restore cellular function. Some suggested directions for potential treatments include cellular therapies, pharmacological agents, treatments targeting age-related molecular mechanisms, and physical therapeutics. Advanced age is the primary risk factor for a fracture, due to the low bone mass and inferior bone quality associated with aging; a better understanding of the dysfunctional behavior of the aging cell will provide a foundation for new treatments to decrease healing time and reduce the development of complications during the extended recovery from fracture healing in the elderly.

Dysfunctional stem and progenitor cells impair fracture healing with age.

Limitations in scaffold material properties, such as sub-optimal degradation time, highlight the need for alternative approaches to engineer  tissues. One emerging solution for fabricating tissue constructs is scaffold-free tissue engineering. To facilitate this approach, three-dimensional (3D) bioprinting technology (Regenova Bio 3D Printer) has been developed to construct complex geometric shapes from discrete cellular spheroids without exogenous scaffolds. Optimizing spheroid fabrication and characterizing cellular behavior in the spheroid environment are important first steps prior to printing larger constructs. Here, we characterized spheroids of immortalized mouse bone marrow stromal cells (BMSCs) that were differentiated to the osteogenic lineage. Immortalized BMSCs were seeded in low attachment 96-well plates in various numbers to generate self-aggregated spheroids either under the force of gravity or centrifugation. Cells were cultured in control or osteogenic media for up to 28 days. Spheroid diameter, roundness and smoothness were measured. Cell viability, DNA content and alkaline phosphatase activity were assessed at multiple time points. Additionally, expression of osteogenic markers was determined using real time qPCR. Spheroids formed under gravity with 20 K, 30 K and 40 K cells had average diameters of 498.5 ± 8.3 μm, 580.0 ± 32.9 μm and 639.2 ± 54.0 μm, respectively, while those formed under 300G centrifugation with the same numbers of cells had average diameters of 362.3 ± 3.5 μm, 433.1 ± 6.4 μm and 491.2 ± 8.0 μm. Spheroids formed via centrifugation were superior to those formed by gravity, as evidenced by better roundness and smoothness and double the retention of DNA (cellular) content. Cells in spheroids exhibited a robust osteogenic response to the differentiation medium, including higher mRNA expression of alkaline phosphatase, collagen type I, and osteocalcin than those cultured in control medium, as well as greater alkaline phosphatase activity. The optimal spheroid fabrication technique from this study was to aggregate 40K cells under 150-300G centrifugation. In future investigations, these spheroids will be 3D printed into larger tissue constructs.

Scaffold-free bioprinting of mesenchymal stem cells using the Regenova printer: Spheroid characterization and osteogenic differentiation.

The Kenzan bioprinting method provides a high-resolution biofabrication process by facilitating the fusion of submillimeter cell aggregates (spheroids) into larger tissue constructs on a needle array that is removed upon spheroid fusion. Although the method is relatively straightforward in principle, Kenzan method bioprinting relies on a complex 3D bioprinter (Regenova Bio 3D Printer, Cyfuse, K.K., Japan) implementing an advanced vision system to verify the microscopic spheroids' geometry and high-precision mechatronics to aseptically manipulate the spheroids into position. Due to the complexity of the operation, the need for aseptic conditions, and the size of the spheroids, proficiency with the Regenova Bio 3D Printer and the Kenzan method requires development of best practices and troubleshooting techniques to ensure a robust print and minimize the use of resources. In addition, managing the construct post-bioprinting both in culture and for surgical implantation requires careful consideration and workflow design. Here, we describe methods for generating a competent tissue construct and optimizing the bioprinting process. Optimization resulted in a 4-fold reduction in print times, a 20-fold reduction in the use of bioprinting nozzles, and more robust constructs. The results and procedures described herein will have potential applications for tissue engineering, research, and clinical uses in the future.

Scaffold-free Bioprinting of Mesenchymal Stem Cells with the Regenova Printer: Optimization of Printing Parameters.

Many material properties of articular cartilage are anisotropic, particularly in the superficial zone where collagen fibers have a preferential direction. However, the anisotropy of cartilage wear had not been previously investigated. The objective of this study was to evaluate the anisotropy of cartilage material behavior in an in vitro wear test. The wear and coefficient of friction of bovine condylar cartilage were measured with loading in directions parallel (longitudinal) and orthogonal (transverse) to the collagen fiber orientation at the articular surface. An accelerated cartilage wear test was performed against a T316 stainless-steel plate in a solution of phosphate buffered saline with protease inhibitors. A constant load of 160 N was maintained for 14000 cycles of reciprocal sliding motion at 4 mm/s velocity and a travel distance of 18 mm in each direction. The contact pressure during the wear test was approximately 2 MPa, which is in the range of that reported in the human knee and hip joint. Wear was measured by biochemically quantifying the glycosaminoglycans (GAGs) and collagen that was released from the tissue during the wear test. Collagen damage was evaluated with collagen hybridizing peptide (CHP), while visualization of the tissue composition after the wear test was provided with histologic analysis. Results demonstrated that wear in the transverse direction released about twice as many GAGs than in the longitudinal direction, but that no significant differences were seen in the amount of collagen released from the specimens. Specimens worn in the transverse direction had a higher intensity of CHP stain than those worn in the longitudinal direction, suggesting more collagen damage from wear in the transverse direction. No anisotropy in friction was detected at any point in the wear test. Histologic and CHP images demonstrate that the GAG loss and collagen damage extended through much of the depth of the cartilage tissue, particularly for wear in the transverse direction. These results highlight distinct differences between cartilage wear and the wear of traditional engineering materials, and suggest that further study on cartilage wear is warranted. A potential clinical implication of these results is that orienting osteochondral grafts such that the direction of wear is aligned with the primary fiber direction at the articular surface may optimize the life of the graft.

Anisotropic properties of articular cartilage in an accelerated in vitro wear test.

To investigate the role of Cacalmodulin-dependent protein kinase 2 (CaMKK2) in post-traumatic osteoarthritis (PTOA).

Systemic inhibition or global deletion of CaMKK2 protects against post-traumatic osteoarthritis.

The objective of this study was to evaluate photochemical crosslinking using Al(III) phthalocyanine chloride tetrasulfonic acid (CASPc) and light with a wavelength of 670 nm as a potential therapy to strengthen articular cartilage and prevent tissue degradation.

A Photochemical Crosslinking Approach to Enhance Resistance to Mechanical Wear and Biochemical Degradation of Articular Cartilage.

A better understanding of molecular events following cartilage injury is required to develop treatments that prevent or delay the onset of trauma-induced osteoarthritis. In this study, alterations to SIRT1 activity in bovine articular cartilage explants were evaluated in the 24 h following a mechanical overload, and the effect of pharmacological SIRT1 activator SRT1720 on acute chondrocyte injury was assessed. SIRT1 enzymatic activity decreased as early as 5 min following the mechanical overload, and remained suppressed for at least 24 h. The chondrocyte injury response, including apoptosis, oxidative stress, secretion of inflammatory mediators, and alterations in cartilage matrix expression, was prevented with pharmacological activation of SIRT1 in a dose-dependent manner. Overall, the results implicate SIRT1 deactivation as a key molecular event in chondrocyte injury following a mechanical impact overload. As decreased SIRT1 signaling is associated with advanced age, these findings suggest that downregulated SIRT1 activity may be common to both age-related and injury-induced osteoarthritis.

Decreased SIRT1 Activity Is Involved in the Acute Injury Response of Chondrocytes to Ex Vivo Injurious Mechanical Overload.

Healthy articular cartilage exhibits remarkable resistance to wear, sustaining mechanical loads and relative motion for decades. However, tissues that replace or repair cartilage defects are much less long lasting. Better information on the compositional and material characteristics that contribute to the wear resistance of healthy cartilage could help guide strategies to replace and repair degenerated tissue. The main objective of this study was to assess the relationship between wear of healthy articular cartilage, its biochemical composition, and its viscoelastic material properties. The correlation of these factors with the coefficient of friction during the wear test was also evaluated. Viscoelastic properties of healthy bovine cartilage were determined via stress relaxation indentation. The same specimens underwent an accelerated, in vitro wear test, and the amount of glycosaminoglycans (GAGs) and collagen released during the wear test were considered measures of wear. The frictional response during the wear test was also recorded. The GAG, collagen and water content and the concentration of the enzymatic collagen crosslink pyridinoline were quantified in tissue that was adjacent to each wear test specimen. Finally, correlation analysis was performed to identify potential relationships between wear characteristics of healthy articular cartilage with its composition, viscoelastic material properties and friction. The findings suggest that stiffer cartilage with higher GAG, collagen and water content has a higher wear resistance. Enzymatic collagen crosslinks also enhance the wear resistance of the collagen network. The parameters of wear, composition, and mechanical stiffness of cartilage were all correlated with one another, suggesting that they are interrelated. However, friction was largely independent of these in this study. The results identify characteristics of healthy articular cartilage that contribute to its remarkable wear resistance. These data may be useful for guiding techniques to restore, regenerate, and stabilize cartilage tissue.

Diane Wagner

Department of Orthopaedic Surgery,
Indiana Center for Musculoskeletal Health

Indiana University School of Medicine

A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit

Diane Wagner

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Orthopaedic Surgery,Indiana Center for Musculoskeletal Health

Indiana University School of Medicine

A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit

Department of Orthopaedic Surgery,
Indiana Center for Musculoskeletal Health