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Contribution of collagen degradation and proteoglycan depletion to cartilage degeneration in primary and secondary osteoarthritis: an in silico study

  • S.A. Elahi
    Correspondence
    Address correspondence and reprint requests to: S.A. Elahi, Department of Movement Sciences, KU Leuven, Tervuursevest 101 – Box 1501, 3001 Leuven, Belgium.
    Affiliations
    Department of Movement Sciences, Human Movement Biomechanics Research Group, KU Leuven, Leuven, Belgium

    Mechanical Engineering Department, Biomechanics Section, Soft Tissue Biomechanics Group, KU Leuven, Leuven, Belgium
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  • R. Castro-Viñuelas
    Affiliations
    Department of Movement Sciences, Human Movement Biomechanics Research Group, KU Leuven, Leuven, Belgium

    Department of Development and Regeneration, Skeletal Biology and Engineering Research Centre, Laboratory of Tissue Homeostasis and Disease, KU Leuven, Leuven, Belgium
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  • P. Tanska
    Affiliations
    Department of Applied Physics, University of Eastern Finland, Kuopio, Finland
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  • R.K. Korhonen
    Affiliations
    Department of Applied Physics, University of Eastern Finland, Kuopio, Finland
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  • R. Lories
    Affiliations
    Department of Development and Regeneration, Skeletal Biology and Engineering Research Centre, Laboratory of Tissue Homeostasis and Disease, KU Leuven, Leuven, Belgium

    Division of Rheumatology, University Hospitals Leuven, Leuven, Belgium
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  • N. Famaey
    Affiliations
    Mechanical Engineering Department, Biomechanics Section, Soft Tissue Biomechanics Group, KU Leuven, Leuven, Belgium
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  • I. Jonkers
    Affiliations
    Department of Movement Sciences, Human Movement Biomechanics Research Group, KU Leuven, Leuven, Belgium

    Department of Development and Regeneration, Skeletal Biology and Engineering Research Centre, Laboratory of Tissue Homeostasis and Disease, KU Leuven, Leuven, Belgium
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Open AccessPublished:January 17, 2023DOI:https://doi.org/10.1016/j.joca.2023.01.004

      Summary

      Objectives

      Current experimental approaches cannot elucidate the effect of maladaptive changes on the main cartilage constituents during the degeneration process in osteoarthritis (OA). In silico approaches, however, allow creating ‘virtual knock-out’ cases to elucidate these effects in a constituent-specific manner. We used such an approach to study the main mechanisms of cartilage degeneration in different mechanical loadings associated with the following OA etiologies: (1) physiological loading of degenerated cartilage, (2) injurious loading of healthy intact cartilage and (3) physiological loading of cartilage with a focal defect.

      Methods

      We used the recently developed Cartilage Adaptive REorientation Degeneration (CARED) framework to simulate cartilage degeneration associated with primary and secondary OA (OA cases (1)–(3)). CARED incorporates numerical description of tissue-level cartilage degeneration mechanisms in OA, namely, collagen degradation, collagen reorientation, fixed charged density loss and tissue hydration increase following mechanical loading. We created ‘virtual knock-out’ scenarios by deactivating these degenerative processes one at a time in each of the three OA cases.

      Results

      In the injurious loading of intact and physiological loading of degenerated cartilage, collagen degradation drives degenerative changes through fixed charge density loss and tissue hydration rise. In contrast, the two later mechanisms were more prominent in the focal defect cartilage model.

      Conclusion

      The virtual knock-out models reveal that injurious loading to intact cartilage and physiological loading to degenerated cartilage induce initial degenerative changes in the collagen network, whereas, in the presence of a focal cartilage defect, mechanical loading initially causes proteoglycans (PG) depletion, before changes in the collagen fibril network occur.

      Keywords

      Introduction

      Progressive cartilage degeneration is a key event in the pathophysiology of osteoarthritis (OA)
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      , thereby contributing to the loss of the cartilage's mechanical integrity and accelerating OA development.
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      . However, some of the early structural alterations, more specifically early PG depletion, is considered to be reversible, in contrast to changes in the collagen network
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      Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity.
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      Collagen damage location in articular cartilage differs if damage is caused by excessive loading magnitude or rate.
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      The previous experimental studies suggest that the contribution of distinct aspects of cartilage degradation may differ dependent on OA etiology: (1) first, normal physiological mechanical loading of degenerated cartilage tissue was associated with ECM changes
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      • Lawless B.M.
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      Matrix degradation in osteoarthritis primes the superficial region of cartilage for mechanical damage.
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      Extracellular matrix content and WNT/β-catenin levels of cartilage determine the chondrocyte response to compressive load.
      and increase in surface roughness. Initiation of PG and collagen degeneration was detected (with the former preceding the latter in earlier disease stages)
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      • Grover L.M.
      Matrix degradation in osteoarthritis primes the superficial region of cartilage for mechanical damage.
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      Decreased metalloproteinase production as a response to mechanical pressure in human cartilage: a mechanism for homeostatic regulation.
      , while no additional cell death was observed
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      • Bonilla A.
      • Benito P.
      • et al.
      Decreased metalloproteinase production as a response to mechanical pressure in human cartilage: a mechanism for homeostatic regulation.
      . Each of these changes impairs the mechanical integrity and ‘softens’ the tissue, hence causing further tissue damage
      • Praxenthaler H.
      • Krämer E.
      • Weisser M.
      • Hecht N.
      • Fischer J.
      • Grossner T.
      • et al.
      Extracellular matrix content and WNT/β-catenin levels of cartilage determine the chondrocyte response to compressive load.
      • Monfort J.
      • Garcia-Giralt N.
      • López-Armada M.J.
      • Monllau J.C.
      • Bonilla A.
      • Benito P.
      • et al.
      Decreased metalloproteinase production as a response to mechanical pressure in human cartilage: a mechanism for homeostatic regulation.
      • Dolzani P.
      • Assirelli E.
      • Pulsatelli L.
      • Meliconi R.
      • Mariani E.
      • Neri S.
      Ex vivo physiological compression of human osteoarthritis cartilage modulates cellular and matrix components.
      associated with increased tissue strains. (2) Second, injurious mechanical loading of intact healthy cartilage was suggested to be associated with collagen fiber breakdown at the cartilage surface with cell death and PG depletion starting from the surface but increasing in depth with time
      • Lin P.M.
      • Chen C.T.
      • Torzilli P.A.
      Increased stromelysin-1 (MMP-3), proteoglycan degradation (3B3-and 7D4) and collagen damage in cyclically load-injured articular cartilage.
      ,
      • Loening A.M.
      • James I.E.
      • Levenston M.E.
      • Badger A.M.
      • Frank E.H.
      • Kurz B.
      • et al.
      Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis.
      • Kurz B.
      • Jin M.
      • Patwari P.
      • Cheng D.M.
      • Lark M.W.
      • Grodzinsky A.J.
      Biosynthetic response and mechanical properties of articular cartilage after injurious compression.
      • Li Y.
      • Frank E.H.
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      • Huang H.H.
      • Grodzinsky A.J.
      Moderate dynamic compression inhibits pro-catabolic response of cartilage to mechanical injury, tumor necrosis factor-α and interleukin-6, but accentuates degradation above a strain threshold.
      . (3) Third, physiological mechanical loading of cartilage with focal defects was proposed to be associated with localized FCD loss and localized cell death being the driving degenerative changes
      • Orozco G.A.
      • Tanska P.
      • Florea C.
      • Grodzinsky A.J.
      • Korhonen R.K.
      A novel mechanobiological model can predict how physiologically relevant dynamic loading causes proteoglycan loss in mechanically injured articular cartilage.
      ,
      • Aisenbrey E.A.
      • Tomaschke A.A.
      • Schoonraad S.A.
      • Fischenich K.M.
      • Wahlquist J.A.
      • Randolph M.A.
      • et al.
      Assessment and prevention of cartilage degeneration surrounding a focal chondral defect in the porcine model.
      .
      To evaluate the individual constituent's contribution to OA onset and progression triggered by different etiologies, the role of individual cartilage components in the degeneration process would need to be tested. This is currently not feasible experimentally. Nevertheless, a confounding understanding, how the different factors that contribute to the development of OA and how they interact in the different OA etiologies, is critical for preventing disease onset and progression, as well as optimizing potential treatments.
      In silico models have excellent potential to overcome these limitations of in vivo and in vitro experiments and elucidate the role of individual constituents and their interactions in cartilage degeneration. Several adaptive finite element (FE) mathematical models that integrate physics-based insights on cartilage structure and mechanics and relate these to changes in specific cartilage constituents have been proposed
      • Orozco G.A.
      • Tanska P.
      • Florea C.
      • Grodzinsky A.J.
      • Korhonen R.K.
      A novel mechanobiological model can predict how physiologically relevant dynamic loading causes proteoglycan loss in mechanically injured articular cartilage.
      ,
      • Tanska P.
      • Julkunen P.
      • Korhonen R.
      A computational algorithm to simulate disorganization of collagen network in injured articular cartilage.
      ,
      • Mononen M.E.
      • Tanska P.
      • Isaksson H.
      • Korhonen R.K.
      A novel method to simulate the progression of collagen degeneration of cartilage in the knee: data from the osteoarthritis initiative.
      • Wilson W.
      • Driessen N.J.B.
      • van Donkelaar C.C.
      • Ito K.
      Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm.
      • Quiroga J.M.P.
      • Wilson W.
      • Ito K.
      • van Donkelaar C.C.
      The effect of loading rate on the development of early damage in articular cartilage.
      • Eskelinen A.S.A.
      • Mononen M.E.
      • Venäläinen M.S.
      • Korhonen R.K.
      • Tanska P.
      Maximum shear strain-based algorithm can predict proteoglycan loss in damaged articular cartilage.
      . We recently introduced and verified a Cartilage Adaptive REorientation Degeneration (CARED) model that integrates previous formulations describing collagen degradation, collagen reorientation and FCD loss
      • Elahi S.A.
      • Tanska P.
      • Korhonen R.K.
      • Lories R.
      • Famaey N.
      • Jonkers I.
      An in silico framework of cartilage degeneration that integrates fibril reorientation and degradation along with altered hydration and fixed charge density loss.
      . Further, the CARED model includes a novel formulation relating PG depletion to consequent FCD loss and tissue hydration, consistent with experimental observations
      • Sah R.L.Y.
      • Doong J.Y.H.
      • Grodzinsky A.J.
      • Plaas A.H.K.
      • Sandy J.D.
      Effects of compression on the loss of newly synthesized proteoglycans and proteins from cartilage explants.
      • Roughley P.J.
      • Lee E.R.
      Cartilage proteoglycans: structure and potential functions.
      • Orozco G.A.
      • Tanska P.
      • Florea C.
      • Grodzinsky A.J.
      • Korhonen R.K.
      A novel mechanobiological model can predict how physiologically relevant dynamic loading causes proteoglycan loss in mechanically injured articular cartilage.
      ,
      • Men Y.T.
      • Li X.M.
      • Chen L.
      • Fu H.
      Experimental study on the mechanical properties of porcine cartilage with microdefect under rolling load.
      , thereby adding a new level to existing models
      • Elahi S.A.
      • Tanska P.
      • Korhonen R.K.
      • Lories R.
      • Famaey N.
      • Jonkers I.
      An in silico framework of cartilage degeneration that integrates fibril reorientation and degradation along with altered hydration and fixed charge density loss.
      . This integrated in silico modeling framework provides the unique opportunity to unravel the interaction between collagen degradation and reorientation, FCD loss and tissue hydration as part of OA onset and progression.
      Here, we fully exploited the potential of the CARED model to study the interaction between the compositional changes underlying cartilage degeneration in different OA-associated triggers by selectively deactivating individual adaptive formulations. Indeed, this in silico approach allows to create ‘virtual knock-out’ cases and evaluate their effect on the onset and progression of cartilage degeneration. More specifically, we focused on the role of tissue hydration increase, FCD loss, collagen degradation and reorientation in cartilage degeneration for three OA etiologies: (1) physiological mechanical loading of degenerated cartilage, (2) injurious mechanical loading of healthy intact cartilage and (3) physiological mechanical loading of cartilage with a focal defect. We hypothesized that the degenerative changes in the collagen network and PG content are differently affected depending on the OA etiology.

      Methods

      In silico modeling framework

      Technical details of the in silico FE based adaptive model (CARED model) are described in Supplementary materials. Briefly, cylindrical cartilage plugs were modeled as fibril reinforced poro-viscoelastic swelling (FRPVES) materials subjected to unconfined compression loading (see Section 2.2 for details on applied loading). This material model has been extensively used in previous studies to simulate cartilage mechanics and the sensitivity of mechanical responses to the individual material parameters has been tested
      • Orozco G.A.
      • Tanska P.
      • Florea C.
      • Grodzinsky A.J.
      • Korhonen R.K.
      A novel mechanobiological model can predict how physiologically relevant dynamic loading causes proteoglycan loss in mechanically injured articular cartilage.
      ,
      • Tanska P.
      • Julkunen P.
      • Korhonen R.
      A computational algorithm to simulate disorganization of collagen network in injured articular cartilage.
      ,
      • Mononen M.E.
      • Tanska P.
      • Isaksson H.
      • Korhonen R.K.
      A novel method to simulate the progression of collagen degeneration of cartilage in the knee: data from the osteoarthritis initiative.
      • Wilson W.
      • Driessen N.J.B.
      • van Donkelaar C.C.
      • Ito K.
      Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm.
      • Quiroga J.M.P.
      • Wilson W.
      • Ito K.
      • van Donkelaar C.C.
      The effect of loading rate on the development of early damage in articular cartilage.
      • Eskelinen A.S.A.
      • Mononen M.E.
      • Venäläinen M.S.
      • Korhonen R.K.
      • Tanska P.
      Maximum shear strain-based algorithm can predict proteoglycan loss in damaged articular cartilage.
      • Elahi S.A.
      • Tanska P.
      • Korhonen R.K.
      • Lories R.
      • Famaey N.
      • Jonkers I.
      An in silico framework of cartilage degeneration that integrates fibril reorientation and degradation along with altered hydration and fixed charge density loss.
      . The FE model accounts for depth-dependent collagen fibril content and orientation, FCD content and water content. Novel integrated adaptive algorithms were developed to iteratively anticipate the interaction between collagen fibril reorientation and degradation (adapted from a theory developed for arterial tissues), together with PG depletion. The adaptive algorithms use the deformation gradient tensor of the elements estimated by the FE simulation to calculate the principal strains and directions of the Green–Lagrangian strain tensor. These values are used to determine elementwise the preferred fibril directions between the directions of the principal strains (for fibril reorientation), strain in fibril direction (for fibril degradation if it passed the threshold of K0,f=10%) and maximum shear strain (for PG depletion if it passed the threshold of K0,PG=30%). The FCD loss and increase in water content are linearly related to the estimated PG depletion. This procedure is repeated in 50 consecutive iterations after which the cartilage contents distribution reaches an equilibrium
      • Elahi S.A.
      • Tanska P.
      • Korhonen R.K.
      • Lories R.
      • Famaey N.
      • Jonkers I.
      An in silico framework of cartilage degeneration that integrates fibril reorientation and degradation along with altered hydration and fixed charge density loss.
      (i.e., no change in collagen, FCD and fluid contents and collagen orientation is observed when the input is kept constant). For more details regarding the FE model, material properties, degeneration processes and threshold values, we refer to Supplementary materials.
      An overview of the workflow in this study is illustrated in Fig. 1:
      Fig. 1
      Fig. 1Workflow. (A) A reference healthy and 3 OA cartilage explant models were created using finite element method and their results were used within (B) the adaptive algorithms of CARED model that account for cartilage reorientation (green color), cartilage degradation (orange color), FCD loss (blue color) and increase in tissue hydration (sky blue color). (C) CARED model was used in a virtual knock-out study to create 5 simulations: complete degeneration response and knock-out simulations by deactivating the different degenerative processes (gray color). (D) To unravel the interaction between the degenerative processes, effects of deactivating them on the studied model parameters (Section ) were determined. (E) The interaction study results were used to define a degeneration cascade for each of the three OA models.
      First, a reference model was created (detailed in Supplementary materials), representative of the degeneration of a healthy cartilage explant with healthy depth-dependent cartilage content. After simulated swelling equilibrium in the tissue was established (free swelling step for 3600 s), physiological mechanical loading of 2 MPa was applied in 0.1 s in unconfined compression
      • Tanska P.
      • Julkunen P.
      • Korhonen R.
      A computational algorithm to simulate disorganization of collagen network in injured articular cartilage.
      ,
      • Eskelinen A.S.A.
      • Mononen M.E.
      • Venäläinen M.S.
      • Korhonen R.K.
      • Tanska P.
      Maximum shear strain-based algorithm can predict proteoglycan loss in damaged articular cartilage.
      • Elahi S.A.
      • Tanska P.
      • Korhonen R.K.
      • Lories R.
      • Famaey N.
      • Jonkers I.
      An in silico framework of cartilage degeneration that integrates fibril reorientation and degradation along with altered hydration and fixed charge density loss.
      • Kłodowski A.
      • Mononen M.E.
      • Kulmala J.P.
      • Valkeapää A.
      • Korhonen R.K.
      • Avela J.
      • et al.
      Merge of motion analysis, multibody dynamics and finite element method for the subject-specific analysis of cartilage loading patterns during gait: differences between rotation and moment-driven models of human knee joint.
      . This loading corresponds to typical cartilage contact pressure and speed during the loading response of the gait cycle. Then, we explored the interaction between the compositional changes at the onset and after 50 iterations of cartilage degeneration progression for three different OA models (Fig. 1):
      • Model 1: physiological mechanical loading of degenerated cartilage with 70% of collagen, FCD and solid contents of healthy cartilage
        • Elahi S.A.
        • Tanska P.
        • Korhonen R.K.
        • Lories R.
        • Famaey N.
        • Jonkers I.
        An in silico framework of cartilage degeneration that integrates fibril reorientation and degradation along with altered hydration and fixed charge density loss.
        ,
        • Ebrahimi M.
        • Turunen M.J.
        • Finnilä M.A.
        • Joukainen A.
        • Kröger H.
        • Saarakkala S.
        • et al.
        Structure-function relationships of healthy and osteoarthritic human tibial cartilage: experimental and numerical investigation.
        .
      • Model 2: injurious mechanical loading of healthy intact cartilage – induced by 4 MPa unconfined compression ramp load in 0.1 s, comparable to injurious loading models presented in the literature
        • Loening A.M.
        • James I.E.
        • Levenston M.E.
        • Badger A.M.
        • Frank E.H.
        • Kurz B.
        • et al.
        Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis.
        ,
        • Elahi S.A.
        • Tanska P.
        • Korhonen R.K.
        • Lories R.
        • Famaey N.
        • Jonkers I.
        An in silico framework of cartilage degeneration that integrates fibril reorientation and degradation along with altered hydration and fixed charge density loss.
        ,
        • Loening A.M.
        • Levenston M.E.
        • James I.E.
        • Nuttall M.E.
        • Gowen M.
        • Grodzinsky A.J.
        • et al.
        Injurious compression of bovine articular cartilage induces chondrocyte apoptosis before detectable mechanical damage.
        ,
        • Quinn T.M.
        • Allen R.G.
        • Schalet B.J.
        • Perumbuli P.
        • Hunziker E.B.
        Matrix and cell injury due to sub-impact loading of adult bovine articular cartilage explants: effects of strain rate and peak stress.
        .
      • Model 3: physiological mechanical loading of healthy cartilage with a focal defect of 20 μm width and 750 μm depth, representative of Internation cartilage regeneration and joint preservation society (ICRS) grade 3
        • Tanska P.
        • Julkunen P.
        • Korhonen R.
        A computational algorithm to simulate disorganization of collagen network in injured articular cartilage.
        ,
        • Elahi S.A.
        • Tanska P.
        • Korhonen R.K.
        • Lories R.
        • Famaey N.
        • Jonkers I.
        An in silico framework of cartilage degeneration that integrates fibril reorientation and degradation along with altered hydration and fixed charge density loss.
        . The focal damage was introduced by modifying the FE model to incorporate the physical damage as a discontinuity of the FE mesh (shown with a red line in Fig. 1(A)).
      Then the virtual knock-out experiments were defined by selectively deactivating the adaptive processes (fibril reorientation, fibril degradation, FCD loss and increase in tissue hydration) and evaluating the effects on cartilage degeneration. To this end, for each of the three OA models the following five simulations were repeated:
      • Simulation 1: complete degeneration response, including all the adaptive processes of Fig. 1, as the reference situation (reference simulation)
      • Simulation 2: degeneration response without fibril reorientation
      • Simulation 3: degeneration response without fibril degradation
      • Simulation 4: degeneration response without FCD loss
      • Simulation 5: degeneration response without hydration increase

      Studied model parameters

      First, the complete degeneration responses (simulation 1 as reference) of the three OA models were compared, by evaluating the degeneration effect on the following model parameters:
      • average collagen density, reflecting the overall collagen fibril degradation.
      • average fibril angle w.r.t. split line direction (x-direction in Fig. 1), reflecting collagen fibril reorientation with respect to the cartilage surface.
      • average FCD content, reflecting FCD loss.
      • average solid fraction, reflecting the increase in tissue hydration. The solid fraction is the fraction of solid volume with respect to total solid and fluid volume in the porous material. The degenerative changes in this parameter in the CARED model were assumed to linearly depend on the changes in PG content. Here, a decrease in solid content is equivalent to an increase in tissue hydration.
      • equilibrium modulus determined based on a stress-relaxation simulation as described in Ref.
        • Elahi S.A.
        • Tanska P.
        • Korhonen R.K.
        • Lories R.
        • Famaey N.
        • Jonkers I.
        An in silico framework of cartilage degeneration that integrates fibril reorientation and degradation along with altered hydration and fixed charge density loss.
        , reflecting overall tissue stiffness.
      More details on the parameters, their definitions and initial values before degeneration can be found in Ref.
      • Elahi S.A.
      • Tanska P.
      • Korhonen R.K.
      • Lories R.
      • Famaey N.
      • Jonkers I.
      An in silico framework of cartilage degeneration that integrates fibril reorientation and degradation along with altered hydration and fixed charge density loss.
      .
      For each of the three OA models, the initial value of each of the above parameters and their value after degeneration (iteration 50 in CARED model) were evaluated. Then, the contribution of different degenerative mechanisms to OA progression in the different OA models was studied by evaluating the change in parameters in the complete degeneration process (simulation 1) normalized to the changes in the virtual knock-out experiments (simulations 2–5). To this end, the changes in the values of the studied parameters after 50 iterations of the reference simulation (using the complete degeneration process: simulation 1) were evaluated and normalized to the observed parameter changes in the four different simulations (simulations 2–5):
      Pnormi,j=Pdegenref,jPinitialjPdegeni,jPinitialj,
      (1)


      where, Pinitialj, Pdegenref,j and Pdegeni,j are the initial parameter measured before degeneration for the jth model (models 1–3), the parameter measured after degeneration for the reference simulation (simulation 1) of the jth model and the parameter measured after degeneration for the ith simulation (simulations 2–5) of the jth model, respectively. Values < 1 represent increased importance, whereas values > 1 represent less importance of the respective adaptive process in the knock-out simulation compared to the reference simulation.

      Results

      Complete degeneration process

      In the reference model (physiological mechanical loading of a healthy cartilage explant), negligible degenerative processes (less than 5%) were found in fibril reorientation and degradation, FCD loss and tissue hydration increase. The effect of the complete degenerative process on the three OA models is presented in Fig. 2, Fig. 4, Fig. 6 as the percentage of normalized changes in the individual model parameters (fibril reorientation, collagen density, FCD content, solid content and equilibrium modulus) after complete degeneration (reference simulation). The changes were normalized to the initial value of the parameters in the respective OA model.
      Fig. 2
      Fig. 2Knock-out study results for the degenerated cartilage model. The percentages of changes in the model parameters after degeneration calculated based on the complete degeneration simulation are indicated in the table. Figure panes indicate the effect of deactivating individual adaptive processes, more specific (A) fibril orientation, (B) fibril degradation, (C) FCD loss and (D) increase in tissue hydration/decrease in solid content. The purple dashed line at the value of 1 corresponds to the observed degenerative responses of the respective processes in the knock-out simulation Individual bar graphs present the degenerative responses for the complete degeneration simulation normalized to the changes in the corresponding knock-out simulation (see Eq. ): values < 1 represent increased importance, whereas values > 1 represent less importance of the respective adaptive process in the knock-out model. The numbers indicated on the bar graphs correspond to the processes indicated in .
      Fig. 3
      Fig. 3The degeneration loop for the degenerated cartilage model was obtained using the interaction study results (bar graphs). The numbers are corresponding to the indicated numbers in bar graphs of . The effect of collagen degradation on different degenerative processes is shown by comparing the complete degeneration response (reference) and the simulation without collagen degradation. Collagen degradation, as the parameter most affecting the strain field in the degenerated cartilage, is the starting point of the cartilage degeneration cascade.
      Fig. 4
      Fig. 4Knock-out study results for the injurious loading model. The percentages of changes in the model parameters after degeneration calculated based on the complete degeneration simulation are indicated in the table. Figure panes indicate the effect of deactivating individual adaptive processes, more specific (A) fibril orientation, (B) fibril degradation, (C) FCD loss and (D) increase in tissue hydration/decrease in solid content. The purple dashed line at the value of 1 corresponds to the observed degenerative responses of the respective processes in the knock-out simulation Individual bar graphs present the degenerative responses for the complete degeneration simulation normalized to the changes in the corresponding knock-out simulation (see Eq. ): values < 1 represent increased importance, whereas values > 1 represent less importance of the respective adaptive process in the knock-out model. The numbers indicated on the bar graphs correspond to the processes indicated in .
      Fig. 5
      Fig. 5The degeneration loop for the injurious loading model was obtained using the interaction study results (bar graphs). The numbers are corresponding to the indicated numbers in bar graphs of . The effect of collagen degradation on different degenerative processes is shown by comparing the complete degeneration response (reference) and the simulation without collagen degradation. Collagen degradation, as the parameter most affecting the strain field under injurious loading, is the starting point of the cartilage degeneration cascade.
      Fig. 6
      Fig. 6Knock-out study results for the focal defect model. The percentages of changes in the model parameters after degeneration calculated based on the complete degeneration simulation are indicated in the table. Figure panes indicate the effect of deactivating individual adaptive processes, more specific (A) fibril orientation, (B) fibril degradation, (C) FCD loss and (D) increase in tissue hydration/decrease in solid content. The purple dashed line at the value of 1 corresponds to the observed degenerative responses of the respective processes in the knock-out simulation Individual bar graphs present the degenerative responses for the complete degeneration simulation normalized to the changes in the corresponding knock-out simulation (see Eq. ): values < 1 represent increased importance, whereas values > 1 represent less importance of the respective adaptive process in the knock-out model. The numbers indicated on the bar graphs correspond to the processes indicated in .

      Effect of deactivating the different degenerative processes

      We aimed to computationally elucidate the role of cartilage degenerative processes in the progression of cartilage degeneration in injurious loading, degenerated cartilage and focal defect models. We exploited the uniqueness of our recently developed CARED model, which integrates various cartilage degenerative processes, previously described in experimental studies into a single model, to go beyond the limits of in vitro and in vivo experimental analysis and created virtual knock-out cases to evaluate the effect of individual adaptive processes on the onset and progression of cartilage degeneration.
      Fig. 2, Fig. 4, Fig. 6 show the interaction effect between degenerative processes (fibril reorientation, collagen degradation, FCD loss and increase in tissue hydration) and the resulting decrease in overall tissue stiffness in the three OA models. To this end, we compared parameter changes induced by the complete degeneration process to the changes after deactivating individual degeneration processes (in the respective OA model (see Eq. (1)). The observed interaction effects, allowed us to define three degeneration cascades specific for degenerated cartilage, injurious loading and focal defect models respectively, shown in Fig. 3, Fig. 5, Fig. 7 and discussed within the following sections.
      Fig. 7
      Fig. 7The degeneration loop for the focal defect model was obtained using the interaction study results (bar graphs). The numbers are corresponding to the indicated numbers in bar graphs of . The effect of increase in water content on change in fibril reorientation behavior is shown by comparing the complete degeneration response (reference) and the simulation without increase in water content. The pronounced local strain around the crack and consequent PG depletion in the cartilage with the focal defect are the starting points of cartilage degeneration progress.

      Degenerated cartilage model

      In the degenerated cartilage model, collagen degradation drives the cartilage degeneration process given the higher effect of collagen degradation on FCD loss and increase in tissue hydration was found compared to the other effects (1–4 in Fig. 2, Fig. 3). This is because collagen fibril degradation increased the local tissue strain (strain field in Fig. 3; simulation without collagen degradation compared to the complete degeneration simulation as reference). The increased local strain had two effects. First, it increased PG depletion that caused a localized increase in FCD loss (FCD loss in Fig. 3; simulation without collagen degradation compared to the complete degeneration simulation as reference). Furthermore, PG depletion caused a non-localized increase in tissue hydration due to a homogeneous initial decrease in the cartilage contents in the degenerated cartilage model. More FCD loss and increase in tissue hydration decreased overall tissue stiffness (5 and 6 in Fig. 2, Fig. 3) and induced cartilage softening. Increased tissue softening increased the strain in fibril direction and consequent fibril degradation, which closed the cartilage degeneration loop (7 in Fig. 2, Fig. 3). Second, increased local strain limited the fibril reorientation (8 in Fig. 2, Fig. 3; simulation without collagen degradation compared to the complete degeneration simulation as reference) thereby increasing the fibril degradation (9 in Fig. 2, Fig. 3). Therefore, the knock-out study indicates that once the collagen degradation occurs a perpetual cascade starts that drives further cartilage degeneration.

      Injurious loading model

      In the injurious loading model, like the degenerated cartilage model, collagen degradation drives the cartilage degeneration process because a higher effect of collagen degradation on FCD loss and increase in tissue hydration was found compared to the other effects (1–4 in Fig. 4, Fig. 5). Therefore, the defined degeneration cascade for the injurious loading model is similar to the degenerated cartilage model (started from collagen degradation; Fig. 5). The only difference is a more localized tissue hydration increase in the injurious loading model due to a more nonhomogeneous increase in local strains compared to the degenerated cartilage model. The localized tissue hydration in the injurious loading model affected the fibril reorientation (6 in Fig. 4, Fig. 5; simulation without an increase in tissue hydration compared to the complete degeneration simulation as reference). Based on these model-based insights, we can therefore conclude that in the injurious loading model, similar to the degenerated cartilage model, collagen degradation can be regarded as the start of a perpetual cascade in cartilage degeneration.

      Focal defect model

      In the focal defect model, the effects of FCD loss and increase in tissue hydration on collagen degradation were slightly higher than the other effects (1–4 in Fig. 6, Fig. 7). This is in clear contrast with the two other models. Therefore, our results propose a different start point for the degeneration process in the focal defect model compared to the two other OA models (compare Fig. 7 with Fig. 3, Fig. 5). Indeed, the presence of the defect increased the local tissue strain in the vicinity of the crack and increased the local PG depletion. The increased PG depletion had two effects. First, a localized increase in tissue hydration in the vicinity of the crack (Fig. 7) limited the fibril reorientation (5 in Fig. 6, Fig. 7; simulation without increase in tissue hydration compared to the complete degeneration simulation as reference) and caused more fibril degradation and FCD loss (6 and 7 in Fig. 6, Fig. 7). The increased fibril degradation and FCD loss decreased the overall tissue stiffness (8 and 9 in Fig. 6, Fig. 7) that in return increased the local strains in the vicinity of the crack and therefore caused more PG depletion. Second, the increased PG depletion further increased the FCD loss, which further decreased the overall tissue stiffness. These results show that local strain increase in the vicinity of the crack and consequent PG depletion is the start point of cartilage degradation cascade in the model with a focal defect contrasting with the collagen damage-driven progression in the two other OA models.

      Discussion

      This study used an innovative virtual knock-out model to investigate the role of different adaptive processes described in literature on cartilage degeneration onset and progression in three different OA etiologies. The knock-out cases do not simulate actual in vitro experimental models. The knock-out models were developed to study the interaction effect of individual degeneration processes, which requires a mathematical description of an integrated model. However, the complete degeneration simulation (simulation 1) can be used to predict the time-dependent degenerative changes caused by different triggers (i.e., defect, injurious loading or OA cartilage with a constituent deficiency). The predictions of the complete degeneration simulation were compared against previously published experimental studies
      • Elahi S.A.
      • Tanska P.
      • Korhonen R.K.
      • Lories R.
      • Famaey N.
      • Jonkers I.
      An in silico framework of cartilage degeneration that integrates fibril reorientation and degradation along with altered hydration and fixed charge density loss.
      . However, it is experimentally impossible to validate the knock-out models by eliminating one individual degenerative mechanism in isolation and studying the effect on the other degenerative mechanisms.

      Comparison to previous in vitro and in silico studies

      In the degenerated cartilage model, our in silico observation suggests a predominant effect of collagen degradation on PG depletion that fits well with observations of in vitro studies in degenerated cartilage
      • Cooke M.E.
      • Lawless B.M.
      • Jones S.W.
      • Grover L.M.
      Matrix degradation in osteoarthritis primes the superficial region of cartilage for mechanical damage.
      ,
      • Monfort J.
      • Garcia-Giralt N.
      • López-Armada M.J.
      • Monllau J.C.
      • Bonilla A.
      • Benito P.
      • et al.
      Decreased metalloproteinase production as a response to mechanical pressure in human cartilage: a mechanism for homeostatic regulation.
      and degenerated cartilage-like constructs
      • Praxenthaler H.
      • Krämer E.
      • Weisser M.
      • Hecht N.
      • Fischer J.
      • Grossner T.
      • et al.
      Extracellular matrix content and WNT/β-catenin levels of cartilage determine the chondrocyte response to compressive load.
      subjected to physiological mechanical loading. In agreement with our results, experimental in vitro studies suggest a reduction in the overall stiffness of a degenerated cartilage tissue subjected to physiological mechanical loading
      • Cooke M.E.
      • Lawless B.M.
      • Jones S.W.
      • Grover L.M.
      Matrix degradation in osteoarthritis primes the superficial region of cartilage for mechanical damage.
      .
      For the injurious loading model, our conclusion regarding the role of collagen degradation as the start point of the cartilage degeneration process agrees with observations in previous studies
      • Stoop R.
      • van der Kraan P.M.
      • Buma P.
      • Hollander A.P.
      • Poole A.R.
      • van den Berg W.B.
      Denaturation of type II collagen in articular cartilage in experimental murine arthritis. Evidence for collagen degradation in both reversible and irreversible cartilage damage.
      ,
      • Mononen M.E.
      • Tanska P.
      • Isaksson H.
      • Korhonen R.K.
      A novel method to simulate the progression of collagen degeneration of cartilage in the knee: data from the osteoarthritis initiative.
      . Experimentally, it has been suggested that abnormal loading is associated with collagen fibril damage at the nanoscale
      • Liang T.
      • Zhang L.L.
      • Xia W.
      • Yang H.L.
      • Luo Z.P.
      Individual collagen fibril thickening and stiffening of annulus fibrosus in degenerative intervertebral disc.
      , fibril reorientation
      • Bleuel J.
      • Zaucke F.
      • Brüggemann G.P.
      • Heilig J.
      • Wolter M.L.
      • Hamann N.
      • et al.
      Moderate cyclic tensile strain alters the assembly of cartilage extracellular matrix proteins in vitro.
      and degradation
      • Loening A.M.
      • James I.E.
      • Levenston M.E.
      • Badger A.M.
      • Frank E.H.
      • Kurz B.
      • et al.
      Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis.
      . Also, injurious compression causes an increase in cartilage tissue hydration
      • Loening A.M.
      • James I.E.
      • Levenston M.E.
      • Badger A.M.
      • Frank E.H.
      • Kurz B.
      • et al.
      Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis.
      and induces loss of superficial and mid-zone PG matrix
      • van Haaften E.E.
      • Ito K.
      • van Donkelaar C.C.
      The initial repair response of articular cartilage after mechanically induced damage.
      . In agreement with our results, one experimental study shows that in cartilage subjected to injurious loading collagen fibril degradation increased PG depletion, while PG depletion had less effect on collagen degradation
      • Kempson G.E.
      • Muir H.
      • Pollard C.
      • Tuke M.
      The tensile properties of the cartilage of human femoral condyles related to the content of collagen and glycosaminoglycans.
      . In another in silico study decrease in tissue stiffness due to solid matrix (mainly composed of PG) damage was promoted by additional collagen damage
      • Hosseini S.M.
      • Wilson W.
      • Ito K.
      • van Donkelaar C.C.
      A numerical model to study mechanically induced initiation and progression of damage in articular cartilage.
      . Similar to our results, this study showed that cartilage tissue softening as a consequence of PG depletion reduced the overall tissue stiffness more than collagen fibril damage.
      Similar to our focal defect model results, experimental studies also report signs of more local degeneration in the cartilage adjacent to the defect, evidenced by reduced staining for PGs within the middle zone
      • Aisenbrey E.A.
      • Tomaschke A.A.
      • Schoonraad S.A.
      • Fischenich K.M.
      • Wahlquist J.A.
      • Randolph M.A.
      • et al.
      Assessment and prevention of cartilage degeneration surrounding a focal chondral defect in the porcine model.
      . This agrees with the hypothesis in some in vitro studies suggesting that as a consequence of the initial cartilage lesion, the FCD (associated with Glycosaminoglycans (GAG) chains of PGs) and tissue swelling may decrease near the injury, reducing the overall tissue stiffness, and weakening the ability of the organized collagen network to resist tensile forces
      • Orozco G.A.
      • Tanska P.
      • Florea C.
      • Grodzinsky A.J.
      • Korhonen R.K.
      A novel mechanobiological model can predict how physiologically relevant dynamic loading causes proteoglycan loss in mechanically injured articular cartilage.
      ,
      • Buckwalter J.A.
      Mechanical injuries of articular cartilage.
      ,
      • Aisenbrey E.A.
      • Tomaschke A.A.
      • Schoonraad S.A.
      • Fischenich K.M.
      • Wahlquist J.A.
      • Randolph M.A.
      • et al.
      Assessment and prevention of cartilage degeneration surrounding a focal chondral defect in the porcine model.
      .

      Comparison of the three OA models

      The driving mechanism in CARED is the mechanical loading that induces a change in the deformation (gradient) of the individual elements of the FE model. Based on the change in the deformation gradient, we simultaneously calculate the change in 1) the principal strain direction (driving factor for collagen reorientation), 2) fibril strain (driving factor for fibril degradation) and 3) maximum shear strain (driving factor for PG depletion). Depending on the magnitude of changes in these parameters, one of these mechanisms can be more pronounced in the degeneration process. Therefore, depending on the deformations caused by the mechanical loading, either of the presented degenerative mechanisms can dominate. This allows the CARED model to result in unique responses in terms of the interactions between the constituents. This can be observed in the results by comparing the knock-out studies of the three OA models. Each of the models resulted in different cascades of degenerative mechanisms induced by the different strain patterns.
      A comparison of the complete degeneration simulation over the three OA models (percentage of changes after complete degeneration simulation given within the tables in Fig. 2, Fig. 4, Fig. 6) shows that in the degenerated cartilage model (primary OA) the changes in collagen orientation and content were more prominent than FCD loss and tissue hydration increase, while in the injurious loading and focal defect models (secondary OA) FCD loss and tissue hydration increase were more pronounced. Nevertheless, a similar degeneration process was observed in degenerated cartilage and injurious loading models, which is different compared to the degeneration process in the focal defect model (Fig. 3, Fig. 5, Fig. 7). The substantial difference between the interactive effects of degeneration processes in the focal defect model and the two other OA models can be explained by the different strain patterns (strain field) around the crack of the focal defect model. The crack causes a local strain concentration that increases the applied deformation to the cartilage solid matrix, mainly composed of PG, thereby accelerating PG depletion, which in turn increases the FCD loss and tissue hydration around the crack. In contrast, in the two other OA conditions, higher deformations over the whole explant due to higher loading (injurious loading model) or decreased stiffness (degenerated cartilage model) increases strain in fibril direction and collagen fibril degradation.
      In the injurious loading model, the increase in tissue hydration increases the fibril reorientation, while in degenerated cartilage increases in tissue hydration mainly decreases tissue stiffness with only minimal change in fibril reorientation (compare 6 in Fig. 3, Fig. 5). This difference between the two models relates to differences in increased tissue hydration in the injurious loading and degenerated cartilage models, respectively: in degenerated cartilage model, the decrease in cartilage contents causes a more homogenous increase in tissue strain and tissue hydration that therefore has minimal effect on the fibril reorientation but causes more decrease in the overall tissue stiffness, whereas in the injurious loading condition, the effect of increased tissue hydration is more localized, thereby impacting local fiber reorientation.
      Excluding the fibril reorientation process from the three OA models substantially increased all the other degenerative changes (fibril reorientation effect in Fig. 2, Fig. 4, Fig. 6). Therefore, fibril reorientation as an acute response by collagen fibrils seems to represents a protective effect in conditions where the cartilage mechanical integrity is already compromised.
      The highest FCD loss and increased tissue hydration were observed in the injurious loading model compared to the two other OA models (percentage of changes after complete degeneration simulation given within the tables in Fig. 2, Fig. 4, Fig. 6). This likely occurs due to the higher compressive tissue deformation in the injurious loading model (compare strain fields in Fig. 3, Fig. 5, Fig. 7). Since cartilage fluid and FCD contents, as a regulator of the fluid content, tolerate the compressive strains
      • Nimeskern L.
      • Utomo L.
      • Lehtoviita I.
      • Fessel G.
      • Snedeker J.G.
      • van Osch G.J.
      • et al.
      Tissue composition regulates distinct viscoelastic responses in auricular and articular cartilage.
      ,
      • Han E.
      • Chen S.S.
      • Klisch S.M.
      • Sah R.L.
      Contribution of proteoglycan osmotic swelling pressure to the compressive properties of articular cartilage.
      , a more compressive load in the injurious loading model caused more FCD loss and increased tissue hydration than the two other OA models.
      It is important to recognize that, in the OA joint environment, besides the mechanical factors, pro-inflammatory cytokines affect the cartilage degenerative process
      • Eskelinen A.S.
      • Tanska P.
      • Florea C.
      • Orozco G.A.
      • Julkunen P.
      • Grodzinsky A.J.
      • et al.
      Mechanobiological model for simulation of injured cartilage degradation via pro-inflammatory cytokines and mechanical stimulus.
      . Therefore, the next step in developing the modeling frameworks can be accounting for the degenerative effects of pro-inflammatory cytokines as suggested for PG depletion
      • Eskelinen A.S.
      • Tanska P.
      • Florea C.
      • Orozco G.A.
      • Julkunen P.
      • Grodzinsky A.J.
      • et al.
      Mechanobiological model for simulation of injured cartilage degradation via pro-inflammatory cytokines and mechanical stimulus.
      in the integrative CARED model. Furthermore, to take the patient-specific cartilage constituents into account, the in silico results can be further supported using in vivo quantitative magnetic resonance imaging (MRI)-based methods that relate the MRI maps to cartilage composition
      • Linka K.
      • Thüring J.
      • Rieppo L.
      • Aydin R.C.
      • Cyron C.J.
      • Kuhl C.
      • et al.
      Machine learning-augmented and microspectroscopy-informed multiparametric MRI for the non-invasive prediction of articular cartilage composition.
      for OA patients.

      Conclusion

      The insights from our in silico knock-out models suggest that in primary OA (initially degenerated cartilage) and in contrast with secondary OA (injurious loading or cartilage with a focal defect) FCD loss and tissue hydration increase are more pronounced than the collagen fibril reorientation and degradation. However, the study of the interaction between degenerative mechanisms suggests that injurious loading and degenerated cartilage cause initial changes in the collagen network, while the existence of a focal cartilage defect initially depletes the PG content before causing changes in the collagen network. Future research should combine these model-based understandings with MRI data to unravel the OA process in vivo and design optimized treatment strategies.

      Author contributions

      SAE: Conception and design; Analysis and interpretation of the data; Drafting of the article; Final approval of the article; Obtaining of funding; Collection and assembly of data.
      RCV: Analysis and interpretation of the data; Drafting of the article; Final approval of the article.
      PT: Conception and design; Analysis and interpretation of the data; Critical revision of the article for important intellectual content; Final approval of the article.
      RK: Conception and design; Analysis and interpretation of the data; Critical revision of the article for important intellectual content; Final approval of the article.
      RL: Conception and design; Analysis and interpretation of the data; Critical revision of the article for important intellectual content; Final approval of the article.
      NF: Conception and design; Analysis and interpretation of the data; Critical revision of the article for important intellectual content; Final approval of the article; Obtaining of funding.
      IJ: Conception and design; Analysis and interpretation of the data; Critical revision of the article for important intellectual content; Final approval of the article; Obtaining of funding.

      Declaration of competing interest

      None.

      Role of the funding source

      This work was supported by Marie Skłodowska-Curie Individual Fellowship (CREATION project: MSCA-IF-2019-893771) and KU Leuven/FWO Happy Joints project (C14/18/077 and G045320N). It is also part of the EOS excellence program Joint-Against OA (G0F8218N). None of the funding sources had a role in the study design, collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication.

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:

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