Establishment of an Ex Vivo Cartilage Degeneration Model by Combined Collagenase and Aggrecanase Treatment and Mechanical Loading

doi:10.21203/rs.3.rs-4682114/v1

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Establishment of an Ex Vivo Cartilage Degeneration Model by Combined Collagenase and Aggrecanase Treatment and Mechanical Loading

https://doi.org/10.21203/rs.3.rs-4682114/v1

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Background

There is a substantial need for ex vivo cartilage degeneration models to assess new emerging cartilage repair strategies. Ex vivo cartilage explant models have the advantages of achieving standardized and reproducible experimental conditions while maintaining the cells in their native tissue environment. This study aimed to establish a bovine cartilage degeneration model to evaluate the safety and efficacy of novel cartilage repair therapies.

Methods

Bovine osteochondral plugs underwent a brief 5-minutes treatment with collagenase to induce mild cartilage degeneration by disrupting the collagen network. To induce a moderate cartilage degeneration, aggrecanase 1 and aggrecanase 2 were additionally applied to the cartilage for 40 minutes post-collagenase treatment to degrade aggrecan.

Results

Observations revealed sulphated glycosaminoglycan loss using both Collagenase Treatment and Collagenase and Aggrecanase Treatment, while chondrocyte viability was maintained. Both treatments resulted in acute inflammation during the initial two days, evidenced by elevated release of IL6 and nitric oxide. Collagenase treatment also increased Neo epitopes of aggrecan compared to the untreated plugs at day 7, suggesting endogenous aggrecanase activation by chondrocytes upon collagen network disruption. The additional effect of mechanical loading on cartilage degeneration was also explored. Mildly degenerated cartilage treated solely with collagenase could withstand 1 hour per day of cyclical load, at 10%-20% compression of cartilage thickness combined with interfacial shear. However, higher compression levels (20%-40% of cartilage thickness) with the same shear stress regimen led to necrosis of surface layer chondrocytes.

Conclusions

This study establishes a useful model for evaluating cartilage repair strategies, and screening anti-catabolic drugs, particularly overload-related osteoarthritis. Combining mild cartilage degeneration with mechanical loading can simulate different degrees of cartilage degeneration and hold potential as a valuable tool in preclinical research.

Cartilage Degeneration Model

Collagenase

Aggrecanase

Osteochondral plugs

Mechanical load

Osteoarthritis (OA) represents a prevalent cartilage degeneration disease that significantly impacts individuals' quality of life globally[1]. Due to the incomplete understanding of the molecular mechanisms driving cartilage degeneration and the lack of effective interventions to slow its progression, total joint replacement surgery remains the sole option for severe OA[2]. Therefore, there is a significant demand for a reliable cartilage degeneration model capable of assessing targeted treatment strategies.

With the incorporation of the principles of the 3Rs (Replacement, Reduction, and Refinement) into national and international animal use regulations, development of new methods that minimize the utilization of animals in research is crucial [3]. Intra-articular injection of collagenase in the knee is commonly applied in mice[4] or rats[5] to induce osteoarthritis in vivo by weakening ligaments and degrading the cartilage collagen network. However, the thinner cartilage of mice and rats, and greater potential for intrinsic healing than humans, limit the translational value of rodent studies [6]. Bovine osteochondral explants present a translational and easily accessible method for assessing cartilage repair[7]. To mimic the in vivo environment, an Ex Vivo platform capable of separating cartilage and bone, can be utilized to culture explants with tissue-specific medium[8].

Apart from collagenases, aggrecanases which belongs to the ‘A Disintegrin and Metalloproteinase with ThromboSpondin motifs’ (ADAMTS) family also plays a significant role in osteoarthritic cartilage[9]. Specifically, aggrecanse 1 (ADAMTS-4) and aggrecanse 2 (ADAMTS-5) are key enzymes in osteoarthritis progression[10]. For inducing a moderate degeneration, our procedure described in this study involves an initial treatment with collagenase for 5 minutes, followed by a subsequent treatment with aggrecanase 1 and aggrecanase 2 for 40 minutes.

Mechanical loading of cartilage induces complex changes, including matrix and cell deformation, fluid flow, and alterations in matrix water content, ion concentration, and fixed charge density. Excessive mechanical loading affects chondrocyte metabolism, causing an imbalance between anabolic and catabolic activities, which depletes matrix components[11]. Therefore, we also investigated the effect of mechanical load including compression and shear in our our enzymatically induced cartilage degeneration model.

In this study, we aimed to establish an ex vivo cartilage degeneration model combining enzyme digestion and mechanical loading using bovine osteochondral explants. The dose and treatment duration of collagenase and aggrecanases were investigated. The cell viability and tissue histology of explants were assessed by live and dead staining and immunohistology. The expression of genes related to cartilage anabolism and inflammation was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the sulphated glycosaminoglycan (sGAG), nitric oxide (NO), and inflammatory cytokine releases into the conditioned medium were quantified. Finally, the effect of compression and shear force on degenerated cartilage was explored.

Materials

Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose, penicillin-streptomycin (Pen/Strep), HEPES, and non-essential amino acids were purchased from Gibco. ITS supplement and fetal bovine serum (FBS) were supplied by Corning. Collagenase II, HBSS, and SuperScript Vilo RT Kit were supplied by ThermoFisher. Recombinant human ADAMTS4 protein (aggrecanase 1) and recombinant human ADAMTS5 protein (aggrecanase 2) were purchased from R&D Systems. Keratanase II was purchased from Glycofinechem. Biotinylated Anti-Mouse IgG [H + L] made in horse, ABC‐complex, and DAB (3,3′‐diaminobenzidine) was supplied by Vector Laboratories. Bovine IL6 Elisa kit was supplied by KINGFISHER. Griess Reagent System was supplied by Promega. TUNEL Assay Kit - HRP-DAB was purchased from Abcam. Silencer™ Cy™3-labeled Negative Control No. 1 siRNA and Quant-iT PicoGreen dsDNA Assay Kits were purchased from Invitrogen. TRI reagent and PolyAcryl carrier were supplied by Molecular Research Center. RNeasy Mini Kit was purchased from Qiagen. TaqMan Gene Expression Master Mix was supplied by Applied Biosystems. All other materials and reagents were purchased from Sigma-Aldrich.

Osteochondral plugs harvest and culture

Osteochondral plugs were isolated from fresh femoral condyles from bovines slaughtered for human consumption. The biopsies were isolated using an 8 mm trephine drill. Biopsies were cut to a bone length of 5 mm, then cultured in a LifeTec Group (Eindhoven, Netherlands) osteochondral culture bioreactor platform. The osteochondral explants were mounted into the inserts using an O-ring positioned exactly at the interface between cartilage and bone, creating two isolated culture compartments: the upper compartment containing the cartilage and the lower compartment containing the bone, each with respective media [8]. Cartilage was cultured with 1.5 mL cartilage medium consisting of DMEM high glucose supplemented with 1% Pen/Strep, 50 µg/mL ascorbic acid, 1% ITS + Premix, 1% non-essential amino acids, and 2.5% HEPES. Bone medium consisted of 1.5 mL DMEM high glucose supplemented with 1% Pen/Strep, 50µg/mL ascorbic acid, 10% FBS, 5mM β-glycerophosphate and 2.5% HEPES.

Induction of Cartilage degeneration

To establish a mild cartilage degeneration, 1 mL 0.1% Collagenase II (285 U/mL) in HBSS was added to the cartilage compartment for 5 minutes at 37°C, then washed with PBS and refreshed with cartilage medium. For a moderate degeneration, the cartilage was treated with 800 µL 2 µg/mL aggrecanase 1 and 2 µg/mL aggrecanase 2 in HBSS for 40 minutes at 37°C after Collagenase II treatment. After 7 days of culture, the cartilage and subchondral bone were separated. Cartilage part and medium were collected for further analysis.

Mechanical loading of osteochondral plug

A previously described cartilage bioreactor system [12] was used to load the osteochondral plugs. Briefly, a ceramic hip ball (32 mm in diameter) was pressed onto the osteochondral plug. Interface motion was generated by oscillation of the ball about an axis perpendicular to the explant axis. Loaded plugs were exposed to ball oscillation of ± 25° at 1 Hz. Simultaneously, dynamic compression was applied at 0.2 mm to 0.4 mm (10–20%) or at 0.4 mm to 0.8 mm (20–40%) sinusoidal strain related to the cartilage thickness in the center of the explant. Mechanical load was performed for 1 hour per day for 1, 2, 3 or 7 days, then cartilage part was collected for histology.

sGAG and DNA measurement

Cartilage was digested overnight at 56°C in 0.5 mg/mL proteinase K. The total sulfated glycosaminoglycan (sGAG) content in cartilage and in the medium were measured using the 1,9-dimethyl-methylene blue (DMMB) colorimetric method [13]. Briefly, 20 µL of digested cartilage samples or 50 µL of conditioned medium were added into each well followed by 200 µL DMMB reagent. Absorbance at 535 nm was immediately read using a Tecan microplate reader. The proteinase K-digested samples were also used for measuring DNA content by the PicoGreen reagent, following the manufacturer’s instructions. Briefly, the samples were added in duplicates (100 µL) into a 96-well white plate. 100 µL PicoGreen working solution was added and the fluorescence of the samples was measured at an excitation wavelength of 485nm and emission of 535nm by using a Tecan reader after incubation for 3 minutes at room temperature.

Safranin O / Fast Green staining

Cartilage was fixed in 10% buffered formalin for two days and dehydrated in an ascending ethanol series. Then the samples were embedded in paraffin and sectioned at 6 µm thickness. Sections were dewaxed and then rehydrated in ethanol. To visualize the nuclei, slides were first stained with Weigert’s iron Haematoxylin for 10 minutes. After that, sections were stained with 0.02% Fast Green in ddH₂O for 6 minutes to reveal collagen deposition, then with 0.1% Safranin O for 12 minutes to show proteoglycan deposition, finally rinsed in ddH₂O, dehydrated using ascending concentrations of ethanol (50%, 70%, 96%, 100%) and mounted in Eukitt mounting medium. Images were acquired on an Olympus BX63 microscope.

Lactate dehydrogenase (LDH) and ethidium homodimer staining

Cartilage was snap frozen in liquid nitrogen and stored at -20°C. Sections with thickness of 10 µm were made by cryosection microtome (Leica, Wetzlar, Germany). An LDH assay was performed on cryostat sections after drying at room temperature. Sections were washed twice with PBS for 5 minutes, then stained with 1µg/mL ethidium homodimer for 45 minutes at room temperature. Sections were then incubated with freshly prepared LDH solution containing 40% Polypep, 2 mM Gly-Gly, 5.4 mg/mL lactic acid, 1.75 mg/mL ß-nicotinamide adenine and 3 mg/mL Nitroblue Tetrazolium pH 8.0, for 3 hours at 37°C. Slides underwent 2 washes with 50°C tap water, followed by 4% formalin. Slides were washed with PBS, followed by washing with water. Coverslips were placed on the slides with aqueous Faramount mounting medium. Images were acquired on an Olympus BX63 microscope.

Immunohistochemistry of COL2A1, ACAN, Neo epitopes of aggrecan

Paraffin sections were dewaxed and rehydrated in ethanol. Endogenous peroxidase activity was blocked by incubation with 0.3% hydrogen peroxide for 30 minutes. Then cartilage sections underwent different antigen retrieval protocols for the separate antibodies, which are shown in Table 1. Afterwards, unspecific antigen was blocked with horse serum for 1 hour at room temperature. Samples were then incubated with primary antibody at 4°C overnight. The sections were washed with PBST, and then incubated with anti-mouse secondary antibody at room temperature for 30 minutes. After washing with PBST, the sections were incubated with ABC‐complex at room temperature for 30 minutes. Following thorough washing with PBST, the sections were incubated with DAB for 4 minutes, followed by counterstaining with hematoxylin. The sections were then washed and dehydrated. Finally, cartilage sections were mounted with Eukitt.

Table 1
Antibody	Company, Catalog	Host	Digestion	Working concentration
COL2A1	ThermoFisher, MA5-12789	Mouse	1mg/mL pepsin in Tris-HCl, pH 2.0 for 10 minutes at 37°C	1/200
ACAN	DSHB, 12/21/1-C-6	Mouse	0.25 U/mL Chondroitinase ABC and 2000 U/mL Hyaluronidase in PBST for 30 minutes at 37°C	4 µg/mL
Neo epitopes of Aggrecanase	ThermoFisher, MA3-16888	Mouse	0.25U/mL Chondroitinase ABC, 0.05 U/mL Endo beta galactosidase, 0.0005 U/mL Keratanase II in PBST for 30 mins at 37°C	1/50

Interleukin-6 and nitric oxide release

Bovine interleukin-6 (IL-6) released by cartilage was measured by using a commercial enzyme-linked immunosorbent assay (ELISA) kit according to manufactures' instructions. Nitrite, a stable product of nitric oxide in aqueous solutions, was measured using the Griess reaction system following the manufacturer's instructions.

TUNEL staining

TUNEL staining was used for detection of cell apoptosis in cartilage sections. Briefly, after dewaxing and dehydration, paraffin sections were stained with a TUNEL assay kit according to the manufacturer's instructions. Images were acquired on an Olympus BX63 microscope.

Gene expression analysis

Cartilage samples were precooled with liquid nitrogen and pulverized before transferring into 1 mL TRI Reagent and 5 µl PolyAcryl carrier, then homogenized with a Retsch tissue lyser. After centrifugation, the supernatant was collected. 0.1 mL 1-Bromo-3-chloropropane (BCP) was added to get the aqueous phase containing RNA, which was purified with RNeasy Mini Kit. Reverse transcription was conducted with Vilo RT Kit. qRT-PCR was performed with TaqMan gene expression master mix on a QuantStudio™ 6 Pro Real-Time PCR system. Primer and probes sequences or gene expression assay numbers are presented in Table 2.

Table 2

Primers and probes used for PCR.
Bovine Gene	Forward primer (5′→3′)	Reverse primer (5′→3′)	Probe (5′→3′)
ACAN	CCA ACG AAA CCT ATG ACG TGT ACT	GCA CTC GTT GGC TGC CTC	ATG TTG CAT AGA AGA CCT CGC CCT CCA T (5′FAM/3′TAMRA)
COL2A1	AAG AAA CAC ATC TGG TTT GGA GAA A	TGG GAG CCA GGT TGT CAT C	CAA CGG TGG CTT CCA CTT CAG CTA TGG (5′FAM/3′TAMRA)
SOX9	AAC GCC GAG CTC AGC AAG	ACG AAC GGC CGC TTC TC	TTC AGC AGT CTC CAG AGC TTG CCC A (5′FAM/3′TAMRA)
IL6	TTC CAA AAA TGG AGG AAA AGG A	TCC AGA AGA CCA GCA GTG GTT	CTT CCA ATC TGG GTT CAA TCA GGC GAT T (5′FAM/3′TAMRA)
ADAMTS4	CCC CAT GTG CAA CGT CAA G	AGT CTC CAC AAA TCT GCT CAG TGA	AGC CCC CGA AGG GCT AAG CGC (5′FAM/3′TAMRA)
ADAMTS5	GAT GGT CAC GGT AAC TGT TTG CT	GCC GGG ACA CAC CGA GTA C	AGG CCA GAC CTA CGA TGC CAG CC (5′FAM/3′TAMRA)
RPLP0	Bt03218086_m1

Primers and probes presented with sequences were custom designed; primers and probes presented with catalogue numbers were from Applied Biosystems.

Statistical analysis

Data are presented as the mean ± standard deviation (SD) of four or five independent experiments. Shapiro–Wilk normality test was used to define whether data were normally distributed in GraphPad Prism 8.1.0 software. Analysis of variance (ANOVA) was conducted to determine differences with normal distribution. Friedman test was conducted to evaluate the differences for non-normally distributed data. Multiple comparison was corrected by Tukey test. P value < 0.05 was considered significant.

Mild and moderate cartilage degeneration

An ex vivo culture platform was applied for osteochondral plugs (Fig. 1A), which aimed to simulate the in vivo environment and enabled supply of tissue specific factors to meet the different needs of bone and cartilage[8]. Mild degeneration was induced by treating cartilage with collagenase II for a duration of 5 minutes (CT group), whereas moderate degeneration was induced by additional digestion using aggrecanase1 and aggrecanase2 for a period of 40 minutes (CAT group).

Compared to the untreated samples, the release of sGAG in the CT and CAT groups primarily occurred within the initial two-day period (Fig. 1B). The ratio of tissue sGAG to DNA decreased in degenerated cartilage both at day 2 and day 7 compared to untreated cartilage (Fig. 1C). The sGAG/DNA ratio remained unchanged between days 2 and 7 for all treatments, consistent with the measured sGAG release slope. This observation was further substantiated by histological analysis using SO-FG staining (Fig. 1D).

Notably, cartilage exhibited high viability in both the CT and CAT groups according to LDH staining (Fig. 1D). In the CT cartilage explants, IHC analysis revealed reduced COL2A1 staining on the cartilage surface but increased COL2A1 in the deep zone compared to the untreated group. In the CAT group, COL2A1 staining was observed to be lower than the untreated group. Elevated levels of ACAN were observed in both the CT and CAT groups. In addition to the CAT explants, the CT group also exhibited the presence of Neo epitopes of aggrecan (Fig. 1D), indicating that upon treatment with collagenase II, cartilage undergoes a process wherein aggrecanases were activated to facilitate the breakdown of Aggrecan spontaneously.

Acute inflammatory responses and gene expressions

The release of IL-6 (Fig. 2A) and the generation of nitric oxide (NO) (Fig. 2B) was significantly higher in both the CT and CAT groups compared to untreated explants during the initial two days of observation. These findings suggest that cartilage underwent an acute inflammatory response during this period, with normal levels of these parameters thereafter. Notably, on day 2, the CT group exhibited a higher production of nitric oxide (NO) compared to the CAT group, indicating a more pronounced inflammation within the CT group. We did not observe changes in the mRNA expression levels of ACAN, COL2A1, and SOX9 in the degeneration groups at both day 2 and day 7, indicating a lack of global cartilage repair response at transcriptional level. Additionally, there were no apparent alterations in the mRNA expression levels of IL6, ADAMTS4 and ADAMTS5.

Impact of compressive forces ranging from 10–20% of cartilage thickness and shear stress at 25 degrees on mild cartilage degeneration

The mild cartilage degeneration model was used to explore the role of mechanical loading in cartilage degeneration due to the activation of endogenous aggrecanase. Cartilage treated with Collagenase II remained alive when exposed to compression of 10–20% of cartilage thickness and ball rotation of +/- 25 degrees, as assessed by LDH staining (Fig. 3). However, COL2A1 decreased within the degenerated and loaded cartilage at day 3 which did not change further in day 7. More ACAN and Neo epitopes of aggrecan were observed in CT group comparing to untreated cartilage.

Influence of compressive forces within the range of 20–40% of cartilage thickness and shear stress at 25 degrees

Cartilage was subjected to compressive forces ranging from 20–40% of cartilage thickness and ball rotation of +/- 25 degrees. In a preliminary study, cell death was observed on the cartilage surface in the CT group after 7 days of loading. We then focused on determining the cause of cell death. LDH staining revealed cell death on the cartilage surface in the CT groups from day 2 (Fig. 4). TUNEL staining further confirmed that this cell death was necrosis rather than apoptosis, indicating the cartilage's incapacity to withstand these mechanical stresses after collagenase treatment. SO-FG staining additionally revealed a greater loss of sGAG when compared to compression within the range of 10–20%. Furthermore, following mechanical loading, degenerated cartilage exhibited reduced COL2A1 staining compared to untreated samples. ACAN and neo epitopes of aggrecan increased in the CT loaded group compared to untreated cartilage. In the CT loaded group, neo epitopes of aggrecan peaked on day 1, declined on day 2, and further decreased on day 3.

The complexity of Osteoarthritis (OA), coupled with an incomplete understanding of its pathophysiology, contributes to the challenges associated with cartilage repair[14]. There is a great need for ex vivo models that allow for assessment of cartilage repair strategies to reduce the number of animal experiments[15]. The objective of this study is to establish a convenient ex vivo cartilage degeneration model capable of assessing the safety and efficacy of novel cartilage regeneration therapies.

Collagenase has previously been employed to induce osteoarthritis in mice or rats by joint instability leading to altered loading [6]. In this study, a short treatment of 5 minutes with collagenase was applied to the cartilage part of bovine osteochondral plugs to induce a mild cartilage degeneration most likely by breaking the superficial collagen network. Osteochondral plugs were cultured in a platform that enabled the separation of cartilage and bone, thereby more accurately mimicking the in vivo environment and facilitating extended culture periods of up to 84 days[8]. To induce a moderate cartilage degeneration, aggrecanase 1 and aggrecanase 2 were applied to cartilage for 40 minutes following collagenase treatment. In our preliminary study, a two-hour treatment with 2 µg/mL aggrecanase1 and 2 µg/mL aggrecanase2 did not result in sGAG loss observed from SO-FG staining (data not shown). This suggests that aggrecanase alone might not effectively interact with aggrecan when the collagen network remains intact, or that aggrecan fragments diffuse out of the matrix only to a limited extent. Therefore, for inducing a moderate degeneration, our proposed procedure involves an initial treatment with collagenase for 5 minutes, followed by a subsequent treatment with aggrecanase1 and aggrecanase2 for 40 minutes.

sGAG loss was observed in both the Collagenase Treatment (CT) and Collagenase and Aggrecanase Treatment (CAT) groups, while chondrocytes remained viable. In both the CT and CAT groups, the cartilage experienced acute inflammation during the initial two days, as evidenced by elevated levels of IL6 and nitric oxide release. Notably, within the CT group, a higher presence of Neo epitopes of aggrecan compared to the untreated group was observed via immunohistochemical staining at day 7. This observation suggests that upon disruption of the collagen network, chondrocytes initiate the activation of endogenous aggrecanases, possibly as part of the cartilage remodeling process. Collagenase subtypes exhibit proteolytic side activities, such as trypsin[16]. A possible mechanism would be pro-ADAMTS4 was activated [17] by the trypsin-like activity of collagenase II. In comparison to the CAT group, treatment with collagenase alone appears to be more favorable as it triggers the intrinsic activity of aggrecanase by chondrocytes. This model provides a mimic of very early mild collagen damage on cartilage.

Notably, we did not observe alterations in mRNA levels of COL2A1, ACAN, SOX9, IL6, ADAMTS4, and ADAMTS5 within the degeneration groups. The mRNA expression of IL6, ADAMTS4, and ADAMTS5 did not correspond with IL6 release and the immunohistochemical staining of aggrecan neo epitope. We propose that this discrepancy may stem from the mRNA level reflecting all layers of cartilage, whereas inflammation or catalytic processes might primarily occur on the cartilage surface, where the histological changes are observed. However, it is also known that the correlation of gene expression and protein production is low due to several intracellular mechanisms[18].

A previous study demonstrated that the addition of inflammatory cytokines IL-1β and TNF-α for 7 days to human osteochondral plugs could induce osteoarthritic characteristics ex vivo [19]. Human cartilage is a superior model for investigating the response of cartilage in joint diseases compared to bovine cartilage. However, a potential limitation of their study arises from the acquisition of human osteochondral plugs, which often exhibit varying degrees of degeneration already, thereby complicating comparisons across different groups. Additionally, bovine osteochondral tissue is easier to obtain therefore making the model available to more laboratories.

Joint tissues demonstrate a high sensitivity to mechanical stimuli, and mechanical loading might be a critical external factor in regulating both cartilage development and long-term functional maintenance[20]. Given the important role of mechanical loading, our investigation also delved into the state of mild cartilage degeneration under varying compression and shear forces. Our findings indicate that chondrocytes in mild degenerated cartilage could survive under conditions of 10–20% compression of cartilage thickness and 25 degrees of shear stress applied hourly per day. However, when subjected to a higher magnitude of compression, specifically 20–40% of cartilage thickness, combined with the same shear stress regimen, the cartilage surface underwent necrosis rather than apoptotic processes beginning on day 2. The combined treatment of collagenase and mechanical loading at 20–40% compression with superimposed shear stress, may serve as a valuable tool for screening OA drugs. Furthermore, this offers insight into methods for protecting from necrosis in osteoarthritis patients subjected to joint overload.

We do not observe cell death in healthy cartilage even under compression levels ranging from 20–40%. However, another study reported that healthy bovine cartilage underwent apoptosis upon compression levels of 4.5MPa, 10MPa or 20MPa (30–50% thickness of cartilage)[21]. The reason might be the absence of the superficial layer of cartilage in their study. They removed the initial 100–400 µm of cartilage tissue by using a sledge microtome to provide a flat surface, followed by the acquisition of two subsequent 1-mm thick cartilage slices. This methodical difference potentially impacts the comparative analysis, as deep cartilage and superficial cartilage respond differently to loading exercise[22].

Under static conditions, decreased COL2A1 staining was observed on the cartilage surface, while an increase was noted in the deep layer in the CT group. This may be due to the diffusion of COL2A1 fragments to the deep zone or variations in COL2A1 expression across cartilage layers. Upon application of 10–20% compression, COL2A1 staining slightly decreased in degenerated cartilage at day 3 but remained unchanged at day 7. This might be attributed to increased COL2A1 production after day 3 or small variation in COL2A1 staining intensity different osteochondral plugs. Notably, reduced COL2A1 staining was consistently observed in the CT group subjected to 20–40% compression.

Under static conditions and 10–20% compression, more aggrecan and aggrecan neo-epitopes were observed in the CT group comparing to untreated cartilage. The neo epitope of aggrecan serves as an indicator of aggrecanase activity. We deduce that aggrecanase activity may facilitate interaction between the ACAN antibody and its antigen. The staining pattern of the neo epitope of aggrecan usually aligns with the intense staining observed in ACAN staining, thus providing partial support for our speculation. At 20–40% compression in CT group, cleaved aggrecan gradually decreased compared to day 1, while ACAN levels remained consistent across all three days. Aggrecanase cleaves ACAN into a few peptides[23]. The peptide containing the neoepitope may be released into the medium, whereas the ACAN antibody stains the remaining portion of the ACAN molecule that is retained within the matrix.

A further aspect of this degeneration model is that it caused acute inflammation of cartilage. However, the inflammation in osteoarthritis is chronic, low-grade, and differs in its clinical progress[24]. A limitation of this study is its focus on chondral tissue responses within an artificial platform and loading setting. Factors such as angiogenesis and cross talk with synovial tissue and fluid could not be adequately considered under the described culture conditions.

In conclusion, we have successfully established a mild degeneration model in bovine cartilage by subjecting it to collagenase treatment for 5 minutes (Fig. 5). Additional multiaxial load exacerbated the degeneration and could also induce cell death. This provides a model for early cartilage damage under mechanical conditions that would be experienced in a patients joint. Moreover, when 20–40% compression is applied, it has the potential to facilitate the screening of therapies for patients with osteoarthritis induced by regular tissue overload such as obese patients.

SD - Standard Deviation

ANOVA - Analysis of Variance

CT - Collagenase II Treated Cartilage

CAT - Collagenase II and Aggrecanase Treated Cartilage

sGAG - Sulfated Glycosaminoglycans

DNA - Deoxyribonucleic Acid

SO-FG - Safranin O and Fast Green

LDH - Lactate Dehydrogenase

IHC - Immunohistochemistry

COL2A1 - Collagen Type II Alpha 1 Chain

ACAN - Aggrecan

IL-6 - Interleukin 6

NO - Nitric Oxide

RT-qPCR - Reverse Transcription Quantitative Polymerase Chain Reaction

ADAMTS4 - A Disintegrin And Metalloproteinase with Thrombospondin Motifs 4

ADAMTS5 - A Disintegrin And Metalloproteinase with Thrombospondin Motifs 5

RPLP0 - Ribosomal Protein Lateral Stalk Subunit P0

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests

Funding

The present study was financially supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 955335. This publication is part of the project LoaD (projectnr. NWA1389.20.009) of the NWA-ORC research programme.

Acknowledgements

Authors' contributions

LW performed the experiments, analyzed the data, and wrote the manuscript. MJS designed the experiments. LBC, SG and MJS reviewed and edited the manuscript. MJS, SG and LBC worked on funding acquisition and supervised the whole work. All authors reviewed the results and approved the final version of the manuscript.

Abramoff B, Caldera FE: Osteoarthritis: Pathology, Diagnosis, and Treatment Options. Med Clin North Am 2020, 104(2):293-311.
Xia B, Di C, Zhang J, Hu S, Jin H, Tong P: Osteoarthritis pathogenesis: a review of molecular mechanisms. Calcif Tissue Int 2014, 95(6):495-505.
Czubala MA, Eilles E, Staubi A, Ipseiz N, Vogt M, Zieglowski L, Ernst L, Tolba RH, Taylor PR, Weiskirchen R: 3R Blackboard: A platform for animal and organ sharing. Lab Anim 2022, 56(3):292-296.
van der Kraan PM: Collagenase-Induced Osteoarthritis in Mice. Methods Mol Biol 2023, 2598:375-379.
Adães S, Mendonça M, Santos TN, Castro-Lopes JM, Ferreira-Gomes J, Neto FL: Intra-articular injection of collagenase in the knee of rats as an alternative model to study nociception associated with osteoarthritis. Arthritis Res Ther 2014, 16(1):R10.
Chu CR, Szczodry M, Bruno S: Animal models for cartilage regeneration and repair. Tissue Eng Part B Rev 2010, 16(1):105-115.
Thudium CS, Engstrom A, Groen SS, Karsdal MA, Bay-Jensen AC: An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants. J Vis Exp 2019(153).
Schwab A, Meeuwsen A, Ehlicke F, Hansmann J, Mulder L, Smits A, Walles H, Kock L: Ex vivo culture platform for assessment of cartilage repair treatment strategies. Altex 2017, 34(2):267-277.
Li T, Peng J, Li Q, Shu Y, Zhu P, Hao L: The Mechanism and Role of ADAMTS Protein Family in Osteoarthritis. Biomolecules 2022, 12(7).
Verma P, Dalal K: ADAMTS-4 and ADAMTS-5: key enzymes in osteoarthritis. J Cell Biochem 2011, 112(12):3507-3514.
Musumeci G: The Effect of Mechanical Loading on Articular Cartilage. Journal of Functional Morphology and Kinesiology 2016, 1(2):154-161.
Wimmer MA, Grad S, Kaup T, Hänni M, Schneider E, Gogolewski S, Alini M: Tribology approach to the engineering and study of articular cartilage. Tissue Eng 2004, 10(9-10):1436-1445.
Ladner YD, Alini M, Armiento AR: The Dimethylmethylene Blue Assay (DMMB) for the Quantification of Sulfated Glycosaminoglycans. Methods Mol Biol 2023, 2598:115-121.
Martel-Pelletier J, Barr AJ, Cicuttini FM, Conaghan PG, Cooper C, Goldring MB, Goldring SR, Jones G, Teichtahl AJ, Pelletier JP: Osteoarthritis. Nat Rev Dis Primers 2016, 2:16072.
Cope PJ, Ourradi K, Li Y, Sharif M: Models of osteoarthritis: the good, the bad and the promising. Osteoarthritis Cartilage 2019, 27(2):230-239.
Miersch C, Stange K, Röntgen M: Effects of trypsinization and of a combined trypsin, collagenase, and DNase digestion on liberation and in vitro function of satellite cells isolated from juvenile porcine muscles. In Vitro Cell Dev Biol Anim 2018, 54(6):406-412.
Tortorella MD, Arner EC, Hills R, Gormley J, Fok K, Pegg L, Munie G, Malfait AM: ADAMTS-4 (aggrecanase-1): N-terminal activation mechanisms. Arch Biochem Biophys 2005, 444(1):34-44.
Fortelny N, Overall CM, Pavlidis P, Freue GVC: Can we predict protein from mRNA levels? Nature 2017, 547(7664):E19-e20.
Li K, Zhang P, Zhu Y, Alini M, Grad S, Li Z: Establishment of an Ex Vivo Inflammatory Osteoarthritis Model With Human Osteochondral Explants. Front Bioeng Biotechnol 2021, 9:787020.
Sun HB: Mechanical loading, cartilage degradation, and arthritis. Ann N Y Acad Sci 2010, 1211:37-50.
Loening AM, James IE, Levenston ME, Badger AM, Frank EH, Kurz B, Nuttall ME, Hung HH, Blake SM, Grodzinsky AJ, Lark MW: Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis. Arch Biochem Biophys 2000, 381(2):205-212.
Wang H, Li Z, Li Q, Sommer S, Chen T, Sun Y, Wei H, Yan F, Lu Y: Comparing the Effect of Mechanical Loading on Deep and Superficial Cartilage Using Quantitative UTE MRI. J Magn Reson Imaging 2023.
Huang K, Wu LD: Aggrecanase and aggrecan degradation in osteoarthritis: a review. J Int Med Res 2008, 36(6):1149-1160.
Robinson WH, Lepus CM, Wang Q, Raghu H, Mao R, Lindstrom TM, Sokolove J: Low-grade inflammation as a key mediator of the pathogenesis of osteoarthritis. Nat Rev Rheumatol 2016, 12(10):580-592.

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Establishment of an Ex Vivo Cartilage Degeneration Model by Combined Collagenase and Aggrecanase Treatment and Mechanical Loading

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials and Methods

Materials

Osteochondral plugs harvest and culture

Induction of Cartilage degeneration

Mechanical loading of osteochondral plug

sGAG and DNA measurement

Safranin O / Fast Green staining

Lactate dehydrogenase (LDH) and ethidium homodimer staining

Immunohistochemistry of COL2A1, ACAN, Neo epitopes of aggrecan

Interleukin-6 and nitric oxide release

TUNEL staining

Gene expression analysis

Statistical analysis

Results

Mild and moderate cartilage degeneration

Acute inflammatory responses and gene expressions

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1