Proteoglycan and collagen contribution to the strain-rate-dependent mechanical behaviour of knee and shoulder cartilage

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Abstract:

The contribution of the proteoglycan to the strain-rate-dependent mechanical behaviour of cartilage tissues has been suggested to decrease with an increase in the strain-rate. On the other hand, the contribution from the collagen network has been suggested to increase as the strain-rate increases. These conclusions are drawn mainly based on numerical studies conducted on high-load-bearing knee cartilage tissues, while experimental evidence of these behaviours have not been demonstrated previously. Further, in contrast to the reported findings on high-load bearing knee cartilage, our previous study on the low-load-bearing kangaroo shoulder cartilage indicated that proteoglycan and collagen contribution remained steady as the strain-rate increases. Therefore, in the present study, we experimentally investigate the contribution of proteoglycan and collagen network to the strain-rate-dependent behaviour of the kangaroo knee cartilage, and plausible reasons for the differences observed in relation to the kangaroo shoulder cartilage. Firstly, in order to quantify the contribution of proteoglycans and collagen network, the indentation testings on normal, proteoglycan, and collagen-degraded kangaroo knee cartilage were conducted at different strain-rates. Then, structural and compositional differences between the kangaroo knee and shoulder cartilage were assessed qualitatively through polarised light microscopy (PLM) imaging and histological staining. Identified differences in the collagen architecture and proteoglycan composition were incorporated in a fibril-reinforced porohyperelastic Finite Element (FE) model with the objective of explaining the mechanisms underlying differences observed between the two tissues. Experimental results on knee cartilage indicated that when the strain-rate increases, proteoglycan contribution decreases while collagen contribution increases, where statistically significant differences were identified at each strain-rate (p < 0.05). PLM images revealed a sizable deep zone in the kangaroo knee cartilage where collagen fibrils were oriented perpendicular to the subchondral bone. On the other hand, no such apparent deep zone was observed in the shoulder cartilage. FE model confirmed that the biomechanical differences observed in the knee and shoulder cartilage are due to the differences in the collagen fibril arrangement in the deep zone. From these results, it can be concluded that in high-load-bearing cartilage tissues, the collagen network in the deep zone assists in increasing the stiffness of tissue with strain-rate and plays a significant role in supporting transient loads. This, in turn, helps protect the solid matrix against large distortions and strains at the subchondral junction, pointing to the importance of the collagen network in deep zone in assisting high-load-bearing cartilage tissues.

Introduction

The response of the articular cartilage tissues to external loading depends on the structural organisation, distribution and composition of its constituents, namely, water-swollen proteoglycans and the collagen network (Oloyede and Broom, 1996). The proteoglycans account for 20–30% of the cartilage's dry weight, while 60–70% is contributed by collagens (Sophia Fox et al., 2009; Maroudas and Freeman, 1973). The cartilage tissues in vivo are routinely subjected to different loading-rates (Oloyede et al., 1992a; Rubin and Lanyon, 1982). The mechanisms underlying the response of cartilage tissues to different loading-rates have been of particular interest for decades due to their importance in developing functional biomaterials capable of facilitating cartilage tissue regeneration (Oloyede and Broom, 1993; Oloyede et al., 1992b; Li and Herzog, 2004a). Therefore, a more in-depth understanding of the contribution of cartilage constituents to its loading-rate-dependent behaviour is imperative.

The behaviour of articular cartilage under different loading-rates has been well documented. It is known that the stiffness of the cartilage dramatically increases as the loading-rate increases from lower end of the physiological strain-rate (10−5 s−1) to higher end of the physiological strain-rate (10−2 s−1). However, as the strain-rates is increase further (>10−1 s−1), stiffness plateaus and does not increase with strain-rate after it has reached an asymptote. This loading-rate-dependent behaviour of articular cartilage is intimately linked to the structural organisation, distribution, and composition of its constituents and their coupled interactions (Li and Herzog, 2004a; Li et al., 2003a; Huang et al., 2003). It is well known that the cartilage responds to the external compressive loading through the containment of the water-swollen proteoglycans by its sophisticated collagen network. Hence, the integrity of the collagen network and the presence of proteoglycans are equally important in determining the compressive load-bearing response of cartilage tissues (Oloyede and Broom, 1996). Studies have found that at physiologically low strain-rates (<10−3 s−1) and at equilibrium, most of the loading is supported by the osmotic pressure generated by the water-swollen proteoglycans coupled with the collagen network (Quiroga et al., 2017; Korhonen et al., 2003; Julkunen et al., 2010). Moreover, as the physiological strain-rate (>10−1 s−1) increases, the contribution of the collagen network starts to become substantial (Quiroga et al., 2017; Julkunen et al., 2010), where superficial collagen also begins to considerably affect the tissue behaviour (Korhonen et al., 2002; Julkunen et al., 2008, 2009). Hence, it appears that as the strain-rate increases, the water-swollen proteoglycan contribution decreases while the collagen network begins to considerably affect the cartilage behaviour. However, specific experimental evidence of these conclusions has not been demonstrated previously. Further, the studies reported in the literature are on high-load-bearing knee cartilage tissues. Contrary to these results, our previous study (Thibbotuwawa et al., 2015a) on low-load-bearing kangaroo shoulder cartilage found that proteoglycans more or less had a similar contribution to the tissue behaviour as the strain-rate increases. Since knee and shoulder cartilage of bipedal animals such as kangaroo experience considerably different loading, it was hypothesised that differences in results found in our previous study and literature might be linked to the structural and compositional differences between the two tissues. Therefore, the main objectives of the present study were to 1) Experimentally quantify the contribution of proteoglycans and the collagen network to strain-rate-dependent behaviour of the knee cartilage tissues and 2) Investigate the reason behind the differences in the proteoglycan and collagen contribution to strain-rate-dependent behaviour in the kangaroo knee and shoulder cartilage.

In order to achieve the aforementioned objectives, proteoglycan and superficial collagen of the kangaroo knee cartilage were chemically degraded and subjected to simultaneous mechanical indentation testing at different strain-rates. The results were compared with our previously reported results on kangaroo shoulder cartilage. Polarised light microscopy (PLM) and histological study were conducted on the kangaroo knee and shoulder cartilage to qualitatively investigate the structural and compositional differences between the two tissues. Identified differences in the collagen architecture and proteoglycan composition were incorporated in a fibril-reinforced porohyperelastic Finite Element (FE) model with the objective of explaining the mechanisms underlying differences observed in proteoglycan and collagen contribution to strain-rate-dependent behaviour in kangaroo knee and shoulder cartilage.

Section snippets

Tissue harvesting and preparation

Immediately upon receiving, 8 mm diameter, visually normal (ICRS (Brittberg et al., 2000) macroscopic score = 0), osteochondral cartilage samples (2–3 mm intact subchondral bone) were harvested from the medial condyle of the femur of ten adult (n = 10, approximately 5-year-old) red kangaroo knee joints using a specially designed stainless steel puncher. The kangaroo knee joints were bought from an abattoir (Game Meat Processing Pvt. Ltd, QLD, Australia) within 24hrs of death. The harvested

Fibril-reinforced porohyperelastic cartilage FE model

Experimental evidence (section 4.3 below) in the present study on high-load-bearing knee cartilage showed a clear difference in the proteoglycan and collagen contribution to strain-rate-dependent behaviour when compared with our previous study on low-load-bearing kangaroo shoulder cartilage. The PLM images (section 4.4 below) showed that the collagen architectural differences in two tissues, specifically the apparent absence of deep zone in the kangaroo shoulder cartilage when compared to

Contribution of proteoglycans to the strain-rate-dependent behaviour of the knee cartilage

Repeated measures ANOVA analysis revealed that both strain-rate and trypsin treatment had a significant effect on the tissue stiffness (p < 0.05). The stiffness increased with an increase in strain-rate irrespective of the trypsin treatment (Fig. 2a). Further, the tissue stiffness was significantly different from each other at each level of the strain-rate (p < 0.05). The average stiffness of the tissues were 0.221 ± 0.059, 0.369 ± 0.071, 0.776 ± 0.307 and 0.986 ± 0.351 MPa for strain-rates of

Summary

A summary of the results of the experimental and numerical investigations conducted in the present study on the high-load-bearing kangaroo knee cartilage tissues and low-load-bearing kangaroo should cartilage tissues is as follows.

  • The 4 h trypsin treatment to degrade proteoglycans reduced the stiffness of the kangaroo knee cartilage at all the tested strain-rates. The percentage reduction in stiffness due to proteoglycan degradation was found to decrease with strain-rate.

  • The 44 h collagenase

Conclusion

The current study experimentally demonstrated that the contribution of proteoglycans to the mechanical behaviour of the high-load-bearing kangaroo knee cartilage decreased, while the contribution of collagen increased with an increase in strain-rates. These results are different to report results on low-load-bearing kangaroo shoulder cartilage. While investigating reasons for observed biomechanical differences between the kangaroo knee and shoulder cartilage, a well-developed deep-zone was

Ethical approval

Kangaroo cartilage tissue was used based on ethical clearance obtained from the Research Ethics Unit of Queensland University of Technology (Approval No: 1200000376).

CRediT authorship contribution statement

Namal Thibbotuwawa: Conceptualization, Methodology, Software, Investigation, Visualization, Writing- Original draft preparation. Sanjleena Singh: Resources, Investigation. YuanTong Gu: Supervision, Writing- Reviewing and Editing, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appered to influence the work reported in this paper.

Acknowledgements

This research was supported by an Australian Research Council (ARC) research grants (DP180103009 and IC190100020). The authors gratefully acknowledge the assistance of the Central Analytical Research Facility at QUT, the technical support provided by Ms. Melissa Johnston and Mr. Len Wilcox at QUT, and the cooperation of Mr. Don Church at Game Meat Processing Pty Ltd in providing kangaroo joints for testing.

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