Bi-layered micro-fibre reinforced hydrogels for articular cartilage regeneration

Abstract

Articular cartilage has limited capacity for regeneration and when damaged cannot be repaired with currently available metallic or synthetic implants. We aim to bioengineer a microfibre-reinforced hydrogel that can capture the zonal depth-dependent mechanical properties of native cartilage, and simultaneously support neo-cartilage formation. With this goal, a sophisticated bi-layered microfibre architecture, combining a densely distributed crossed fibre mat (superficial tangential zone, STZ) and a uniform box structure (middle and deep zone, MDZ), was successfully manufactured via melt electrospinning and combined with a gelatin-methacrylamide hydrogel. The inclusion of a thin STZ layer greatly increased the composite construct's peak modulus under both incongruent (3.2-fold) and congruent (2.1-fold) loading, as compared to hydrogels reinforced with only a uniform MDZ structure. Notably, the stress relaxation response of the bi-layered composite construct was comparable to the tested native cartilage tissue. Furthermore, similar production of sulphated glycosaminoglycans and collagen II was observed for the novel composite constructs cultured under mechanical conditioning w/o TGF-ß1 supplementation and in static conditions w/TGF-ß1 supplementation, which confirmed the capability of the novel composite construct to support neo-cartilage formation upon mechanical stimulation. To conclude, these results are an important step towards the design and manufacture of biomechanically competent implants for cartilage regeneration. STATEMENT OF SIGNIFICANCE: Damage to articular cartilage results in severe pain and joint disfunction that cannot be treated with currently available implants. This study presents a sophisticated bioengineered bi-layered fibre reinforced cell-laden hydrogel that can approximate the functional mechanical properties of native cartilage. For the first time, the importance of incorporating a viable superficial tangential zone (STZ) - like structure to improve the load-bearing properties of bioengineered constructs, particularly when in-congruent surfaces are compressed, is demonstrated. The present work also provides new insights for the development of implants that are able to promote and guide new cartilaginous tissue formation upon physiologically relevant mechanical stimulation.

Keywords: Cartilage regeneration; Fibre-reinforced hydrogels; Melt electrowriting; Osteoarthritis; Superficial tangential zone.

Fig. 1

Design and 3D printing of fibre reinforced hydrogels with a superficial tangential zone. A) Design approach and sequential melt electrowriting of the bi-layered fibre scaffolds. SEM images of printed STMDZ fibre constructs from B) side view and C) bottom view. Detail of D) angle-ply fibre deposition at STZ and E) accurately stacked fibres at MDZ. F) Stereoscopic image of a representative GelMA gel reinforced with the STMDZ microfibre scaffold.

Fig. 2

Effect of superficial tangential zone on engineered hydrogel-fibre composites mechanical behavior. A) Schematics of the loading methodologies used: unconfined compression geometry (congruent type-loading), and indentation methodology (in-congruent type-loading). B) Representative stress relaxation curve of tested constructs, showing the fast and slow relaxation phase, and corresponding exponential model fit (RMSE < 0.05) with determined mechanical parameters. C) Comparison of time-dependent mechanical properties: apparent peak modulus, peak stress: equilibrium stress ratio, and time constants, short decay (τ₁) and long decay (τ₂) for C) congruent-type loading and D) incongruent-type loading of gel alone, superficial tangential zone (STZ), middle and deep zone (MDZ), and superficial tangential and middle and deep zone reinforced constructs (STMDZ). ^*Indicates a significant difference, p = 0.05, and NS indicates non-significant difference.

Fig. 3

Effect of superficial tangential zone on articular cartilage mechanical behavior A) Osteochondral plugs isolated from porcine knee joints before (intact) and after superficial tangential zone removal (w/o STZ). Safranin-O stain displays an increase in GAG content with cartilage depth. Comparison of time-dependent mechanical properties of intact and w/o STZ cartilage samples under B) congruent and C) in-congruent type loading. ^*Indicates a significant difference, p = 0.05, and NS indicates nonsignificant difference, p > 0.25. p values within 0.05 and 0.25 are also considered significant and specific values are indicated.

Fig. 4

Neo-cartilage formation in gelMA hydrogel reinforced constructs. A) Cell viability and distribution in MDZ and STMDZ hydrogel composites at day 1 of static and dynamic loading conditioning in base medium w/o TGF-ß1. Viable cells are stained in green and dead cells in red. B) GAG content normalized to DNA of the static and dynamically loaded constructs at day 1 and 28. C) Schematic representation of the dynamic compression bioreactor. Cross section of the single station bioreactor system and stimulation units. D) Histological analysis of dynamic loaded constructs at day 1 and 28. Some MEW fibres are indicated with #. Scale bars represent 400 μm and 50 μm. NS indicates non-significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)