Elsevier

Acta Biomaterialia

Volume 95, 1 September 2019, Pages 297-306
Acta Biomaterialia

Full length article
Bi-layered micro-fibre reinforced hydrogels for articular cartilage regeneration

https://doi.org/10.1016/j.actbio.2019.06.030Get rights and content

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.

Introduction

The inability to treat articular cartilage damage has driven intensive efforts in orthopedic research over the last few decades. Currently, the standard clinical treatment for joint degeneration is a total joint replacement using metallic prostheses, which lack biologically adaptive properties and thus have a limited life span [1]. A proposed alternative is to replace damaged cartilage with a bioengineered regenerative implant. Ideally, such an implant would promote new tissue formation, while simultaneously mimicking the biomechanical properties of cartilage. This entails the use of degradable scaffold materials as carriers and/or delivery vehicles for mature (or progenitor) and autologous (or allogeneic) chondrocytes capable of cartilage formation [2], [3], [4]. Although promising, the bioengineered constructs typically exhibit inferior strength and stiffness compared to native cartilage and feature isotropic cell distribution, as opposed to the zonal cell distribution and composition seen in healthy cartilage.

Native cartilage is comprised of four main components: chondrocytes, proteoglycan macromolecules (PGs), collagen type II fibrils and water. These are distributed heterogeneously in three distinct zones: the superficial tangential (STZ), middle (MD) and deep zone (DZ) [5]. From a functional perspective the cartilage is often simplified into two principal zones: the STZ (representing 10–20% of cartilage thickness) [6] which support tensile loads, distributes compressive loads and ensure frictional properties; and the middle and deep zone (80–90% of cartilage thickness) which together are responsible for the support of compressive loads by ensuring a high osmotic pressure and low permeability. In particular, it has been shown that the tightly packed and tangential oriented collagen fibres present in the STZ zone are key to ensuring the normal mechanical function of cartilage. The fibres act to distribute axial loads laterally through inducing tensile stress parallel to the surface and thus recruiting a larger volume of underlying tissue to support the load than just that immediately below the contact surface [7].

A few studies have attempted to fabricate multi-layered scaffolds to capture the complexity of native cartilage. Predominantly, the manufacturing approaches have relied on solution electrospinning of fibre materials [8], gas foaming or particulate template of porous polymeric structures [9], densification of collagen matrices [10], or extrusion-based 3D printing of cell laden gels [11]. Each manufacturing technique offered its own advantages, however none can (yet) fully replicate the structural organization and functional properties of native cartilage. More recently, we reported on the reinforcement of soft cell-laden hydrogels with organized micro-fibre scaffold obtained by direct melt electrospinning (MEW) [12], [13], [14]. Although abundant matrix formation was observed in these novel composite constructs, they were still not able to ensure adequate mechanical integrity after implantation nor guide zonal tissue formation. We hypothesize that this may have been due to a lack of a superficial tangential zone (STZ) -like structure. In fact, clinical studies have revealed that the disruption of the superficial zone collagen fibres during the early stages of articular cartilage degeneration appears to be the main mechanism for cartilage softening and its subsequent progressive mechanical deterioration [15], [16]. Therefore, the incorporation of a viable superficial tangential zone into tissue engineered constructs appears fundamental for proper mechanical function.

We aim to develop and characterize a bi-layered fibre reinforced cell-laden hydrogel construct that captures the functional properties of both the STZ and MDZ zones of native cartilage. Specifically, we have designed and 3D printed different micro-fibre scaffold architectures to reinforce a gelatin–methacrylamide (GelMA) hydrogel system, i.e. a densely distributed crossed fibre mat (STZ); fibres printed in a uniform box structure (MDZ); and a combination with a construct height consisting of a upper layer of 10% STZ and a lower layer of 90% MDZ (STMDZ). The time-dependent mechanical response of these composite constructs was characterized under unconfined compression and macro-indentation to mimic congruent and incongruent joint loading. Osteochondral cores of porcine knee joints with and without a superficial layer were also characterized under the same loading conditions to elucidate the biomimetic nature of the engineered constructs. A final aim was to investigate whether the novel STMDZ composite constructs could support and direct chondrogenesis under dynamic mechanical conditioning.

Section snippets

Fibre scaffolds fabrication via melt electrowriting

A custom-built MEW printer was used, as described previously [13]. Briefly, GMP-PCL (PURASORB PC 12, Corbion Inc., Netherlands) was melted in a 3 cc glass syringe at 85 °C and extruded through a 23 G spinneret connected to a high voltage source (LNC 10000-5 pos, Heinzinger Electronic GmbH, Germany). Electrified polymer jets were collected in a layer-by-layer fashion onto a grounded computer-controlled collector plate. To allow homogeneous collection of fibres, the key MEW parameters,

Fabrication of bilyared fibre reinforced hydrogels

Fibre scaffolds with a well-defined bi-layered organization were successfully manufactured by melt electrowriting (Fig. 1 and supplementary information, Movie S1). SEM images show the tangentially oriented and angle-plied fibres in the STZ (Fig. 1B), and the consistently stacked fibres in a box-like microstructure in the MDZ (Fig. 1C). All scaffolds exhibited an accurate fibre placement without significant fibre distortion or deviation in fibre diameters (≈20 µm) as shown in Fig. 1D and E. By

Discussion

The prime objective of this study was to engineer a regenerative implant that could capture the zonal mechanical properties of articular cartilage. In an attempt to mimic these properties, we have fabricated a composite construct that combined a bi-layered fibre scaffold with a hydrogel. To reproduce the superficial zone, we have printed a dense mat of crossed diagonal fibres. To reproduce the middle and deep zones, we have printed a box-like structure of multiple stacked fibres, as we

Conclusion

In conclusion, we have demonstrated that it is possible to manufacture a well-defined and sophisticated bi-layered fibre structure that can approximate the functional properties of both the STZ and MDZ zones of native cartilage. The inclusion of a thin superficial tangential zone reinforcing layer greatly improved the load-bearing properties the micro-fibre reinforced hydrogels, particularly under incongruent compressive loads. Our results also demonstrate that the new composite construct is

Acknowledgements

The authors gratefully thank the strategic alliance University Medical Center Utrecht – Eindhoven University of Technology and the European Research Council (ERC) consolidator grant 3D-JOINT (#6474426) for their financial support. In addition, the authors are very grateful to Inge Dokter for all the support with the cell harvesting, in vitro culture and characterization; to Sylvia van Kogelenber and to Joost H van Duijn for their technical support with the fibre scaffold design and 3D printing

References (41)

  • N. Sakai et al.

    A functional effect of the superficial mechanical properties of articular cartilage as a load bearing system in a sliding condition

    Biosurface Biotribol.

    (2016)
  • A.R. Gannon et al.

    The role of the superficial region in determining the dynamic properties of articular cartilage

    Osteoarthritis Cartilage

    (2012)
  • R.L. Mauck et al.

    The role of cell seeding density and nutrient supply for articular cartilage tissue engineering with deformational loading

    Osteoarthritis Cartilage

    (2003)
  • N.J. Steinmetz et al.

    Mechanical loading regulates human MSC differentiation in a multi-layer hydrogel for osteochondral tissue engineering

    Acta Biomater.

    (2015)
  • H. Lee et al.

    Mechanical confinement regulates cartilage matrix formation by chondrocytes

    Nat. Mater.

    (2017)
  • I.J. Wallace et al.

    Knee osteoarthritis has doubled in prevalence since the mid-20th century

    Proc. Natl Acad. Sci. USA

    (2017)
  • I. Chien Liao et al.

    Composite three-dimensional woven scaffolds with interpenetrating network hydrogels to create functional synthetic articular cartilage

    Adv. Funct. Mater.

    (2013)
  • A. Mellati et al.

    Microengineered 3D cell-laden thermoresponsive hydrogels for mimicking cell morphology and orientation in cartilage tissue engineering

    Biotechnol. Bioeng.

    (2017)
  • A.J.S. Fox et al.

    The basic science of articular cartilage: structure, composition, and function

    Sports Health.

    (2009)
  • S. Camarero-Espinosa et al.

    Articular cartilage: from formation to tissue engineering

    Biomater. Sci.

    (2016)
  • Cited by (0)

    Part of the Cell and Tissue Biofabrication Special Issue, edited by Professors Guohao Dai and Kaiming Ye.

    View full text