Full length articleBi-layered micro-fibre reinforced hydrogels for articular cartilage regeneration☆
Graphical abstract
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)
- et al.
The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells
Acta Biomater.
(2017) - et al.
Functional anatomy of articular cartilage under compressive loading quantitative aspects of global, local and zonal reactions of the collagenous network with respect to the surface integrity
Osteoarthr. Cartil.
(2002) - et al.
Combinatorial scaffold morphologies for zonal articular cartilage engineering
Acta Biomater.
(2014) - et al.
Cyclic compression of cartilage/bone explants in vitro leads to physical weakening, mechanical breakdoan of collagen and release of matrix fragments
J. Orthop. Res.
(2002) - et al.
Textile processes for engineering tissues with biomimetic architectures and properties
Trends Biotechnol.
(2016) - et al.
Functionally graded multilayer scaffolds for in vivo osteochondral tissue engineering
Acta Biomater.
(2018) - et al.
Rational design and fabrication of multiphasic soft network composites for tissue engineering articular cartilage: a numerical model-based approach
Chem. Eng. J.
(2018) - et al.
Macro-, micro- and ultrastructural investigation of how degeneration influences the response of cartilage to loading
J. Mech. Behav. Biomed. Mater.
(2012) - et al.
New insights into the role of the superficial tangential zone in influencing the microstructural response of articular cartilage to compression
Osteoarthritis Cartilage
(2010) - et al.
Importance of the superficial tissue layer for the indentation stiffness of articular cartilage
Med. Eng. Phys.
(2002)
A functional effect of the superficial mechanical properties of articular cartilage as a load bearing system in a sliding condition
Biosurface Biotribol.
The role of the superficial region in determining the dynamic properties of articular cartilage
Osteoarthritis Cartilage
The role of cell seeding density and nutrient supply for articular cartilage tissue engineering with deformational loading
Osteoarthritis Cartilage
Mechanical loading regulates human MSC differentiation in a multi-layer hydrogel for osteochondral tissue engineering
Acta Biomater.
Mechanical confinement regulates cartilage matrix formation by chondrocytes
Nat. Mater.
Knee osteoarthritis has doubled in prevalence since the mid-20th century
Proc. Natl Acad. Sci. USA
Composite three-dimensional woven scaffolds with interpenetrating network hydrogels to create functional synthetic articular cartilage
Adv. Funct. Mater.
Microengineered 3D cell-laden thermoresponsive hydrogels for mimicking cell morphology and orientation in cartilage tissue engineering
Biotechnol. Bioeng.
The basic science of articular cartilage: structure, composition, and function
Sports Health.
Articular cartilage: from formation to tissue engineering
Biomater. Sci.
Cited by (0)
- ☆
Part of the Cell and Tissue Biofabrication Special Issue, edited by Professors Guohao Dai and Kaiming Ye.