Engineering Neo-fibrocartilage with Enhanced Maturation and Integration
Characteristics
Eleftherios A. Makris, MD, Regina F. MacBarb, BS, Nikolaos K. Paschos, MD, PhD, Jerry C. Hu, PhD, Kyriacos A. Athanasiou, PhD.
University of California Davis, Davis, CA, USA.
Disclosures:
E.A. Makris: None. R.F. MacBarb: None. N.K. Paschos: None. J.C. Hu: None. K.A. Athanasiou: None.
Introduction: Fibrocartilages, such as knee meniscus, intervertebral disc, and temporomandibular joint (TMJ) disc, lack an
intrinsic ability to self-repair following degradation. With a current lack of treatment options for fibrocartilage pathologies,
tissue engineering of fibrocartilage implants that mimic the complex structure of native tissue holds great potential as a longterm treatment option for patients1. Despite recent advances, the poor biomechanical properties of engineered implants and
their inability to fully integrate with native tissue upon implantation limit their potential for clinical application1,2. It is,
therefore, important that additional treatment modalities be evaluated to specifically target 1) the maturation and extracellular
matrix (ECM) organization of neotissues, and 2) the integration potential of engineered implants. Past work has shown that
treating self-assembled fibrocartilage with both chondroitinase-ABC (C-ABC) and transforming growth factor-β1 (TGF-β1)
enhances the tensile properties of engineered tissue3. Other work has found treatment of self-assembled articular cartilage with
the enzyme lysyl oxidase (LOX) to enhance overall construct properties and promote integration with native tissue through the
development of pyridinoline (PYR) collagen crosslinks4. This study sought to employ a combination of these stimuli to enhance
the tensile properties of neotissue and also as a novel way to foster integration with native tissue. Overall, it was hypothesized
that combined treatment of LOX+C-ABC+TGF-β1 would 1) enhance neotissue tensile properties through collagen enhancement
and PYR crosslink formation, 2) promote time-dependent maturation of the ECM of the neotissue, and 3) foster integration
between engineered and native tissue.
Methods: Articular chondrocytes and meniscus cells from juvenile knee joints were isolated and seeded into 5 mm diameter, 2%
agarose wells at 4.5 M cells/well at a 50:50 cell ratio to form the neofibrocartilage implants2. For all constructs, four treatment
levels were tested: control, LOX, C-ABC+TGF-β1, and their combination. Exogenous LOX was applied continuously at 0.15 ng/ml
from t = 7 - 21 d; C-ABC was administered at 2 U/mL for 4 hr at t = 7 and 21 d in addition to TGF-β1 administered continuously at
10 ng/ml for the entire culture duration. Constructs were grown until either t = 6 or 12 wk to assess the maturation potential of
the applied treatments. To test the ability of LOX + C-ABC + TGF-β1 to enhance construct maturation, neotissue was assessed
biochemically, biomechanically, and histologically, as well as via high performance liquid chromatography (HPLC) for PYR content
and scanning electron microscopy (SEM) to visualize and quantify the matrix at both t = 6 and 12 wks. To test the integration
potential of this combination of stimuli, native-to-engineered assemblies were formed by first obtaining 3 mm diameter biopsies
from neofibrocartilage at t = 6 wk, press-fitting them into same size central defects in 6 mm diameter explants (porcine-derived
TMJ disc fibrocartilage), and culturing them for an additional 6 wk. For this portion of the study, exogenous LOX was applied
continuously at 0.15 ng/ml from t = 7 - 21 d on neofibrocartilage alone, and again from t = 35 - 49 d following formation of the
native-to-engineered assembly, while C-ABC and TGF-β1 were applied as previously described. Native-to-native assemblies were
also created and grown for 6 wk to determine the ability of LOX to promote integration between native fibrocartilages. To assess
the integration of the assembly interfaces, histology and tensile testing were performed 6 wk post-integration.
Results: For the maturation portion of the study, all constructs presented with flat, uniform morphology at both t = 6 and 12 wk.
Histologically, collagen staining was more uniform and denser in C-ABC+TGF-β1 and LOX+C-ABC+TGF-β1 at both time points.
Biochemical analysis found the LOX+C-ABC+TGF-β1 treatment to significantly increase construct collagen content per wet weight
by 205% over controls at t = 6 wk, with similar results at t = 12 wk. GAG content was significantly greater in control and LOX
groups at t = 6 wk, and significantly increased further in both groups by t = 12 wk. Mechanical testing at t = 6 wk found the
LOX+C-ABC+TGF-β1 treatment to significantly increase both the Young’s modulus and ultimate tensile strength 3.0- and 2.2-fold,
respectively, that of controls, which were further enhanced at t = 12 wk (Fig. 1A). Compressive testing showed both the
relaxation modulus and instantaneous modulus to be significantly greater in control and LOX at both time points. HPLC found
LOX and LOX + C-ABC + TGF-β1 treated constructs to have 95% and 166% increases in PYR/WW, respectively, over that of
controls at t = 6 weeks (Fig. 1B), with further enhancements by t = 12 wk. SEM, on the other hand, showed both C-ABC + TGF-β1
and LOX + C-ABC + TGF-β1 constructs to have significantly enhanced collagen fibril diameters and densities over controls at both
time points. In terms of the integration study, both LOX and LOX + C-ABC + TGF-β1 were found histologically to promote fusion
at the interface in native-to-engineered assemblies, while controls and C-ABC + TGF-β1 showed little to no integration (Fig. 1C).
Biomechanical evaluation revealed the integration interface of native-to-engineered assembles to have 4.7-fold greater tensile
stiffness and strength compared to controls (Fig. 1D). Tensile testing further found all LOX-treated native-to-engineered
assemblies to have 10-fold greater tensile stiffness and strength compared to LOX-treated native-to-native assemblies.
Discussion: The fibrocartilages of the human body lack an intrinsic ability to self-repair following disease- or injury-induced
degradation. Over time, such fibrocartilage pathologies can lead to total joint dysfunction if left untreated. Current solutions to
such pathologies remain ineffective, meriting development of alternative solutions. The present study suggests a promising
treatment via the use of self-assembled fibrocartilage employing LOX+C-ABC+TGF-β1. Furthermore, this study demonstrates
that LOX is a potent agent for enhancing integration between native-to-engineered surfaces, due to the critical role played by
PYR crosslinks at the interface of integration.
Significance: The significance of this study lies in the development of methods that not only promote maturation of
neofibrocartilage, but that also encourage integration of engineered implants with native tissues. This is critical to the
translation of such work, as the poor integration potential of implants currently hinder their success in the clinic. Thus, through
the promotion of a more functionally mature and crosslink-stabilized ECM, combination of LOX + C-ABC + TGF-β1 proves to be a
great step toward overcoming some of the major current limitations of engineered fibrocartilage implants.
Acknowledgments: This study was funded by NIH R01DE019666.
References: 1Makris, E. A., Hadidi, P. & Athanasiou, K. A. The knee meniscus: structure-function, pathophysiology, current repair
techniques, and prospects for regeneration. Biomaterials 32, 7411-7431, (2011).
2Kandel, R., Roberts, S. & Urban, J. P. Tissue engineering and the intervertebral disc: the challenges. European spine journal :
official publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the
Cervical Spine Research Society 17 Suppl 4, (2008).
3MacBarb, R. F., Makris, E. A., Hu, J. C. & Athanasiou, K. A. A chondroitinase-ABC and TGF-beta1 treatment regimen for
enhancing the mechanical properties of tissue-engineered fibrocartilage. Acta biomaterialia 9, 4626-4634,
doi:10.1016/j.actbio.2012.09.037 (2013).
4Athens, A. A., Makris, E. A. & Hu, J. C. Induced collagen cross-links enhance cartilage integration. PloS one 8, e60719, (2013).
ORS 2014 Annual Meeting
Poster No: 0469