Accepted Manuscript Engineering of Hyaline Cartilage with a Calcified Zone Using Bone Marrow Stromal Cells Whitaik David Lee, BASc, Mark B. Hurtig, DVM, Robert M. Pilliar, PhD, William L. Stanford, PhD, Rita A. Kandel, MD PII:
S1063-4584(15)01130-9
DOI:
10.1016/j.joca.2015.04.010
Reference:
YJOCA 3459
To appear in:
Osteoarthritis and Cartilage
Received Date: 7 January 2015 Accepted Date: 8 April 2015
Please cite this article as: Lee WD, Hurtig MB, Pilliar RM, Stanford WL, Kandel RA, Engineering of Hyaline Cartilage with a Calcified Zone Using Bone Marrow Stromal Cells, Osteoarthritis and Cartilage (2015), doi: 10.1016/j.joca.2015.04.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Engineering of Hyaline Cartilage with a Calcified Zone Using Bone Marrow Stromal Cells Whitaik David Lee, BASc1; Mark B. Hurtig, DVM2; Robert M. Pilliar, PhD1,3; William L. Stanford, PhD1,4,*; Rita A. Kandel, MD1,5,*
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Institute of Biomaterials and Biomedical Engineering, University of Toronto; 164 College St., Toronto, Ontario M5S 3G9 Canada 2
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Ontario Veterinary College, University of Guelph; 50 McGulvray Lane, Guelph, Ontario N1G 2W1 Canada 3
Faculty of Dentistry, University of Toronto; 124 Edward St., Toronto, Ontario M5G 1G6 Canada 4
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Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute & Departments of Cellular & Molecular Medicine and Biochemistry, Microbiology and Immunology, University of Ottawa 501 Smyth Road, Box 511. Ottawa, Ontario K1H 8L6 Canada Department of Pathology and Laboratory Medicine & Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto; 600 University Ave., Toronto, Ontario M5G 1X5 Canada
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* Co-corresponding authors
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Correspondence Information:
Rita A. Kandel, MD Dept. of Pathology and Laboratory Medicine Mount Sinai Hospital 600 University Avenue Toronto, Ontario M5G 1X5 Canada Phone: +1 (416) 586-8516 Fax: +1 (416) 586-8719 Email: [email protected]
William L. Stanford, PhD Sprott Centre for Stem Cell Research Ottawa Health Research Institute 501 Smyth Road, Box 511 Ottawa, Ontario K1H 8L6 Canada Phone: +1 (613) 737-8899 x75495 Fax: +1 (613) 739-6294 Email: [email protected]
Running title (40 characters maximum): TE cartilage with a calcified interface
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Abstract
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Objective: In healthy joints, a zone of calcified cartilage (ZCC) provides the mechanical
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integration between articular cartilage and subchondral bone. Recapitulation of this
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architectural feature should serve to resist the constant shear force from the movement of
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the joint and prevent the delamination of tissue-engineered cartilage. Previous approaches
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to create the ZCC at the cartilage-substrate interface have relied on strategic use of
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exogenous scaffolds and adhesives, which are susceptible to failure by degradation and
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wear. In contrast, we report a successful scaffold-free engineering of ZCC to integrate
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tissue-engineered cartilage and a porous biodegradable bone substitute, using sheep bone
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marrow stromal cells (BMSCs) as the cell source for both cartilaginous zones.
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Design: BMSCs were predifferentiated to chondrocytes, harvested and then grown on a
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porous calcium polyphosphate substrate in the presence of triiodothyronine (T3). T3 was
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withdrawn, and additional predifferentiated chondrocytes were placed on top of the
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construct and grown for 21 days.
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Results: This protocol yielded two distinct zones: hyaline cartilage that accumulated
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proteoglycans and collagen type II, and calcified cartilage adjacent to the substrate that
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additionally accumulated mineral and collagen type X. Constructs with the calcified
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interface had comparable compressive strength to native sheep osteochondral tissue and
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higher interfacial shear strength compared to control without a calcified zone.
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Conclusion: This protocol improves on the existing scaffold-free approaches to cartilage
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tissue engineering by incorporating a calcified zone. Since this protocol employs no
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xenogeneic material, it will be appropriate for use in preclinical large-animal studies.
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Introduction
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In a synovial joint, articular cartilage bears both compressive and shear forces to facilitate
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weight bearing and movement. Once damaged, these mechanical forces contribute to the
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progression of cartilage damage, resulting in loss of cartilage and subchondral bone
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remodelling. Tissue engineering is widely regarded as a promising technology to generate
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constructs that could replace damaged cartilage and bone [1]. A wide range of
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osteochondral tissue engineering strategies are under investigation [2], including our
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effort to produce biphasic constructs that consist of scaffold-free cartilage tissue formed
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by articular chondrocytes or bone marrow stromal cells (BMSCs) on porous bone
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substitutes (calcium polyphosphate) [3, 4]. Calcium polyphosphate is a biodegradable
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ceramic with excellent load-bearing and osseointegrative properties [5, 6]. In our design,
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ingrowth of bone into calcium polyphosphate holds the biphasic construct in place and
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provides support against compressive force on tissue-engineered cartilage. However, to
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resist shear force, a mechanically competent interface between the cartilage and its
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substrate is needed. Preventing cartilage delamination is a common challenge shared by
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many cartilage repair strategies [7, 8].
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Healthy cartilage tissue interfaces with bone in vivo through a zone of calcified
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cartilage, which enhances mechanical integration by redistributing forces acting at the
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interface [9]. BMSCs are suitable for creating calcified cartilage, because they can be
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differentiated to produce cartilage tissues that mineralize their matrix [10]. While
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chondrocytes from the deep zone of articular cartilage can also yield calcified cartilage in
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vitro [11], BMSCs can be obtained without donor site morbidity and expanded to
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clinically relevant numbers. Furthermore, in vivo studies have shown that BMSC-derived
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cartilage can maintain their non-calcified phenotype after being implanted in a joint
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cavity [12, 13], demonstrating the potential of BMSCs to produce both calcified and non-
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calcified cartilage. However, to use BMSCs as a single source of cells to create cartilage
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tissues with a calcified cartilaginous interface, limiting mineralization of BMSC-derived
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cartilage to the interface will be crucial for the success of these implants.
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Using BMSCs as the cell source, we report the first successful engineering of an osteochondral-like construct that incorporates a zone of calcified cartilage at the
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cartilage-calcium polyphosphate interface without the use of a scaffold as the cartilage
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tissue forms on top of a substrate. A stepwise culturing strategy enabled the selective
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activation of BMSC-derived chondrocytes to induce mineralization only at the interface,
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while avoiding mineralization in the contiguous cartilage tissue, thus conferring localized
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calcified and hyaline zones to the engineered cartilage tissue. This approach is suitable
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for clinical use as the BMSCs were expanded in media supplemented with autologous
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serum and the construct cultured in serum-free, defined media, so this strategy can be
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directly translated to in vivo preclinical studies.
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Materials and methods
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Chondrogenic predifferentiation of BMSCs in membrane cultures
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Sheep BMSCs were isolated and expanded from bone marrow aspirates taken from ewes
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aged between 2 and 5 years as previously described [4] using autologous serum (see
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Supplementary Methods). To differentiate BMSCs to chondrocytes, 2.0×106 BMSCs
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were seeded onto 12mm cell culture inserts (0.2µm pore size; Millipore, Billerica, MA)
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that were coated with collagen type IV (50µg/insert; Sigma-Aldrich, Oakville, ON,
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Canada). Cells were cultured in a defined chondrogenic media composed of high-glucose
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Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Burlington, ON,
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Canada), 1× insulin-transferrin-selenium plus (ITS+) cell culture supplement (BD
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Biosciences, Bedford, MA), 100nM dexamethasone (Sigma-Aldrich), 100µg/mL ascorbic
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acid (Sigma-Aldrich) and 10ng/mL transforming growth factor-β3 (TGF-β3; R&D
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Systems, Minneapolis, MN). For the first 72 hours, chondrogenic media was additionally
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supplemented with 10µM blebbistatin (Cayman Chemical, Ann Arbor, MI) to inhibit
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spontaneous detachment of cells. Media was changed every 2-3 days. After 3 weeks of
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culture, cells (herein referred to as predifferentiated chondrocytes) were isolated by
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digesting the tissue in 0.5% (w/v) collagenase (Roche Diagnostics, Indianapolis, IN) for 2
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hours at 37ºC.
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Preparation of porous calcium polyphosphate substrates with a hydroxyapatite coating
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Porous calcium polyphosphate disks of 4mm diameter and 2mm height were prepared
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from gravity-sintered 75-150µm calcium polyphosphate particles as previously described
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[14]. Disks were incubated in phosphate-buffered saline (PBS) for 1 week. Then, a thin,
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submicron hydroxyapatite layer was deposited over the disks using a sol-gel processing
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method. This hydroxyapatite layer was intended to serve as a ‘barrier’ coating inhibiting
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the rate of release of calcium polyphosphate degradation products during in vitro
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cartilage formation [15]. The sol was prepared with triethyl phosphite and calcium nitrate
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tetrahydrate (Sigma-Aldrich) as previously described [16]. Disks were dipped into the sol
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gel sideways for 8 seconds and withdrawn at a speed of 20cm/min using a stepper motor-
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driven actuator, air-dried for 10 minutes and annealed for 15 minutes at 210ºC. To ensure
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a continuous barrier coating, coated disks were dipped again, annealed at 500ºC for 20
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minutes and gradually cooled. The coated disks were placed in Tygon tubing to create a
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well-like structure and subsequently sterilized by γ-irradiation (2.5 MRad).
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Optimizing the mineralizing culture condition for predifferentiated chondrocytes
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To find a culture condition in which predifferentiated chondrocytes would form calcified
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cartilage tissue, predifferentiated chondrocytes were placed on the top surface of porous
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calcium polyphosphate substrates (1.5×106 cells/substrate) and cultured in basal construct
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media composed of DMEM, 1× ITS+, 100µg/mL ascorbic acid, 50µg/mL L-proline and
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10mM β-glycerophosphate (Sigma-Aldrich). To determine which condition would induce
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mineralization, 100nM dexamethasone, 3nM triiodothyronine (T3) or 100nM retinoic
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acid (all Sigma-Aldrich) were added to cultures. The cultures were harvested at various
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times up to 21 days for further study.
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Tissue culture of multiphasic constructs
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Predifferentiated chondrocytes were placed on the top surface of porous calcium
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polyphosphate substrates (1.0×106 cells/substrate) and cultured in basal construct media,
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with or without 3nM T3. T3 was withdrawn at day 4. At day 5, additional
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predifferentiated chondrocytes were placed on top of the existing constructs (1.5×106
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cells/construct). The multi-layered constructs were maintained under the same culture
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conditions for up to 21 days. This protocol is shown in Figure 1.
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Visualization and quantification of alkaline phosphate (ALP) activity
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To visualize ALP activity, tissues were removed from their substrates and fixed in 10%
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neutral buffered formalin for 1 hour. Then, samples were incubated in 30% (w/v) sucrose
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overnight at 4ºC and snap-frozen in Tissue-Tek OCT compound (Sakura Finetek,
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Torrance, CA). 6µm cross-section cryosections were cut and mounted on silanized glass
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slides. Sections were incubated in azo dye (Naphthol AS-MX phosphate and Fast Blue
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BB salt, both Sigma-Aldrich) for 10 minutes, counterstained with eosin, dried and
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mounted with coverslips. To quantify the ALP activity, cells were isolated from tissues
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by digestion in 0.5% collagenase for 2 hours and lysed by sonication for 15 minutes in
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0.2M Tris pH 7.4 buffer. Activity levels were quantified with p-nitrophenol phosphate
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(Sigma-Aldrich) and measuring the absorbance at 405nm against a standard curve of p-
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nitrophenol [11]. Values were normalized to the lysate’s DNA content (see Biochemical
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Analysis).
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Gene expression analysis
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Predifferentiated chondrocytes (1.5×106 cells/substrate) were cultured on porous calcium
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polyphosphate substrates in basal construct media with 4 days of 3nM T3. T3 was
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removed after 4 days, and the cells were grown in basal construct media only for the
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remainder of the 21 days of culture. Controls were cells that were untreated and grown in
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the same media. Tissues were removed from their substrates at day 4 and 21 and
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homogenized in 1mL TRIzol reagent (Life Technologies) by bead milling. Total RNA
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was then extracted and reverse-transcribed using SuperScript VILO cDNA Synthesis Kit
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(Life Technologies). Quantitative PCR was performed using a LightCycler 96 Real-Time
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PCR System (Roche Diagnostics) and SYBR GreenER master mix (Life Technologies)
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with gene-specific primers (Supplementary Table 1).
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Micro-computed tomography (µCT) imaging
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Constructs were imaged using a Skyscan 1174v2 µCT scanner (Bruker, Belgium).
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Scanning was performed at 50kV and 800µA through a 0.25mm aluminum shield with a
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voxel size of 6.9µm. After reconstruction, cross-sectional tomographs were obtained with
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the software provided by the manufacturer.
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Histological analysis of whole constructs
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Constructs were fixed in 10% neutral buffered formalin overnight and infiltrated using
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the Osteo-Bed bone embedding kit (Polysciences Inc., Warrington, PA). Sections were
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cut and ground to ~50µm thickness, stained with toluidine blue and light green and
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visualized under light microscopy.
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Histological analysis of cartilaginous tissues
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Tissue mineral and proteoglycan accumulation was visualized by staining histological
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sections (see Supplementary Methods) with von Kossa and toluidine blue. Orientation of
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collagen fibrils was visualized by polarized light microscopy of trichrome-stained
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sections. Visualization for collagens types I, II and X were visualized by
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immunofluorescence (see Supplementary Methods).
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Biochemical analysis of extracellular matrix and mineral accumulation
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Tissues were digested in 40µg/mL papain (Sigma-Aldrich) and DNA, glycosaminoglycan
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and hydroxyproline contents were quantified as previously described [17]. To measure
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mineral accumulation, the tissues were lyophilized, dry weights measured, and then
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digested in 3N hydrochloric acid at 90ºC for 2 hours. The pH was adjusted to 4.0 with
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1.5M acetate buffer. Phosphate content was determined using the heteropoly blue assay
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and measuring the absorbance at 620nm. Calcium content was determined using the o-
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cresolphthalein complexone assay and measuring the absorbance at 570nm.
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Mechanical testing of multiphasic constructs
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On day 21 of culture, the equilibrium compressive modulus of the multiphasic construct’s
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cartilaginous tissue was determined by stress relaxation testing on the Mach-1 mechanical
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tester (Bio-Momentum, Laval, Canada) with a 0.65mm-diameter indenter as previously
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described [4]. Interfacial shear strength was determined by applying a force at the
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interface region of these samples using a specially designed jig attached to an Instron
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universal testing machine as previously described [15] (see Supplementary Methods).
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1 Statistical testing
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All values are expressed as mean ± 95% confidence intervals (CI). Each experiment was
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carried out with three donor animals (N=3). Univariate analysis of variance and Tukey
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post-hoc testing were used to analyze ALP activity levels on days 0-4, accumulation of
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GAG, total collagen and mineral contents, as well as the compressive moduli of the
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constructs. The interfacial shear strength data of the constructs were analyzed using t test.
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To account for the wide animal-to-animal variability, ALP activity levels on days 7, 10
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and 14 were each analyzed using paired t test. Similarly, gene expression levels on days 4
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and 21 were log-transformed and analyzed using paired t test.
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Results
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Generation of calcified cartilage at the calcium polyphosphate interface with
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predifferentiated chondrocytes
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To engineer a multiphasic construct that incorporates a zone of calcified cartilage at the
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cartilage-calcium polyphosphate interface using predifferentiated chondrocytes, we first
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sought to establish a culture condition in which predifferentiated chondrocytes could be
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cultured on porous calcium polyphosphate substrates to form mineralized cartilage tissue.
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Triiodothyronine (T3) [18], retinoic acid [19] and dexamethasone [10] have all been
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identified as potential inducers of chondrocyte mineralization in vitro. We cultured
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predifferentiated chondrocytes on calcium polyphosphate substrates with either 3nM T3
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or 100µM retinoic acid treatment in the presence or absence of dexamethasone for 1
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week, and then quantified the alkaline phosphate activity of the cells as an indicator of
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chondrocyte mineralization potential [20, 21]. ALP activity was the highest in
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predifferentiated chondrocytes cultured in the absence of dexamethasone with the T3
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treatment (Figure 2A). While ALP activity was also increased with the retinoic acid
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treatment, continued culture did not yield cartilaginous tissues on calcium polyphosphate
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after 3 weeks (data not shown). Therefore, treatment of predifferentiated chondrocytes
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with T3 was selected for further study.
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When 2×106 predifferentiated chondrocytes were cultured on porous calcium
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polyphosphate substrates for 3 weeks, the mineralized zone was found in the top of the
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tissue (Figure 2B). Between this mineralized zone and the substrate was an intermediary
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zone of non-mineralized cartilage, composed of cells with round morphology and
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extracellular matrix rich in proteoglycans. Changing the concentration of T3 treatment
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did not change the location of the mineralized zone (data not shown). However, when the
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number of initially seeded predifferentiated chondrocytes was decreased, the distance
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between the cartilage-substrate interface and the zone of mineralized cartilage also
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decreased (Figure 2C & 2D). At an initial seeding of 1×106 predifferentiated
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chondrocytes, the zone of mineralized cartilage formed consistently adjacent to the
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interface (Figure 2C & 2D, arrows).
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Short-term treatment of predifferentiated chondrocytes with T3 was sufficient to stimulate
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terminal differentiation
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ALP activity levels of predifferentiated chondrocytes were characterized over a 2-week
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culture period. T3-treated cells had a significantly increased ALP activity by day 4
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(Figure 3A). Even though the T3 treatment was withdrawn at day 4, the ALP activity
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level of cells at day 7 was significantly higher than those that had been continuously
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treated with T3, and were comparable at days 10 and 14 (Figure 3B).
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To confirm that the 4-day T3 treatment was sufficient to induce terminal
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differentiation of predifferentiated chondrocytes, gene expression levels were examined.
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As early as 4 days, collagen type X (Col10a1) expression was significantly higher in the
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T3-treated cells compared to the untreated control (Figure 3G). By 21 days, T3-treated
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cells expressed less collagen type II (Col2a1), aggrecan (Agc1) and Sox9, but greater
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Col10a1 and osteopontin (Spp1) compared to the non-treated controls (Figure 3C-J). This
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confirmed that the 4-day T3 treatment of predifferentiated chondrocytes had altered the
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differentiation of these cells.
3 T3-treated predifferentiated chondrocytes did not induce ALP activity in non-T3-treated
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predifferentiated chondrocytes
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Based on this observation, a two-stage culture protocol was developed (Figure 1). To
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grow an interfacial zone of mineralized cartilage, 1.0×106 predifferentiated chondrocytes
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were first seeded on porous calcium polyphosphate substrates, which was the number of
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cells that had been previously determined to be optimal for creating the mineralized
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cartilage at the interface, and cultured in the presence of T3 (Figure 2C & 2D). T3 was
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withdrawn at 4 days of culture, then 1.5×106 predifferentiated chondrocytes were seeded
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on top of the existing tissue one day later and cultured in the presence of β-
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glycerophosphate for up to 21 days to generate a zone of non-mineralized cartilage. To
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confirm that the T3-treated cells would not stimulate the overlaid, non-T3-treated cells to
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also mineralize, the tissues on calcium polyphosphate were harvested 5 days after the
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addition of the top layer of cells (10 days of culture in total) and cryosectioned.
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Histological analysis demonstrated that ALP activity was present only in the calcifying
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bottom layer (Figure 4), demonstrating the lack of mineralization potential in the top
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layer even though β-glycerophosphate was present.
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Characterization of the multiphasic constructs
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Constructs were grown for 21 days using the two-stage culture protocol with or without
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the 4-day T3 treatment of the interfacial layer and characterized using various methods.
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Histological examination of cartilage tissue on the calcium polyphosphate showed two
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tissue zones. The upper zone of non-mineralized cartilage was hyaline cartilage, rich in
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type II collagen and proteoglycan (Figure 5A, B, D). The interfacial zone was calcified
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cartilage, the mineral staining with von Kossa staining and the extracellular matrix
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staining with toluidine blue and type X collagen (Figure 5A, B, E). Ingrowth of tissue
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into upper parts of the porous calcium polyphosphate substrate was also observed, with
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the mineral clusters directly in contact with the calcium polyphosphate particles (Figure
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5A). No type I collagen was detected in either zone (Figure 5C). In contrast, control
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constructs not treated with T3 did not show a bizonal composition as there was no zone
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of calcified cartilage, and there was an uneven distribution of extracellular matrix (Figure
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5F-I). Differential collagen alignment in the tissue was detected using polarized light
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microscopy. In the superficial aspect, collagen was aligned parallel to the surface.
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Elsewhere, collagen was observed both pericellularly and aligned orthogonal to the
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surface. Collagen, parallel to the surface, was also observed in areas within the bottom
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layer of tissue (Figure 5J). T3-treated constructs accumulated less proteoglycans (Figure
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6A) and total collagens (Figure 6B) compared to those on untreated constructs, but had
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amounts comparable to native sheep cartilage. Tissues on T3-treated constructs
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accumulated calcium and phosphate, consistent with calcification, while untreated
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constructs did not (Figure 6C).
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T3-treated tissues had a comparable equilibrium compressive modulus to that of
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osteochondral tissue obtained from native sheep femoral condyles (Figure 7A, p = 0.15).
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The untreated constructs had a significantly higher equilibrium compressive modulus
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compared to that of the T3-treated tissues, possibly due to their greater extracellular
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matrix accumulation (Figure 6A, 6B). However, T3-treated constructs could withstand a
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stronger shear force (Figure 7B) and absorb more energy (Figure 7C) than untreated
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constructs. Histological assessment of the failed constructs confirmed that failure
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occurred at the interface (data not shown). This demonstrated that the mineralized tissue
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at the interface (Figure 5A) enhanced the interfacial shear strength.
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Discussion
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A mechanically competent interface between the cartilage and its substrate is required to
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resist the shear force generated by the movement of the joint. Previous studies have
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sought to recreate the zone of calcified cartilage that exists between hyaline cartilage and
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bone to reinforce this interface with limited success [22]. To overcome this problem, we
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successfully tissue engineered osteochondral-like constructs with BMSCs as the single
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cell source, in which cartilage tissue and a porous bone substitute substrate were formed
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with an interfacial zone of calcified cartilage without using any exogenous scaffolds in
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the cartilage tissue. The two-stage culture protocol enabled the selective stimulation of
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predifferentiated chondrocytes to form calcified cartilage only at the interface. Treatment
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of the initially seeded cells with T3 induced the terminal differentiation of cells. This
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resulted in a zone of calcified cartilage, which, in addition to mineral, also contained
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collagens type II and X and proteoglycans. These features were seen only in the part of
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the tissue that was treated with mineralization-inducing T3, which was adjacent to (and
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integrated with) the porous calcium polyphosphate substrate. The interfacial shear
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strength of constructs with the calcified cartilaginous interface was increased
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significantly compared to those without it, functionally validating its intended effect. The
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hyaline quality of non-mineralized cartilage was demonstrated by the accumulation of
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collagen type II and the lack of mineral or collagen type I. Further study is required to
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determine whether this osteochondral-like tissue formation will occur on substrates other
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than hydroxyapatite-coated calcium polyphosphate substrate.
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Thus far, approaches to induce selective mineralization of BMSC-derived
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cartilage and create this interface include delivery of bioactive signals either locally via
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microspheres [23] or by using gradients generated by a bioreactor [24] to undifferentiated
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BMSCs seeded in scaffolds. In another approach, BMSCs predifferentiated to
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chondrocytes and osteoblasts are seeded into appropriate locations within scaffolds [25,
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26]. For both strategies, use of exogenous scaffolds is necessary, which has the potential
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to hinder host integration and tissue regeneration [27]. Matching the rate of scaffold
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degeneration to the rate of tissue accumulation remains a challenge when employing
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scaffolds [22, 28]. Synthetic materials also accumulate wear over time, and may provide
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a focal point for shear failure if not replaced with new tissue. On the other hand, if the
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rate of scaffold degradation exceeds the rate of cellular remodelling, the mechanical
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integrity is compromised and the construct would fail under load. A scaffold-free
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approach obviates this issue altogether; however, this necessitated an alternative strategy
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to specify the location of the calcified interface.
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The development of our two-stage culture protocol was first enabled by the
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observation that BMSC-derived predifferentiated chondrocytes formed mineralized
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cartilage at the tissue’s superficial aspect with mineral-inducing stimulus. Given that T3
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was delivered to the tissue through culture media, and that the location of calcified
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cartilage did not change with tissue thickness, this effect was likely due to T3 diffusion
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into the tissue. As there was no scaffold present, selective delivery of T3 to the interface
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was difficult to achieve with media alone. To circumvent this, we seeded a small number
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of cells on the calcium polyphosphate in the presence of T3. This generated thin
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cartilaginous tissue, forcing mineralization to take place juxtaposed to the porous calcium
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polyphosphate substrate. This enabled the subsequent formation of a continuous layer of
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non-mineralized cartilage formed by the same cell type in the absence of T3. The
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presence of collagen types II, X and proteoglycans in the calcified interface, as well as
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the absence of collagen type I, is an important finding in our study. Taken together with
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the finding that T3-treated cells expressed hypertrophic chondrocyte makers, this
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demonstrates that the interface is cartilaginous, not osseous, successfully recapitulating
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characteristics of the native articular cartilage-subchondral bone interface.
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Histology showed that the layer of non-mineralized cartilage successfully fused to the underlying T3-treated tissue. Interestingly, fissures were observed if the intended
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interfacial tissue (i.e., the first layer) was not treated with T3, and the total thickness of
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cartilage tissues on these constructs was increased, with an uneven distribution of the
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extracellular matrix. There are two possible explanations for these results. One is that the
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manner that the interfacial layer grows may affect integration, as BMSC-derived
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chondrocytes share many characteristics with proliferating cartilage [29] that grows
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appositionally. In development, Indian hedgehog, FGF and BMP signaling pathways
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regulate this appositional growth[30]; however, in an in vitro system, absence of such
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complex regulation may allow the interfacial layer to expand laterally. The presence of
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tubing around the constructs prevented lateral expansion by confinement, which could
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have resulted in lateral mechanical stress and caused the tissue to buckle and shear. This
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was not the case for T3-stimulated interfacial layer, however, whose behaviour may more
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resemble terminal hypertrophic chondrocytes, as evidenced by their mineralized matrix.
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Terminal hypertrophic chondrocytes do not proliferate and eventually undergo apoptosis
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upon vascularization of matrix and bone formation [31]. Alternatively, the T3 treatment
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may have affected tissue fusion. Pellets of human MSCs undergoing chondrogenesis have
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been shown to accumulate tenascin along the outer surface that prevented them from
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fusing with one another [32]. The fissure between the two layers of non-mineralizing
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cartilage may stem from a similar boundary-setting phenomenon, whether driven by
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tenascin accumulation or otherwise.
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Sheep gait studies estimate the maximum anterioposterior shear stress in a stifle
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joint to be about 500kPa [33, 34], which is equivalent to approximately 9N of peak shear
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force in our study. However, using our methodology the peak shear force of native
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cartilage interface could not be measured, as it withstood the maximum load (80N)
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without shear failure. In healthy cartilage, collagen fibrils run orthogonally through the
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tidemark plane, which would confer a high resistance against failure in our mode of
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study. Although our multiphasic construct possesses inferior interfacial shear strength
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compared to articular cartilage, it still represented a four-fold improvement over the non-
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mineralized control, and sheep gait studies suggest that this improvement is adequate for
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preventing delamination.
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Importantly, this protocol can be expanded to generate large, anatomically shaped
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osteochondral-like constructs as replacements for damaged joint tissues. Protocols have
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been developed to generate ~108 BMSCs with chondrogenic potential in a good
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manufacturing practices-complaint manner [35, 36], which can be used to generate
17
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sufficiently large numbers of chondrocytes to cover a large articular surface. We have
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previously shown that calcium polyphosphate powders can be used in additive
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manufacturing methods to generate substrates of required shape and size [37]. Calcium
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polyphosphate formed in this way exhibited the same degree of osseointegration after
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implantation as those produced conventionally [38]. As such, preclinical studies are
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warranted to determine if these osteochondral-like constructs with a mineralized
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cartilaginous interface could repair joint defects in vivo.
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Using BMSCs as a single cell source, an osteochondral-like construct comprised of
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hyaline cartilage and porous bone substitute was created with a zone of calcified cartilage
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interfacing them. The novel, two-step cell culture strategy employed no exogenous
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scaffolds, just a substrate on which to form tissue. The presence of calcified cartilage
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increased the shear load that the construct could withstand at the interface. As no
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xenogeneic material was used at any point during culture, this strategy can be directly
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translated into preclinical studies to determine whether these constructs can repair joint
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defects in vivo.
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Acknowledgments
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We thank Eugene Hu for preparing the porous calcium polyphosphate substrates. We also
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thank Dr. Jian Wang and Nancy Valiquette for their technical assistance. This work was
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supported by the United States Department of Defense (to R.A.K., W81XWH-10-1-0787)
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and the Natural Sciences and Engineering Research Council of Canada (to W.L.S.,
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RGPIN 293170-11). W.L.S. is supported by a Canadian Research Chair.
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Author Contributions
Study conception and design: WDL, WLS, RAK.
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Provision of study materials: MBH, RMP.
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Analysis and interpretation of data: WDL, RMP, RAK.
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Drafting of article: WDL, WLS, RAK.
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Critical revision of the article: WDL, RMP, WLS, RAK.
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Final approval of the article: WDL, WLS, RAK.
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Role of the Funding Sources
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None of the listed bodies played any role in this study.
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Conflict of Interest
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No competing financial interests exist for any of the listed authors.
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Figure Legends
2 Figure 1. Line diagram of the tissue culture protocol used for forming scaffold-free
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multiphasic osteochondral-like constructs.
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Figure 2. Predifferentiated chondrocytes formed cartilaginous tissues on porous calcium
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polyphosphate substrates with a zone of mineralized cartilage. (A) ALP activity of
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predifferentiated chondrocytes cultured for 7 days with 3nM triiodothyronine (T3) or
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100nM retinoic acid (RA) with or without dexamethasone (Dex). Mean ± 95% CI, n=3,
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nd: not detected. * p = 0.043 between +T3 –Dex and +T3 +Dex; p = 0.045 between +T3
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–Dex and +RA –Dex. (B) Von Kossa and toluidine blue-stained sections of cartilaginous
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tissue formed by culturing 2×106 predifferentiated chondrocytes on the substrates with
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3nM T3 for 3 weeks, showing mineralized zone occurring at the superficial aspect of
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cartilage. Asterisk denotes the original location of the substrate. Scale bar = 200µm. (C)
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Cross-sectional µCT tomographs and (D) and histology of 3-week-old constructs with
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decreasing numbers of seeded cells: 2×106 (left), 1.5×106 (middle) and 1.0×106 (right)
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predifferentiated chondrocytes. In (D) vertical bars denote non-mineralized cartilage
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(purple), mineralized zone (green) and the substrate (grey). Arrows indicate the lack of
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separation between the mineralized zone and the substrate. Scale bar = 1mm (C) and
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200µm (D).
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Figure 3. Short term T3 treatment of predifferentiated chondrocytes was sufficient to
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stimulate terminal differentiation. Predifferentiated chondrocytes were treated with 3nM
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T3 for the first 4 days. In selected cultures, T3 treatment was withdrawn after 4 days
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(WD). (A) ALP activity during the first 4 days. * p < 0.001 between days 0-4 & 2-4. (B)
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ALP activity of WD, compared to cells in which T3 treatment was continued (+) for up to
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14 days. * p = 0.014. (C-J) Gene expression analysis of WD on days 4 and 21 compared
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to non-T3-treated controls (–). * p = 0.013 (Col2a1 on day 21); p = 0.031 (Agc1 on day
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21); p = 0.009 (Sox9 on day 21); p = 0.003 (Col10a1 on days 4 & 21); p = 0.025 (Spp1 on
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day 4) and p = 0.002 (Spp1 on day 21). Data points are color-coded for each animal
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throughout. Results are expressed as mean ± 95% CI, n=3.
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Figure 4. Layer of predifferentiated chondrocytes treated with T3 did not induce the
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overlaid layer of non-treated predifferentiated chondrocytes to activate ALP activity.
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1x106 cells were cultured on porous calcium polyphosphate substrates with (A) or
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without (B) 3nM T3 for 4 days. On day 5, 1.5x106 cells were seeded on top of these
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cells/tissue and cultured without T3. Cryosections of tissues from day 10 multiphasic
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constructs were stained for ALP activity. The blue staining indicative of ALP was seen
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only in the mineralizing layer (vertical bar). Asterisk denotes the location of where the
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substrate would have been located prior to its removal, scale bar = 200µm.
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Figure 5. A two-step culture protocol of predifferentiated chondrocytes on porous
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calcium polyphosphate substrates produced cartilage tissue with a mineralized zone at the
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interface. (A) Day 21 constructs with 4-day T3 treatment were processed uncalcified,
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sectioned and stained to reveal the cartilaginous (purple), mineral (green) and substrate
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particles (brown). (B-I) Tissues were excised from the substrate and examined
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histologically for the distribution of mineral and proteoglycan accumulation (B, F), by
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immunofluorescent staining for the accumulation of different types of collagen (C-E, G-
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I). Constructs with T3-treated interfacial layer are denoted as +T3, and untreated (no T3)
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control constructs are denoted as –T3. Scale bars = 200µm. (J) Differential collagen fiber
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orientations on tissues was seen by polarized light microscopy. Scale bar = 100µm.
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Figure 6. Accumulation of extracellular matrix in T3-treated (+T3) tissues was less
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compared to those in untreated (–T3) tissues, but comparable to native articular cartilage.
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(A) Accumulated proteoglycan and (B) total collagen were quantified and normalized by
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their DNA content. (A) * p = 0.001, (B) * p = 0.012 between +T3 and –T3; p = 0.037
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between +T3 and Native. (C) Accumulation of mineral in tissue was quantified by
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measuring the calcium and phosphate contents, normalized by tissue dry weight. ** p
S1063-4584(15)01130-9
DOI:
10.1016/j.joca.2015.04.010
Reference:
YJOCA 3459
To appear in:
Osteoarthritis and Cartilage
Received Date: 7 January 2015 Accepted Date: 8 April 2015
Please cite this article as: Lee WD, Hurtig MB, Pilliar RM, Stanford WL, Kandel RA, Engineering of Hyaline Cartilage with a Calcified Zone Using Bone Marrow Stromal Cells, Osteoarthritis and Cartilage (2015), doi: 10.1016/j.joca.2015.04.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Engineering of Hyaline Cartilage with a Calcified Zone Using Bone Marrow Stromal Cells Whitaik David Lee, BASc1; Mark B. Hurtig, DVM2; Robert M. Pilliar, PhD1,3; William L. Stanford, PhD1,4,*; Rita A. Kandel, MD1,5,*
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Institute of Biomaterials and Biomedical Engineering, University of Toronto; 164 College St., Toronto, Ontario M5S 3G9 Canada 2
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Ontario Veterinary College, University of Guelph; 50 McGulvray Lane, Guelph, Ontario N1G 2W1 Canada 3
Faculty of Dentistry, University of Toronto; 124 Edward St., Toronto, Ontario M5G 1G6 Canada 4
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Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute & Departments of Cellular & Molecular Medicine and Biochemistry, Microbiology and Immunology, University of Ottawa 501 Smyth Road, Box 511. Ottawa, Ontario K1H 8L6 Canada Department of Pathology and Laboratory Medicine & Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto; 600 University Ave., Toronto, Ontario M5G 1X5 Canada
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* Co-corresponding authors
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Correspondence Information:
Rita A. Kandel, MD Dept. of Pathology and Laboratory Medicine Mount Sinai Hospital 600 University Avenue Toronto, Ontario M5G 1X5 Canada Phone: +1 (416) 586-8516 Fax: +1 (416) 586-8719 Email: [email protected]
William L. Stanford, PhD Sprott Centre for Stem Cell Research Ottawa Health Research Institute 501 Smyth Road, Box 511 Ottawa, Ontario K1H 8L6 Canada Phone: +1 (613) 737-8899 x75495 Fax: +1 (613) 739-6294 Email: [email protected]
Running title (40 characters maximum): TE cartilage with a calcified interface
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Abstract
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Objective: In healthy joints, a zone of calcified cartilage (ZCC) provides the mechanical
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integration between articular cartilage and subchondral bone. Recapitulation of this
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architectural feature should serve to resist the constant shear force from the movement of
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the joint and prevent the delamination of tissue-engineered cartilage. Previous approaches
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to create the ZCC at the cartilage-substrate interface have relied on strategic use of
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exogenous scaffolds and adhesives, which are susceptible to failure by degradation and
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wear. In contrast, we report a successful scaffold-free engineering of ZCC to integrate
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tissue-engineered cartilage and a porous biodegradable bone substitute, using sheep bone
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marrow stromal cells (BMSCs) as the cell source for both cartilaginous zones.
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Design: BMSCs were predifferentiated to chondrocytes, harvested and then grown on a
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porous calcium polyphosphate substrate in the presence of triiodothyronine (T3). T3 was
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withdrawn, and additional predifferentiated chondrocytes were placed on top of the
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construct and grown for 21 days.
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Results: This protocol yielded two distinct zones: hyaline cartilage that accumulated
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proteoglycans and collagen type II, and calcified cartilage adjacent to the substrate that
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additionally accumulated mineral and collagen type X. Constructs with the calcified
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interface had comparable compressive strength to native sheep osteochondral tissue and
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higher interfacial shear strength compared to control without a calcified zone.
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Conclusion: This protocol improves on the existing scaffold-free approaches to cartilage
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tissue engineering by incorporating a calcified zone. Since this protocol employs no
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xenogeneic material, it will be appropriate for use in preclinical large-animal studies.
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Introduction
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In a synovial joint, articular cartilage bears both compressive and shear forces to facilitate
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weight bearing and movement. Once damaged, these mechanical forces contribute to the
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progression of cartilage damage, resulting in loss of cartilage and subchondral bone
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remodelling. Tissue engineering is widely regarded as a promising technology to generate
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constructs that could replace damaged cartilage and bone [1]. A wide range of
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osteochondral tissue engineering strategies are under investigation [2], including our
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effort to produce biphasic constructs that consist of scaffold-free cartilage tissue formed
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by articular chondrocytes or bone marrow stromal cells (BMSCs) on porous bone
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substitutes (calcium polyphosphate) [3, 4]. Calcium polyphosphate is a biodegradable
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ceramic with excellent load-bearing and osseointegrative properties [5, 6]. In our design,
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ingrowth of bone into calcium polyphosphate holds the biphasic construct in place and
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provides support against compressive force on tissue-engineered cartilage. However, to
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resist shear force, a mechanically competent interface between the cartilage and its
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substrate is needed. Preventing cartilage delamination is a common challenge shared by
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many cartilage repair strategies [7, 8].
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Healthy cartilage tissue interfaces with bone in vivo through a zone of calcified
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cartilage, which enhances mechanical integration by redistributing forces acting at the
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interface [9]. BMSCs are suitable for creating calcified cartilage, because they can be
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differentiated to produce cartilage tissues that mineralize their matrix [10]. While
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chondrocytes from the deep zone of articular cartilage can also yield calcified cartilage in
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vitro [11], BMSCs can be obtained without donor site morbidity and expanded to
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clinically relevant numbers. Furthermore, in vivo studies have shown that BMSC-derived
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cartilage can maintain their non-calcified phenotype after being implanted in a joint
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cavity [12, 13], demonstrating the potential of BMSCs to produce both calcified and non-
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calcified cartilage. However, to use BMSCs as a single source of cells to create cartilage
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tissues with a calcified cartilaginous interface, limiting mineralization of BMSC-derived
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cartilage to the interface will be crucial for the success of these implants.
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Using BMSCs as the cell source, we report the first successful engineering of an osteochondral-like construct that incorporates a zone of calcified cartilage at the
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cartilage-calcium polyphosphate interface without the use of a scaffold as the cartilage
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tissue forms on top of a substrate. A stepwise culturing strategy enabled the selective
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activation of BMSC-derived chondrocytes to induce mineralization only at the interface,
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while avoiding mineralization in the contiguous cartilage tissue, thus conferring localized
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calcified and hyaline zones to the engineered cartilage tissue. This approach is suitable
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for clinical use as the BMSCs were expanded in media supplemented with autologous
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serum and the construct cultured in serum-free, defined media, so this strategy can be
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directly translated to in vivo preclinical studies.
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Materials and methods
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Chondrogenic predifferentiation of BMSCs in membrane cultures
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Sheep BMSCs were isolated and expanded from bone marrow aspirates taken from ewes
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aged between 2 and 5 years as previously described [4] using autologous serum (see
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Supplementary Methods). To differentiate BMSCs to chondrocytes, 2.0×106 BMSCs
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were seeded onto 12mm cell culture inserts (0.2µm pore size; Millipore, Billerica, MA)
7
that were coated with collagen type IV (50µg/insert; Sigma-Aldrich, Oakville, ON,
8
Canada). Cells were cultured in a defined chondrogenic media composed of high-glucose
9
Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Burlington, ON,
10
Canada), 1× insulin-transferrin-selenium plus (ITS+) cell culture supplement (BD
11
Biosciences, Bedford, MA), 100nM dexamethasone (Sigma-Aldrich), 100µg/mL ascorbic
12
acid (Sigma-Aldrich) and 10ng/mL transforming growth factor-β3 (TGF-β3; R&D
13
Systems, Minneapolis, MN). For the first 72 hours, chondrogenic media was additionally
14
supplemented with 10µM blebbistatin (Cayman Chemical, Ann Arbor, MI) to inhibit
15
spontaneous detachment of cells. Media was changed every 2-3 days. After 3 weeks of
16
culture, cells (herein referred to as predifferentiated chondrocytes) were isolated by
17
digesting the tissue in 0.5% (w/v) collagenase (Roche Diagnostics, Indianapolis, IN) for 2
18
hours at 37ºC.
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Preparation of porous calcium polyphosphate substrates with a hydroxyapatite coating
21
Porous calcium polyphosphate disks of 4mm diameter and 2mm height were prepared
22
from gravity-sintered 75-150µm calcium polyphosphate particles as previously described
23
[14]. Disks were incubated in phosphate-buffered saline (PBS) for 1 week. Then, a thin,
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submicron hydroxyapatite layer was deposited over the disks using a sol-gel processing
2
method. This hydroxyapatite layer was intended to serve as a ‘barrier’ coating inhibiting
3
the rate of release of calcium polyphosphate degradation products during in vitro
4
cartilage formation [15]. The sol was prepared with triethyl phosphite and calcium nitrate
5
tetrahydrate (Sigma-Aldrich) as previously described [16]. Disks were dipped into the sol
6
gel sideways for 8 seconds and withdrawn at a speed of 20cm/min using a stepper motor-
7
driven actuator, air-dried for 10 minutes and annealed for 15 minutes at 210ºC. To ensure
8
a continuous barrier coating, coated disks were dipped again, annealed at 500ºC for 20
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minutes and gradually cooled. The coated disks were placed in Tygon tubing to create a
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well-like structure and subsequently sterilized by γ-irradiation (2.5 MRad).
11
Optimizing the mineralizing culture condition for predifferentiated chondrocytes
13
To find a culture condition in which predifferentiated chondrocytes would form calcified
14
cartilage tissue, predifferentiated chondrocytes were placed on the top surface of porous
15
calcium polyphosphate substrates (1.5×106 cells/substrate) and cultured in basal construct
16
media composed of DMEM, 1× ITS+, 100µg/mL ascorbic acid, 50µg/mL L-proline and
17
10mM β-glycerophosphate (Sigma-Aldrich). To determine which condition would induce
18
mineralization, 100nM dexamethasone, 3nM triiodothyronine (T3) or 100nM retinoic
19
acid (all Sigma-Aldrich) were added to cultures. The cultures were harvested at various
20
times up to 21 days for further study.
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Tissue culture of multiphasic constructs
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Predifferentiated chondrocytes were placed on the top surface of porous calcium
2
polyphosphate substrates (1.0×106 cells/substrate) and cultured in basal construct media,
3
with or without 3nM T3. T3 was withdrawn at day 4. At day 5, additional
4
predifferentiated chondrocytes were placed on top of the existing constructs (1.5×106
5
cells/construct). The multi-layered constructs were maintained under the same culture
6
conditions for up to 21 days. This protocol is shown in Figure 1.
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Visualization and quantification of alkaline phosphate (ALP) activity
9
To visualize ALP activity, tissues were removed from their substrates and fixed in 10%
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neutral buffered formalin for 1 hour. Then, samples were incubated in 30% (w/v) sucrose
11
overnight at 4ºC and snap-frozen in Tissue-Tek OCT compound (Sakura Finetek,
12
Torrance, CA). 6µm cross-section cryosections were cut and mounted on silanized glass
13
slides. Sections were incubated in azo dye (Naphthol AS-MX phosphate and Fast Blue
14
BB salt, both Sigma-Aldrich) for 10 minutes, counterstained with eosin, dried and
15
mounted with coverslips. To quantify the ALP activity, cells were isolated from tissues
16
by digestion in 0.5% collagenase for 2 hours and lysed by sonication for 15 minutes in
17
0.2M Tris pH 7.4 buffer. Activity levels were quantified with p-nitrophenol phosphate
18
(Sigma-Aldrich) and measuring the absorbance at 405nm against a standard curve of p-
19
nitrophenol [11]. Values were normalized to the lysate’s DNA content (see Biochemical
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Analysis).
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Gene expression analysis
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Predifferentiated chondrocytes (1.5×106 cells/substrate) were cultured on porous calcium
2
polyphosphate substrates in basal construct media with 4 days of 3nM T3. T3 was
3
removed after 4 days, and the cells were grown in basal construct media only for the
4
remainder of the 21 days of culture. Controls were cells that were untreated and grown in
5
the same media. Tissues were removed from their substrates at day 4 and 21 and
6
homogenized in 1mL TRIzol reagent (Life Technologies) by bead milling. Total RNA
7
was then extracted and reverse-transcribed using SuperScript VILO cDNA Synthesis Kit
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(Life Technologies). Quantitative PCR was performed using a LightCycler 96 Real-Time
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PCR System (Roche Diagnostics) and SYBR GreenER master mix (Life Technologies)
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with gene-specific primers (Supplementary Table 1).
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Micro-computed tomography (µCT) imaging
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Constructs were imaged using a Skyscan 1174v2 µCT scanner (Bruker, Belgium).
14
Scanning was performed at 50kV and 800µA through a 0.25mm aluminum shield with a
15
voxel size of 6.9µm. After reconstruction, cross-sectional tomographs were obtained with
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the software provided by the manufacturer.
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Histological analysis of whole constructs
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Constructs were fixed in 10% neutral buffered formalin overnight and infiltrated using
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the Osteo-Bed bone embedding kit (Polysciences Inc., Warrington, PA). Sections were
21
cut and ground to ~50µm thickness, stained with toluidine blue and light green and
22
visualized under light microscopy.
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Histological analysis of cartilaginous tissues
2
Tissue mineral and proteoglycan accumulation was visualized by staining histological
3
sections (see Supplementary Methods) with von Kossa and toluidine blue. Orientation of
4
collagen fibrils was visualized by polarized light microscopy of trichrome-stained
5
sections. Visualization for collagens types I, II and X were visualized by
6
immunofluorescence (see Supplementary Methods).
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Biochemical analysis of extracellular matrix and mineral accumulation
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Tissues were digested in 40µg/mL papain (Sigma-Aldrich) and DNA, glycosaminoglycan
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and hydroxyproline contents were quantified as previously described [17]. To measure
11
mineral accumulation, the tissues were lyophilized, dry weights measured, and then
12
digested in 3N hydrochloric acid at 90ºC for 2 hours. The pH was adjusted to 4.0 with
13
1.5M acetate buffer. Phosphate content was determined using the heteropoly blue assay
14
and measuring the absorbance at 620nm. Calcium content was determined using the o-
15
cresolphthalein complexone assay and measuring the absorbance at 570nm.
17
Mechanical testing of multiphasic constructs
18
On day 21 of culture, the equilibrium compressive modulus of the multiphasic construct’s
19
cartilaginous tissue was determined by stress relaxation testing on the Mach-1 mechanical
20
tester (Bio-Momentum, Laval, Canada) with a 0.65mm-diameter indenter as previously
21
described [4]. Interfacial shear strength was determined by applying a force at the
22
interface region of these samples using a specially designed jig attached to an Instron
23
universal testing machine as previously described [15] (see Supplementary Methods).
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1 Statistical testing
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All values are expressed as mean ± 95% confidence intervals (CI). Each experiment was
4
carried out with three donor animals (N=3). Univariate analysis of variance and Tukey
5
post-hoc testing were used to analyze ALP activity levels on days 0-4, accumulation of
6
GAG, total collagen and mineral contents, as well as the compressive moduli of the
7
constructs. The interfacial shear strength data of the constructs were analyzed using t test.
8
To account for the wide animal-to-animal variability, ALP activity levels on days 7, 10
9
and 14 were each analyzed using paired t test. Similarly, gene expression levels on days 4
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and 21 were log-transformed and analyzed using paired t test.
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Results
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Generation of calcified cartilage at the calcium polyphosphate interface with
3
predifferentiated chondrocytes
4
To engineer a multiphasic construct that incorporates a zone of calcified cartilage at the
5
cartilage-calcium polyphosphate interface using predifferentiated chondrocytes, we first
6
sought to establish a culture condition in which predifferentiated chondrocytes could be
7
cultured on porous calcium polyphosphate substrates to form mineralized cartilage tissue.
8
Triiodothyronine (T3) [18], retinoic acid [19] and dexamethasone [10] have all been
9
identified as potential inducers of chondrocyte mineralization in vitro. We cultured
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predifferentiated chondrocytes on calcium polyphosphate substrates with either 3nM T3
11
or 100µM retinoic acid treatment in the presence or absence of dexamethasone for 1
12
week, and then quantified the alkaline phosphate activity of the cells as an indicator of
13
chondrocyte mineralization potential [20, 21]. ALP activity was the highest in
14
predifferentiated chondrocytes cultured in the absence of dexamethasone with the T3
15
treatment (Figure 2A). While ALP activity was also increased with the retinoic acid
16
treatment, continued culture did not yield cartilaginous tissues on calcium polyphosphate
17
after 3 weeks (data not shown). Therefore, treatment of predifferentiated chondrocytes
18
with T3 was selected for further study.
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When 2×106 predifferentiated chondrocytes were cultured on porous calcium
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polyphosphate substrates for 3 weeks, the mineralized zone was found in the top of the
22
tissue (Figure 2B). Between this mineralized zone and the substrate was an intermediary
23
zone of non-mineralized cartilage, composed of cells with round morphology and
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extracellular matrix rich in proteoglycans. Changing the concentration of T3 treatment
2
did not change the location of the mineralized zone (data not shown). However, when the
3
number of initially seeded predifferentiated chondrocytes was decreased, the distance
4
between the cartilage-substrate interface and the zone of mineralized cartilage also
5
decreased (Figure 2C & 2D). At an initial seeding of 1×106 predifferentiated
6
chondrocytes, the zone of mineralized cartilage formed consistently adjacent to the
7
interface (Figure 2C & 2D, arrows).
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Short-term treatment of predifferentiated chondrocytes with T3 was sufficient to stimulate
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terminal differentiation
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ALP activity levels of predifferentiated chondrocytes were characterized over a 2-week
12
culture period. T3-treated cells had a significantly increased ALP activity by day 4
13
(Figure 3A). Even though the T3 treatment was withdrawn at day 4, the ALP activity
14
level of cells at day 7 was significantly higher than those that had been continuously
15
treated with T3, and were comparable at days 10 and 14 (Figure 3B).
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To confirm that the 4-day T3 treatment was sufficient to induce terminal
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differentiation of predifferentiated chondrocytes, gene expression levels were examined.
19
As early as 4 days, collagen type X (Col10a1) expression was significantly higher in the
20
T3-treated cells compared to the untreated control (Figure 3G). By 21 days, T3-treated
21
cells expressed less collagen type II (Col2a1), aggrecan (Agc1) and Sox9, but greater
22
Col10a1 and osteopontin (Spp1) compared to the non-treated controls (Figure 3C-J). This
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confirmed that the 4-day T3 treatment of predifferentiated chondrocytes had altered the
2
differentiation of these cells.
3 T3-treated predifferentiated chondrocytes did not induce ALP activity in non-T3-treated
5
predifferentiated chondrocytes
6
Based on this observation, a two-stage culture protocol was developed (Figure 1). To
7
grow an interfacial zone of mineralized cartilage, 1.0×106 predifferentiated chondrocytes
8
were first seeded on porous calcium polyphosphate substrates, which was the number of
9
cells that had been previously determined to be optimal for creating the mineralized
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cartilage at the interface, and cultured in the presence of T3 (Figure 2C & 2D). T3 was
11
withdrawn at 4 days of culture, then 1.5×106 predifferentiated chondrocytes were seeded
12
on top of the existing tissue one day later and cultured in the presence of β-
13
glycerophosphate for up to 21 days to generate a zone of non-mineralized cartilage. To
14
confirm that the T3-treated cells would not stimulate the overlaid, non-T3-treated cells to
15
also mineralize, the tissues on calcium polyphosphate were harvested 5 days after the
16
addition of the top layer of cells (10 days of culture in total) and cryosectioned.
17
Histological analysis demonstrated that ALP activity was present only in the calcifying
18
bottom layer (Figure 4), demonstrating the lack of mineralization potential in the top
19
layer even though β-glycerophosphate was present.
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Characterization of the multiphasic constructs
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Constructs were grown for 21 days using the two-stage culture protocol with or without
23
the 4-day T3 treatment of the interfacial layer and characterized using various methods.
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Histological examination of cartilage tissue on the calcium polyphosphate showed two
2
tissue zones. The upper zone of non-mineralized cartilage was hyaline cartilage, rich in
3
type II collagen and proteoglycan (Figure 5A, B, D). The interfacial zone was calcified
4
cartilage, the mineral staining with von Kossa staining and the extracellular matrix
5
staining with toluidine blue and type X collagen (Figure 5A, B, E). Ingrowth of tissue
6
into upper parts of the porous calcium polyphosphate substrate was also observed, with
7
the mineral clusters directly in contact with the calcium polyphosphate particles (Figure
8
5A). No type I collagen was detected in either zone (Figure 5C). In contrast, control
9
constructs not treated with T3 did not show a bizonal composition as there was no zone
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of calcified cartilage, and there was an uneven distribution of extracellular matrix (Figure
11
5F-I). Differential collagen alignment in the tissue was detected using polarized light
12
microscopy. In the superficial aspect, collagen was aligned parallel to the surface.
13
Elsewhere, collagen was observed both pericellularly and aligned orthogonal to the
14
surface. Collagen, parallel to the surface, was also observed in areas within the bottom
15
layer of tissue (Figure 5J). T3-treated constructs accumulated less proteoglycans (Figure
16
6A) and total collagens (Figure 6B) compared to those on untreated constructs, but had
17
amounts comparable to native sheep cartilage. Tissues on T3-treated constructs
18
accumulated calcium and phosphate, consistent with calcification, while untreated
19
constructs did not (Figure 6C).
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T3-treated tissues had a comparable equilibrium compressive modulus to that of
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osteochondral tissue obtained from native sheep femoral condyles (Figure 7A, p = 0.15).
23
The untreated constructs had a significantly higher equilibrium compressive modulus
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compared to that of the T3-treated tissues, possibly due to their greater extracellular
2
matrix accumulation (Figure 6A, 6B). However, T3-treated constructs could withstand a
3
stronger shear force (Figure 7B) and absorb more energy (Figure 7C) than untreated
4
constructs. Histological assessment of the failed constructs confirmed that failure
5
occurred at the interface (data not shown). This demonstrated that the mineralized tissue
6
at the interface (Figure 5A) enhanced the interfacial shear strength.
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Discussion
2
A mechanically competent interface between the cartilage and its substrate is required to
3
resist the shear force generated by the movement of the joint. Previous studies have
4
sought to recreate the zone of calcified cartilage that exists between hyaline cartilage and
5
bone to reinforce this interface with limited success [22]. To overcome this problem, we
6
successfully tissue engineered osteochondral-like constructs with BMSCs as the single
7
cell source, in which cartilage tissue and a porous bone substitute substrate were formed
8
with an interfacial zone of calcified cartilage without using any exogenous scaffolds in
9
the cartilage tissue. The two-stage culture protocol enabled the selective stimulation of
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predifferentiated chondrocytes to form calcified cartilage only at the interface. Treatment
11
of the initially seeded cells with T3 induced the terminal differentiation of cells. This
12
resulted in a zone of calcified cartilage, which, in addition to mineral, also contained
13
collagens type II and X and proteoglycans. These features were seen only in the part of
14
the tissue that was treated with mineralization-inducing T3, which was adjacent to (and
15
integrated with) the porous calcium polyphosphate substrate. The interfacial shear
16
strength of constructs with the calcified cartilaginous interface was increased
17
significantly compared to those without it, functionally validating its intended effect. The
18
hyaline quality of non-mineralized cartilage was demonstrated by the accumulation of
19
collagen type II and the lack of mineral or collagen type I. Further study is required to
20
determine whether this osteochondral-like tissue formation will occur on substrates other
21
than hydroxyapatite-coated calcium polyphosphate substrate.
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Thus far, approaches to induce selective mineralization of BMSC-derived
2
cartilage and create this interface include delivery of bioactive signals either locally via
3
microspheres [23] or by using gradients generated by a bioreactor [24] to undifferentiated
4
BMSCs seeded in scaffolds. In another approach, BMSCs predifferentiated to
5
chondrocytes and osteoblasts are seeded into appropriate locations within scaffolds [25,
6
26]. For both strategies, use of exogenous scaffolds is necessary, which has the potential
7
to hinder host integration and tissue regeneration [27]. Matching the rate of scaffold
8
degeneration to the rate of tissue accumulation remains a challenge when employing
9
scaffolds [22, 28]. Synthetic materials also accumulate wear over time, and may provide
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a focal point for shear failure if not replaced with new tissue. On the other hand, if the
11
rate of scaffold degradation exceeds the rate of cellular remodelling, the mechanical
12
integrity is compromised and the construct would fail under load. A scaffold-free
13
approach obviates this issue altogether; however, this necessitated an alternative strategy
14
to specify the location of the calcified interface.
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The development of our two-stage culture protocol was first enabled by the
17
observation that BMSC-derived predifferentiated chondrocytes formed mineralized
18
cartilage at the tissue’s superficial aspect with mineral-inducing stimulus. Given that T3
19
was delivered to the tissue through culture media, and that the location of calcified
20
cartilage did not change with tissue thickness, this effect was likely due to T3 diffusion
21
into the tissue. As there was no scaffold present, selective delivery of T3 to the interface
22
was difficult to achieve with media alone. To circumvent this, we seeded a small number
23
of cells on the calcium polyphosphate in the presence of T3. This generated thin
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cartilaginous tissue, forcing mineralization to take place juxtaposed to the porous calcium
2
polyphosphate substrate. This enabled the subsequent formation of a continuous layer of
3
non-mineralized cartilage formed by the same cell type in the absence of T3. The
4
presence of collagen types II, X and proteoglycans in the calcified interface, as well as
5
the absence of collagen type I, is an important finding in our study. Taken together with
6
the finding that T3-treated cells expressed hypertrophic chondrocyte makers, this
7
demonstrates that the interface is cartilaginous, not osseous, successfully recapitulating
8
characteristics of the native articular cartilage-subchondral bone interface.
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Histology showed that the layer of non-mineralized cartilage successfully fused to the underlying T3-treated tissue. Interestingly, fissures were observed if the intended
12
interfacial tissue (i.e., the first layer) was not treated with T3, and the total thickness of
13
cartilage tissues on these constructs was increased, with an uneven distribution of the
14
extracellular matrix. There are two possible explanations for these results. One is that the
15
manner that the interfacial layer grows may affect integration, as BMSC-derived
16
chondrocytes share many characteristics with proliferating cartilage [29] that grows
17
appositionally. In development, Indian hedgehog, FGF and BMP signaling pathways
18
regulate this appositional growth[30]; however, in an in vitro system, absence of such
19
complex regulation may allow the interfacial layer to expand laterally. The presence of
20
tubing around the constructs prevented lateral expansion by confinement, which could
21
have resulted in lateral mechanical stress and caused the tissue to buckle and shear. This
22
was not the case for T3-stimulated interfacial layer, however, whose behaviour may more
23
resemble terminal hypertrophic chondrocytes, as evidenced by their mineralized matrix.
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Terminal hypertrophic chondrocytes do not proliferate and eventually undergo apoptosis
2
upon vascularization of matrix and bone formation [31]. Alternatively, the T3 treatment
3
may have affected tissue fusion. Pellets of human MSCs undergoing chondrogenesis have
4
been shown to accumulate tenascin along the outer surface that prevented them from
5
fusing with one another [32]. The fissure between the two layers of non-mineralizing
6
cartilage may stem from a similar boundary-setting phenomenon, whether driven by
7
tenascin accumulation or otherwise.
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Sheep gait studies estimate the maximum anterioposterior shear stress in a stifle
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joint to be about 500kPa [33, 34], which is equivalent to approximately 9N of peak shear
11
force in our study. However, using our methodology the peak shear force of native
12
cartilage interface could not be measured, as it withstood the maximum load (80N)
13
without shear failure. In healthy cartilage, collagen fibrils run orthogonally through the
14
tidemark plane, which would confer a high resistance against failure in our mode of
15
study. Although our multiphasic construct possesses inferior interfacial shear strength
16
compared to articular cartilage, it still represented a four-fold improvement over the non-
17
mineralized control, and sheep gait studies suggest that this improvement is adequate for
18
preventing delamination.
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Importantly, this protocol can be expanded to generate large, anatomically shaped
21
osteochondral-like constructs as replacements for damaged joint tissues. Protocols have
22
been developed to generate ~108 BMSCs with chondrogenic potential in a good
23
manufacturing practices-complaint manner [35, 36], which can be used to generate
17
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sufficiently large numbers of chondrocytes to cover a large articular surface. We have
2
previously shown that calcium polyphosphate powders can be used in additive
3
manufacturing methods to generate substrates of required shape and size [37]. Calcium
4
polyphosphate formed in this way exhibited the same degree of osseointegration after
5
implantation as those produced conventionally [38]. As such, preclinical studies are
6
warranted to determine if these osteochondral-like constructs with a mineralized
7
cartilaginous interface could repair joint defects in vivo.
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8 Conclusion
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Using BMSCs as a single cell source, an osteochondral-like construct comprised of
11
hyaline cartilage and porous bone substitute was created with a zone of calcified cartilage
12
interfacing them. The novel, two-step cell culture strategy employed no exogenous
13
scaffolds, just a substrate on which to form tissue. The presence of calcified cartilage
14
increased the shear load that the construct could withstand at the interface. As no
15
xenogeneic material was used at any point during culture, this strategy can be directly
16
translated into preclinical studies to determine whether these constructs can repair joint
17
defects in vivo.
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Acknowledgments
2
We thank Eugene Hu for preparing the porous calcium polyphosphate substrates. We also
3
thank Dr. Jian Wang and Nancy Valiquette for their technical assistance. This work was
4
supported by the United States Department of Defense (to R.A.K., W81XWH-10-1-0787)
5
and the Natural Sciences and Engineering Research Council of Canada (to W.L.S.,
6
RGPIN 293170-11). W.L.S. is supported by a Canadian Research Chair.
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Author Contributions
Study conception and design: WDL, WLS, RAK.
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Provision of study materials: MBH, RMP.
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Analysis and interpretation of data: WDL, RMP, RAK.
12
Drafting of article: WDL, WLS, RAK.
13
Critical revision of the article: WDL, RMP, WLS, RAK.
14
Final approval of the article: WDL, WLS, RAK.
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Role of the Funding Sources
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None of the listed bodies played any role in this study.
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Conflict of Interest
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No competing financial interests exist for any of the listed authors.
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Figure Legends
2 Figure 1. Line diagram of the tissue culture protocol used for forming scaffold-free
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multiphasic osteochondral-like constructs.
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Figure 2. Predifferentiated chondrocytes formed cartilaginous tissues on porous calcium
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polyphosphate substrates with a zone of mineralized cartilage. (A) ALP activity of
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predifferentiated chondrocytes cultured for 7 days with 3nM triiodothyronine (T3) or
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100nM retinoic acid (RA) with or without dexamethasone (Dex). Mean ± 95% CI, n=3,
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nd: not detected. * p = 0.043 between +T3 –Dex and +T3 +Dex; p = 0.045 between +T3
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–Dex and +RA –Dex. (B) Von Kossa and toluidine blue-stained sections of cartilaginous
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tissue formed by culturing 2×106 predifferentiated chondrocytes on the substrates with
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3nM T3 for 3 weeks, showing mineralized zone occurring at the superficial aspect of
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cartilage. Asterisk denotes the original location of the substrate. Scale bar = 200µm. (C)
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Cross-sectional µCT tomographs and (D) and histology of 3-week-old constructs with
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decreasing numbers of seeded cells: 2×106 (left), 1.5×106 (middle) and 1.0×106 (right)
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predifferentiated chondrocytes. In (D) vertical bars denote non-mineralized cartilage
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(purple), mineralized zone (green) and the substrate (grey). Arrows indicate the lack of
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separation between the mineralized zone and the substrate. Scale bar = 1mm (C) and
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200µm (D).
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Figure 3. Short term T3 treatment of predifferentiated chondrocytes was sufficient to
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stimulate terminal differentiation. Predifferentiated chondrocytes were treated with 3nM
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T3 for the first 4 days. In selected cultures, T3 treatment was withdrawn after 4 days
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(WD). (A) ALP activity during the first 4 days. * p < 0.001 between days 0-4 & 2-4. (B)
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ALP activity of WD, compared to cells in which T3 treatment was continued (+) for up to
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14 days. * p = 0.014. (C-J) Gene expression analysis of WD on days 4 and 21 compared
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to non-T3-treated controls (–). * p = 0.013 (Col2a1 on day 21); p = 0.031 (Agc1 on day
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21); p = 0.009 (Sox9 on day 21); p = 0.003 (Col10a1 on days 4 & 21); p = 0.025 (Spp1 on
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day 4) and p = 0.002 (Spp1 on day 21). Data points are color-coded for each animal
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throughout. Results are expressed as mean ± 95% CI, n=3.
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Figure 4. Layer of predifferentiated chondrocytes treated with T3 did not induce the
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overlaid layer of non-treated predifferentiated chondrocytes to activate ALP activity.
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1x106 cells were cultured on porous calcium polyphosphate substrates with (A) or
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without (B) 3nM T3 for 4 days. On day 5, 1.5x106 cells were seeded on top of these
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cells/tissue and cultured without T3. Cryosections of tissues from day 10 multiphasic
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constructs were stained for ALP activity. The blue staining indicative of ALP was seen
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only in the mineralizing layer (vertical bar). Asterisk denotes the location of where the
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substrate would have been located prior to its removal, scale bar = 200µm.
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Figure 5. A two-step culture protocol of predifferentiated chondrocytes on porous
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calcium polyphosphate substrates produced cartilage tissue with a mineralized zone at the
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interface. (A) Day 21 constructs with 4-day T3 treatment were processed uncalcified,
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sectioned and stained to reveal the cartilaginous (purple), mineral (green) and substrate
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particles (brown). (B-I) Tissues were excised from the substrate and examined
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histologically for the distribution of mineral and proteoglycan accumulation (B, F), by
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immunofluorescent staining for the accumulation of different types of collagen (C-E, G-
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I). Constructs with T3-treated interfacial layer are denoted as +T3, and untreated (no T3)
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control constructs are denoted as –T3. Scale bars = 200µm. (J) Differential collagen fiber
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orientations on tissues was seen by polarized light microscopy. Scale bar = 100µm.
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Figure 6. Accumulation of extracellular matrix in T3-treated (+T3) tissues was less
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compared to those in untreated (–T3) tissues, but comparable to native articular cartilage.
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(A) Accumulated proteoglycan and (B) total collagen were quantified and normalized by
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their DNA content. (A) * p = 0.001, (B) * p = 0.012 between +T3 and –T3; p = 0.037
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between +T3 and Native. (C) Accumulation of mineral in tissue was quantified by
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measuring the calcium and phosphate contents, normalized by tissue dry weight. ** p
