Cell Tissue Res (2006) 323: 127–135 DOI 10.1007/s00441-005-0050-y
REGULAR A RTICLE
Chisa Hidaka . Christina Cheng . Deborah Alexandre . Madhu Bhargava . Peter A. Torzilli
Maturational differences in superficial and deep zone articular chondrocytes Received: 14 December 2004 / Accepted: 27 June 2005 / Published online: 31 August 2005 # Springer-Verlag 2006
Abstract To examine whether differences in chondrocytes from skeletally immature versus adult individuals are important in cartilage healing, repair, or tissue engineering, superficial zone chondrocytes (SZC, from within 100 μm of the articular surface) and deep zone chondrocytes (DZC, from 30%–45% of the deepest un-mineralized part of articular cartilage) were harvested from immature (1–4 months) and young adult (18–36 months) steers and compared. Cell size, matrix gene expression and protein levels, integrin levels, and chemotactic ability were measured in cells maintained in micromass culture for up to 7 days. Regardless of age, SZC were smaller, had a lower type II to type I collagen gene expression ratio, and higher gene expression of SZ proteins than their DZC counterparts. Regardless of zone, chondrocytes from immature steers had higher levels of Sox 9 and type II collagen gene expression. Over 7 days in culture, the SZC of immature steers had the highest rate of proliferation. Phenotypically, the SZC of immature and adult steers were more stable than their respective DZC. Cell surface α5 and α2 integrin subunit levels were higher in the SZC of immature than of adult steers, whereas β1 integrin subunit levels were similar. Both immature and adult SZC were capable of chemotaxis in response to fetal bovine serum or basic fibroblast growth factor. Our data indicate that articular chonThis work was supported in part by the National Chapter of the Arthritis Foundation, the Ira DeCamp Fellowship for Musculoskeletal Research of the Hospital for Special Surgery, the Institute for Sports Medicine Research in New York, and the National Institute of Health grant AR045748 and was conducted in a facility constructed with support from the Research Facilities Improvement Program (grant no. C06-RR12538-01) of the National Center for Research Resources, National Institutes of Health. C. Hidaka (*) . C. Cheng . D. Alexandre . M. Bhargava . P. A. Torzilli Laboratory for Soft Tissue Research, Musculoskeletal Integrity Program, Hospital for Special Surgery, New York, NY, USA e-mail:
[email protected] Tel.: +1-212-7742384 Fax: +1-212-2492373
drocytes vary in the different zones of cartilage and with the age of the donor. These differences may be important for cartilage growth, tissue engineering, and/or repair. Keywords Chondrocyte . Migration . De-differentiation . Integrin . Maturation . Bovine
Introduction Although intra-articular injuries are clinically problematic in patients of any age, better healing appears to occur in skeletally immature individuals for reasons that are not well understood (Mankin 1964; Campbell 1969; Wei and Messner 1997, 1999; Namba et al. 1998; Hunziker 2002; Martin and Buckwalter 2003). In adults, injured cartilage remains quiescent and/or slowly degrades. To induce a repair response, cells must be brought into the defect either by drilling into the subchondral bone or by implantation of chondrocytes and/or other cells and tissues (Mankin 1964; Campbell 1969; Hunziker 2002). These strategies have clinical efficacy, but no available treatment can lead to the regeneration of tissue whose organization and function approaches those of native articular cartilage (Mankin 1964; Campbell 1969; Hunziker 2002). One area that has recently received attention for potentially enhancing cell-based cartilage repair is the use of zonal sub-populations of articular chondrocytes (Kim et al. 2003; Klein et al. 2003; Waldman et al. 2003). Although articular chondrocytes are often considered as being homogeneous, several studies have reported significant phenotypic differences between “surface zone chondrocytes” (SZC), isolated from close to the articular surface, versus “deep zone chondrocytes” (DZC), isolated from deeper regions near the subchondral plate (Aydelotte and Kuettner 1988; Aydelotte et al. 1988; Archer et al. 1990; Siczkowski and Watt 1990; Zanetti et al. 1985; Darling et al. 2004). By using SZC and DZC, researchers have generated layered constructs with organizational features similar to normal articular cartilage (Kim et al. 2003; Klein et al. 2003). However, these constructs were engineered from chondro-
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cytes harvested from immature animals (steers), whereas the studies characterizing differences between SZC and DZC were performed with cells harvested from mature animals (Aydelotte and Kuettner 1988; Aydelotte et al. 1988; Archer et al. 1990; Siczkowski and Watt 1990; Zanetti et al. 1985; Darling et al. 2004). Because cartilage healing is more vigorous in skeletally immature individuals, and because the boundary between articular cartilage and the epiphyseal growth plate may be indistinct in such individuals, we have designed this study to compare SZC and DZC from immature and adult steers. In addition to several phenotypic characteristics previously shown to be different between SZC and DZC, such as cell size, proliferation, and matrix production, we have also examined de-differentiation, cell migration, and integrin expression, as these may be important for cell-based cartilage repair. We have found significant differences in these parameters between the SZC and DZC; these differences are related to the age of the donor.
Materials and methods Preparation of SZC and DZC Chondrocytes were isolated from the femoral condyles of immature (1–4 mo) and young adult (18–36 mo) steers (Aydelotte and Kuettner 1988; Aydelotte et al. 1988). Briefly, superficial (SZ) and deep (DZ) zone cartilage was dissected with a scalpel, and chondrocytes were isolated by overnight digestion in 0.1% bacterial collagenase (type II, Worthington Biochemical, Lakewood, N.J.) in Dulbecco’s modified essential medium (DMEM) and 5% fetal bovine serum (FBS; both from GibcoInvitrogen, Carlsbad, Calif.). The SZ was defined as the 100 μm (or less) of tissue closest to the articular surface as measured by using a custom device with a ±2.5 mm displacement transducer (resolution 1 μm). After initial thickness measurements, SZ was defined as being less than 4% or 10% of the wet weight of immature and adult cartilage, respectively. The DZ was defined as 45% or less of the cartilage closest to the subchondral bone in cartilage from steers of either age. The middle zone was weighed and then discarded. Assays (see below) were performed on SZC and DZC immediately after isolation (day 0) and after 4 and 7 days of micromass culture (5×105 cells per 10 μl drop) in “complete media” consisting of DMEM supplemented with 10% FBS, 1× antibiotic/antimycotic solution (GibcoInvitrogen), and 25 μg/ml ascorbate (Sigma, St. Louis, Mo.). Cell number was determined at each time point by enzymatically releasing cells from the wells and counting them on a hemocytometer.
Coulter counter (Beckmann Coulter, Miami, Fl.). Histograms were used to determine an average cell size for each sub-population. Gene expression Total RNA was isolated from chondrocytes (RNeasy Mini Kit, Qiagen, Valencia, Calif.), and equal concentrations were reverse-transcribed (First Strand cDNA Synthesis Kit, Fermentas, Hanover, Md.). Quantitative polymerase chain reaction (PCR) was performed by using a MyiQ Singlecolor Real Time PCR detection system (BioRad, Hercules, Calif.). Primers were designed by using Primer Express software Version 2.0.0 (Applied Biosystems, Foster City, Calif.) and were based on bovine sequences (GenBank); homology was confirmed against the bovine genome. Primer sets included: type II collagen (5′-GCA TTG CCT ACC TGG ACG AA-3′ forward; 5′-GAA CCT GCT GTT GCC CTC AG-3′ reverse), type IA collagen (5′-ATC CCA GTC AAG AAC TGG TAC AGA A-3′ forward; 5′-CTG GGT ACC ACC GTT GAT AGT TT-3′ reverse), Sox 9 (5′ACC AGT ACC CGC ACC TGC-3′ forward; 5′- AAC GGC CGC TTC TCG C-3′ reverse); aggrecan core protein (5′-GTG AAT CCC AAA ACG CCA CT-3′ forward; 5′CGG CGT AGC ACT TGT CCA G-3′ reverse), SZ protein (SZP, 5′-TGC AAC GGT AGG CCA GTA GAT-3′ forward; 5′-CAT CCA GAA ATA ATG ACC TCG AAA T-3′ reverse), and the house-keeping gene β-actin (5′- CTG CGG CAT TCA CGA AAC TA -3′ forward; 5′- ACC GTG TTG GCG TAG AGG TC -3′ reverse). Relative expression levels were calculated based on ΔCT, viz., the difference in threshold cycle between the gene of interest and the housekeeping gene β-actin. Statistical analyses were performed on the raw threshold cycle numbers for each gene of interest for each sample. Matrix proteins The amount of type II collagen in culture supernatants was measured by enzyme-linked immunosorbant assay (ELISA; Chondrex, Redmond, Wash.) by using serial dilutions of bovine type II collagen (Southern Biotechnology, Birmingham, Ala.) as a standard. Supernatants were collected on days 4 and 7 after addition of 62.5 μg/ml βaminopropylnitrile (Sigma) during the final 18 h of the culture period to prevent cross-linking of newly formed collagen. Total proteoglycan (PG) content in the supernatant and in micromass cultures digested with papain (Sigma) was measured by using the dimethyl-methylene blue dye-binding assay (Farndale et al. 1986). All measurements were normalized to total cell number.
Cell size distribution Integrins Cell size distributions were determined on day 0 by counting the number of cells with diameters ranging between 6 μm and 16 μm at 0.5-μm intervals with a
The presence of α2, α5, and β1 integrin subunits on the surface of chondrocytes was determined by flow cytometry
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Statistical analysis Each assay was performed at least twice with triplicate samples of chondrocytes pooled from two or more steers of each age. In all, stifles (joint next to the hock) from 10 steers of each age were used. For all measurements (except for cell size and real-time PCR), the results were reported as a mean ± standard deviation (SD), and comparisons were made by analysis of variance (ANOVA) with posthoc testing if differences were detected. Cell sizes were compared by using the Kruskal-Wallis test, with further
Tissue thickness and weight The SZ cartilage slices were approximately 80 μm thick with a coefficient of variation of ±15%, confirming the reproducibility of our manual dissection. The SZ comprised 3.0±0.2% of the total wet weight of tissue from immature steers and 6.3±0.4% of tissue from adult steers, the differences arising from the greater thickness of cartilage from the immature versus adult steers. The DZ comprised 33.8±1.1% of the total wet weight of tissue from immature steers and 47.8±1.7% of the total wet weight of adult steers. Cell size Immature SZC were smaller than those from adult, with 51% of immature but only 26% of adult SZC having a diameter of
