Glycosaminoglycan profiles of repair tissue formed following autologous chondrocyte implantation differ from control cartilage

Abstract

Currently, autologous chondrocyte implantation (ACI) is the most commonly used cell-based therapy for the treatment of isolated femoral condyle lesions of the knee. A small number of centres performing ACI have reported encouraging long-term clinical results, but there is currently a lack of quantitative and qualitative biochemical data regarding the nature of the repair tissue. Glycosaminoglycan (GAG) structure influences physiological function and is likely to be important in the long-term stability of the repair tissue. The objective of this study was to use fluorophore-assisted carbohydrate electrophoresis (FACE) to both quantitatively and qualitatively analyse the GAG composition of repair tissue biopsies and compare them with age-matched cadaveric controls. We used immunohistochemistry to provide a baseline reference for comparison. Biopsies were taken from eight patients (22 to 52 years old) 1 year after ACI treatment and from four cadavers (20 to 50 years old). FACE quantitatively profiled the GAGs in as little as 5 microg of cartilage. The pattern and intensity of immunostaining were generally comparable with the data obtained with FACE. In the ACI repair tissue, there was a twofold reduction in chondroitin sulphate and keratan sulphate compared with age-matched control cartilage. By contrast, there was an increase in hyaluronan with significantly shorter chondroitin sulphate chains and less chondroitin 6-sulphate in repair tissue than control cartilage. The composition of the repair tissue thus is not identical to mature articular cartilage.

Figure 1

Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis. (a) Representative FACE gel of hyaluronidase and chondroitinase digestion products from a 30-year-old cadaveric control. Lanes A and B contain a mixture of predefined fluorotagged saccharide standards. Lane A: (1) ΔDiHA, (2) ΔDi6S, (3) ΔDi4S, (4) ΔDi2S, (5) ΔDi4,6S, (6) ΔDi2,6S, (7) ΔDi2,4S, (8) ΔTri2,4,6S. Lane B: (9) N-acetylgalactosamine (GalNAc), (10) ΔDi0S, (11) GalNAc6S, (12) GalNAc4S, (13) 4-/6-sulphated GalNAc. Samples were digested with hyaluronidase (from Streptococcus dysgalactiae) (lane C) and chondroitinase ABC (lane D). (b) Representative FACE gel of chondroitin sulphate termini products from a 22-year-old cadaveric control (lane B) and a 22-year-old autologous chondrocyte implantation (ACI) patient (lane C). Lane A contains a mixture of predefined fluorotagged saccharide standards. Lane A: (1) GalNAc, (2) GalNAc6S, (3) GalNAc4S, (4) GalNAc4,6S, (5) GalNAc2,4,6S. (c) Representative FACE gel of keratanase II and endo-β-galactosidase digestion products obtained from full-depth cartilage of a 30-year-old cadaveric control (lanes E and F) and a 27-year-old ACI patient (lanes G and H). Lanes A to D contain a mixture of predefined fluorotagged saccharide standards. Lanes A to D: (1) Galβ1,2 [fucα1,3]GlcNAc6S, (2) Galβ1,4GlcNAc6S, (3) Gal6Sβ1,4glcNAc6S, (4) NeuAα2,3Gal6Sβ1,4GlcNAc6S, (5) GlcNAcβ1,3Gal, (6) GlcNAc6Sβ1,3Gal. ΔDi0S, unsulphated chondroitin sulphate disaccharide; ΔDi4S, chondroitin-4 sulphate disaccharide; ΔDi6S, chondroitin-6 sulphate disaccharide; ΔDiHA, hyaluronan disaccharide.

Figure 2

Fluorophore-assisted carbohydrate electrophoresis analysis of hyaluronidase, chondroitinase, keratanase II, and endo-β-galactosidase digestion products. Quantitation of levels of (a,b) hyaluronan and chondroitin sulphate (CS) disaccharides, (c,d) nonreducing terminal sugars of CS, and (e,f) keratan sulphate in autologous chondrocyte implantation (ACI) repair and cadaveric control tissues. Data are expressed as micrograms of disaccharide per milligram of wet weight of cartilage tissue. For each biopsy, the data are presented as the mean ± standard deviation of the replicate measurements. Differences between the ACI repair and cadaveric tissues were analysed using the Mann-Whitney U test (p < 0.05%). ΔDi0S, unsulphated chondroitin sulphate disaccharide; ΔDi4S, chondroitin-4 sulphate disaccharide; ΔDi6S, chondroitin-6 sulphate disaccharide; ΔDiHA, hyaluronan disaccharide; GalNAc, N-acetylgalactosamine.

Figure 3

Determination of the averaged chondroitin sulphate chain size by fluorophore-assisted carbohydrate electrophoresis analysis and ratio of ΔDi6S/ΔDi4S. Comparison of (a) number averaged chondroitin sulphate chain size and (b) ratio of ΔDi6S/ΔDi4S in autologous chondrocyte implantation (ACI) repair and cadaveric control tissues. For each biopsy, the data are presented as the mean ± standard deviation of the replicate measurements. Differences between the repair and control tissue were analysed using the Mann-Whitney test (*p < 0.05%). ΔDi4S, chondroitin-4 sulphate disaccharide; ΔDi6S, chondroitin-6 sulphate disaccharide.

Figure 4

Proportions of chondroitin sulphate (CS), hyaluronan (HA), and keratan sulphate (KS) in the biopsies. Proportions of HA, CS, and KS in biopsies from (a) patients 1 year after autologous chondrocyte implantation and (b) controls. Each glycosaminoglycan (GAG), with its constituent disaccharide and monosaccharide component, is expressed as a total percentage of GAGs analysed by fluorophore-assisted carbohydrate electrophoresis.

Figure 5

Control tissue stained with haematoxylin and eosin. Representative sections from the medial femoral condyle of (a) a 30-year-old and (b) a 50-year-old to illustrate the surface layer in cartilage taken. The arrows indicate chondrocyte clusters. The bars represent 25 μm.

Figure 6

Immunohistochemistry to assess glycosaminoglycan distribution. Cartilage sections immunohistochemically stained for (a,b) chondroitin-4-sulphate and (c,d) chondroitin-6-sulphate. Repair tissue from autologous chondrocyte implantation (ACI)-treated patients with the highest (a) (22-year-old) and lowest (b) (52-year-old) levels of chondroitin-4-sulphate measured by fluorophore-assisted carbohydrate electrophoresis (FACE). Biopsies of cartilage from a 22 yr old ACI patient (c) and its age-matched control (d) immunostained for chondroitin 6-sulphate. The pattern of immunostaining in repair tissue from the ACI-treated patient (c) with the highest levels of chondroitin-6-sulphate as measured by FACE demonstrated a pericellular pattern, similar to that seen in the age-matched cadaveric control tissue but there was also more widespread matrix staining (d).