Altered Hypertrophic Chondrocyte Kinetics in Gdf-5 Deficient Murine Growth Plates

INTRODUCTION The growth/differentiation factors (GDFs) are a subgroup of the Bone Morphogenetic Proteins (BMPs) best known for their role in joint formation and chondrogenesis [1,2]. Mice deficient in one of these signaling proteins, GDF-5, exhibit numerous skeletal abnormalities, including shortened limb bones [3]. Because longitudinal bone growth is achieved via the process of endochondral ossification, it is likely that GDF-5 plays a part in modulating this process, although the precise nature of its role remains to be determined. The aim of this study was two-fold. First, we sought to determine whether GDF-5 deficiency would alter the growth rate in growth plates from the long bones of brachypod (bp) mice. Second, we asked whether the effects of GDF-5 deficiency on long bone growth plates are dependent on anatomical location. METHODS Two groups of ten, five-week old healthy female mice were used to assess growth plate stereologic parameters and chondrocyte kinetics in the proximal tibia and humerus. Experimental animals consisted of GDF-5 -/brachypod mice, while phenotypically normal heterozygous (+/-) littermates served as controls. At five weeks of age, all animals received IP injections of calceine at 10mg/kg and were euthanized exactly four days later. Calceine labels were used to determine the growth rate of each individual growth plate. All experimental procedures were approved by the appropriate IACUC. One-micron thick serial vertical sections were cut parallel to the long axis of the growth plate and used for image acquisition and stereologic analyses of proximal tibial and humeral growth plates after standard fixation and epon embedding [4]. Alternate serial sections were either left unstained or stained with toluidine blue. Zone heights were determined by delineating the top of the growth plate, the junctions between each zone, as well as the chondrosseous junction. Using point-counting methods on highly magnified images (800X), cell area fraction was estimated in the proliferative and hypertrophic zones. Mean cell volume (MCV) was estimated in these same zones using point-sampled intercepts measured on cells lying on grid intersection points with a different grid angle used for each field of view [5]. Cell populations in the proliferative and hypertrophic zones were estimated by counting chondrocyte numbers in a reference volume of a cylinder of 1mm diameter and height equal to the height of the zone being analyzed [6,7]. Growth rate was measured in microns per day by measuring the distance between the center of the fluorescent calceine label and the chondrosseous junction. Lastly, chondrocyte kinetic parameters were obtained by making the assumption of steady-state cell activity in the growth plate over a 24-hour period [6]. Using published techniques, the number of cells lost per day and the phase duration of the proliferative and hypertrophic phases were also determined [7]. Data were analyzed using a two-factor ANOVA with genotype and site as the two factors. A cut-off value of p < 0.05 was chosen for statistical significance. RESULTS Histologically, there were no grossly discernable distinctions in the morphology of GDF-5 deficient versus control growth plates at either location. Statistically, no significant interaction between genotype and growth plate site was detected for any of the parameters under investigation, indicating that GDF-5 deficiency did not differentially affect one growth plate site more than the other (Table 1). Based on the results of the two-factor ANOVA, genotype was found to significantly affect growth rate (reduced in mutants), hypertrophic zone height (increased in mutants), and hypertrophic phase duration (increased in mutants) (Table 1). The number of cells lost per day was lower in mutants, although the effect of genotype was not statistically significant (p = 0.08; Table 1). For the purposes of quantifying the magnitude of the effect of genotype on growth rate parameters, each site was analyzed independently (Tables 2 & 3). GDF-5 deficient proximal tibial growth plates exhibited a statistically significant 14% reduction in growth rate as well as a 25% increase in hypertrophic cell phase duration (p < 0.05; Table 1). Similar trends were observed in the proximal humeral growth plate, but none of the differences were statistically significant between mutants and controls when this site was analyzed alone (Table 3). Table 1: ANOVA Results for Growth Plate Parameters p-value Parameter: genotype site gene x site Growth Rate (μm/day) 0.0225* < 0.0001* 0.1676 Total height (μm) 0.2226 < 0.0001* 0.2333 RZ height (μm) 0.9140 0.2970 0.8290 PZ height (μm) 0.8433 < 0.0001* 0.0590 PZ MCV (μm) 0.2193 0.5645 0.6288 HZ height (μm) 0.0489* 0.0001* 0.8504 HZ MCV (μm) 0.7685 0.0003* 0.9726 Cells lost/day 0.0802 0.0058* 0.9460 PZ cells/cylinder (mm) 0.6192 0.0009* 0.8551 PZ phase duration (hr) 0.1435 0.7029 0.9856 HZ cells/cylinder (mm) 0.3978 0.6219 0.2259 HZ phase duration (hr) 0.0010* 0.0020* 0.2589 * p < 0.05; RZ = Resting Zone; PZ = Proliferative Zone; HZ = Hypertrophic Zone Table 2: Proximal Tibial Growth Plate Stereological Parameters Parameter GDF-5 -/(n = 10) GDF-5 +/(n = 10) Difference (%) Growth Rate (μm/day) 68 (11) 79 (11) -14%* Total height (μm) 203.3 (9.9) 203.2 (11.5) <1% RZ height (μm) 31.9 (4.0) 32.3 (3.7) <1% PZ height (μm) 92.1 (6.1) 95.9 (7.1) -4% PZ MCV (μm) 713 (88) 658 (100) 8% HZ height (μm) 79.4 (6.3) 75.1 (6.8) 6% HZ MCV (μm) 3040 (434) 3090 (478) -2% Cells lost/day 7530 (1631) 8320 (1256) -9% PZ cells/cylinder (mm) 31800(4909) 32900(4851) -3% PZ phase duration (hr) 106.4 (23.6) 96.7 (16.6) 10% HZ cells/cylinder (mm) 8760 (1318) 7830 (877) 12% HZ phase duration (hr) 28.7 (4.8) 23.0 (3.3) 25%* * p < 0.05 Table 3: Proximal Humeral Growth Plate Stereological Parameters Parameter GDF-5 -/(n = 10) GDF-5 +/(n = 10) Difference (%) Growth Rate (μm/day) 55 (7) 58 (8) -5% Total height (μm) 187.9 (9.5) 179.4 (12.6) 5% RZ height (μm) 33.2 (2.4) 33.1 (2.4) <1% PZ height (μm) 84.1 (6.5) 79.3 (8.1) 6% PZ MCV (μm) 679 (62.4) 655 (135.8) 4% HZ height (μm) 70.7 (6.2) 67.1 (5.4) 5% HZ MCV (μm) 2530 (212) 2560 (448) -1% Cells lost/day 6170 (1074) 7020 (1677) -12% PZ cells/cylinder (mm) 26200(4105) 26700(6451) -2% PZ phase duration (hr) 104.0 (20.3) 94.0 (22.3) 11% HZ cells/cylinder (mm) 7990 (1211) 8160 (1958) -2% HZ phase duration (hr) 31.4 (3.6) 28.4 (3.4) 11%