Stereological and serial section analysis of chondrocytic enlargement in the proximal tibial growth plate of the rat.
код для вставкиСкачатьTHE ANATOMICAL RECORD 239:255-268 (1994) Stereological and Serial Section Analysis of Chondrocytic Enlargement in the Proximal Tibia1 Growth Plate of the Rat G.J. BREUR, J. TURGAI, B.A. VANENKEVORT, C.E. FARNUM, AND N.J. WILSMAN Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin, 53706; Department of Anatomy, Cornell University, Ithaca, New York 14853 (Farnum) ABSTRACT Background: It has been suggested that within the growth plate, the final volume and shape of hypertrophic chondrocytes are important variables in determining the rate of longitudinal bone growth. To better understand the organization and regulation of chondrocytic hypertrophy as related to longitudinal bone growth, the beginning and end, and the location and magnitude of chondrocytic volume and shape changes during the hypertrophic process were defined in the proximal tibia1 growth plate of 35-day-old rats. Methods: In this study we used two different approaches, a stereological analysis of chondrocytes in unbiasedly defined, narrow growth plate strata, and a serial section reconstruction and measurement of individual cells. In both experiments chondrocytes were preserved using optimal chemical fixation. Proliferating chondrocytes were identified using bromodeoxyruidine labelling, and the rate of longitudinal bone growth was determined using oxytetracycline labelling. Results: In both studies, immediately following cell division in the proliferative zone, chondrocytic volume gradually increased toward the midpoint of the growth plate. During this phase of about 30 hours, approximately 20% of the final cell volume was obtained. During the following 20 hours the remaining 80% was acquired. The estimated rate of cell volume increased changed from approximately 50 pm3/hr during the first 30 hours to about 800 pm3/hr during the last 20 hours. The increase in cell volume resulted in an increase in both the vertical and the horizontal chondrocytic diameters. Cell parameters did not change during the final five hours of the maturation process. Conclusions: In this study we demonstrated that chondrocytic enlargement starts immediately following cell division in the proliferative zone, and that chondrocytic enlargement consists of two morphologically distinguishable phases. The transition point between the first and the second phase of chondrocytic enlargement corresponded with the junction between the proliferative zone and the maturation zone. 0 1994 Wiley-Liss, Inc. Key words: Growth plate, Chondrocytes,Enlargement, Cell shape, Hypertrophy, Rats The major portion of the elongation of long bones is contributed by cartilage discs (growth plates), located near the end of long bones. During periods of active bone growth, growth plates form cartilage that is converted into metaphyseal bone (endochondral ossification). The formation of new cartilage is a well organized process, as evidenced by the columnar arrangement of chondrocytes and the polar morphology of the growth plates (Ham and Cormack, 1984). Chondrocytes at the epiphyseal end of the columns actively divide (proliferation), while more distally, towards the metaphyseal side of the growth plate, cells increase in size and diameters (hypertrophy) (Buckwalter e t al., 1986; Cruz-Orive and 0 1994 WILEY-LISS, INC Hunziker 1986; Hunziker et al., 1987; Hunziker and Schenk, 1989; Kember, 1960,1972,1973,1978;Kember and Walker 1971; Walker and Kember, 1972). Finally, chondrocytes die abruptly during the conversion of the newly formed cartilage into metaphyseal bone (Brighton et al., 1973, 1982; Farnum and Wilsman, 1987, 1989; Hunziker and Schenk, 1989; Schenk, 1980). Received November 15, 1993; accepted February 10, 1994. Dr. Breur’s present address is Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907-1248. Address reprint requests there. 256 G.J. BREUR ET AL. The rate of longitudinal bone growth is a function of the rate of cell division in the columns and the height (cell diameter parallel to the long axis of the bone) of juxta-metaphyseal hypertrophic chondrocytes (Hunziker and Schenk, 1989; Kember, 1960, 1972, 1973, 1978; Thorngren and Hansson, 1973a,b, 1981; Sissons, 1955). It has been suggested, based on the large variety of the number of newly produced chondrocytes in growth plates growing at different rates and the relatively minor observed changes in the mean height of juxta-metaphyseal chondrocytes, that the rate of new cell production in the proliferative zone is the principal determinant of the rate of longitudinal bone growth (Kember and Walker, 1971; Kember, 1978; Moss-Salentijn, 1974; Nilsson et al., 1986; Thorngren and Hansson, 1973a,b). However, recent studies have indicated that the role of chondrocytic hypertrophy may not be as passive as previously suggested. It has been demonstrated that the volume of hypertrophic chondrocytes may be up to 10 times larger than the volume of proliferative chondrocytes, and chondrocytic height may increase four- to fivefold between the proliferative zone and the hypertrophic zone (Buckwalter et al., 1986; Hunziker et al., 1987; Hunziker and Schenk, 1989). Furthermore, recent studies of chondrocyte performance in the proximal tibial growth plate of mice and rats at different ages suggested that changes in the rate of bone growth are accompanied by changes in the size of hypertrophic chondrocytes (Buckwalter and Mower, 1987; Hunziker and Schenk, 1989). In a recent study, including 16 growth plates spread among four locations and two species a t two different ages, we found a strong correlation (rat = 0.98; pig = 0.83) and a positive linear relationship between the rate of longitudinal bone growth and the volume of hypertrophic chondrocytes (Breur et al., 1991). A similar correlation and relationship was found in rabbits (Kuhn et al., 1993). These studies suggest that in growth plates, chondrocytic enlargement plays a major role in the determination of the rate of longitudinal bone growth. The process of chondrocytic enlargement a s i t relates to longitudinal bone growth is characterized by a n increase in cell volume and a modulation of cell shape. In the proliferative zone, cells have a flattened, oblate spheroid shape, while in the hypertrophic zone enlarged chondrocytes are more rounded and have obtained a more sphere-like shape, or a prolate spheroid or superspheroid shape (Buckwalter e t al., 1986, 1987; Cruz-Orive and Hunziker, 1986; Hunziker et al., 1987; Hunziker and Schenk, 1989). Even though the process of chondrocytic enlargement is generally referred to as hypertrophy, several workers have demonstrated that the increase in cell volume is mainly the result of cellular swelling (absorption of water), rather than cellular hypertrophy (increase in cell organelles) (Brighton e t al., 1973,1982; Buckwalter et al., 1986; Hunziker et al., 1987). The regulation of chondrocytic volume and chondrocytic shape is poorly understood. It has been hypothesized that chondrocytic shape is modulated by transphyseal collagen fibrils in the longitudinal septa of the growth plate and the periosteal-perichondrial complex (matrix directed cell swelling) (Buckwalter and Sjolund, 1989). However, experimental evidence and evidence obtained from pseudoachondroplastic growth plates suggest that other matrix constituents also may be involved in the modulation of chondrocytic shape (Breur et al., 1990, 1992; Dietz et al., 1992; Engfeldt, 1969a; Westerborn, 1961). Recently, a positive relationship between the final volume of hypertrophic chondrocytes and the rate of longitudinal bone growth was demonstrated (Breur et al., 1991; Kuhn et al., 1993). We considered this evidence that the final volume of hypertrophic chondrocytes may be regulated. However, it is unknown where in the growth plate or how chondrocytic volume is regulated. A problem in the study of the organization of chondrocytic hypertrophy is the lack of a uniform definition of the point where cellular enlargement begins. In defining zones of growth plates, investigators often have used criteria based on cell size or cell shape (Brighton, 1978; Buckwalter et al., 1985, 1986; Holtrop, 1972a,b; Hunziker e t al., 1987; Hunziker and Schenk, 1989; Scott and Pease, 1956). Traditionally, i t was thought that chondrocytic enlargement was restricted to the growth plate zones containing large rounded cells (maturation and hypertrophic zone), and that the zone containing small flattened cells (proliferative zone) was solely composed of young proliferating chondrocytes (Ham and Cormack, 1984). However, it has been demonstrated in several studies of growth plates from different locations that chondrocytic proliferation only takes place in the proximal portion of the proliferative zone, at the epiphyseal ends of the columns (Farnum and Wilsman, 1993; Kember, 1960, 1972, 1973; Kember and Walker, 1971; Walker and Kember, 1972). The function of chondrocytes in the proliferative zone distal to the actively proliferating cells is unknown, but in two recent stereological studies they were, though largely ignored, implicitly considered a s enlarging, hypertrophying chondrocytes (Buckwalter et al., 1986; Hunziker et al., 1987; Hunziker and Schenk, 1989). In previous stereologic studies of the growth plate, growth plate zones were defined using subjectively applied cell morphological criteria (Buckwalter et al., 1986; Hunziker et al., 1987; Hunziker and Schenk, 1989). Also, the analyzed zones were relatively thick, thus perhaps obscuring more subtle changes taking place within the analyzed zones. Another limitation of the previous studies is the fact that the reported changes in cell volume and cell shape were not related to time, and i t may be that the reported changes in cell volume and cell shape do not reflect changes in the rate of cell volume increase and the rate of cell shape modulation. Analysis of the rate of chondrocytic volume and shape changes is important, because rapid rate transitions may signal points where mechanisms regulating chondrocytic enlargement act. The goal of this study is (a)to determine where chondrocytic enlargement commences, and (b) to analyze chondrocytic volume and shape changes in relation to time. We studied the process of chondrocytic enlargement in the proximal tibial growth plate of 35-day-old rats and used two different approaches. First, in eight unbiasedly defined, narrow strata of the growth plate, using stereological techniques, we measured changes in mean cellular volume, and mean horizontal and mean vertical chondrocytic profile diameters. In addition, we calculated the rate by which these parameters changed. A unique aspect of this study is defining and ENLARGEMENT OF GROWTH PLATE CHONDROCYTES 257 Measurement of Rate of Longitudinal Growth dividing the growth plate into many thin strata. As the width of each stratum becomes narrower, we have the ability to integrate changes from stratum to stratum. With the second approach, volumes and diameters of individual chondrocytes within columns were measured using serial section techniques. Interpretations of serial section studies are always limited by a small sample size; however, used in combination with a n integrative stereological analysis, the analysis by serial sections is complementary. In both experiments, bromodeoxyuridine (BrdU) labelling was used to identify proliferating chondrocytes, and oxytetracycline (OCT) bone labelling was used to estimate the rate of longitudinal bone growth. All chondrocytes were preserved using optimal chemical fixation. Following thawing of the frozen growth plates, growth plates were cut in four equal parts. Of each part, an approximately 250 pm thick vertical slab was cut with a razor blade. These slabs, representing a systematic sampling of the entire growth plate, were put on glass slides, mounted in glycerine, and viewed under epifluorescent light. On each of the four 250 mm thick slabs, the distance between the 2 fluorescent oxytetracycline labels was measured a t 4 unbiasedly determined locations along the growth plate and the mean of these measurements was considered the longitudinal bone growth per 24 hours (t(bg/day)). ANIMALS, MATERIALS, AND M E T H O D S Irnrnunocytochernical Identification of Proliferating Chondrocytes Animals, Tissue Collection, and Tissue Processing For the stereological analysis of unbiasedly defined, narrow growth plate strata, six 35-day-old male hooded rats (weight 90-120 g) were selected. Oxytetracycline hydrochloride (1-2 mgikg IP) was administered 48 and 24 hours before euthanasia. Thirty minutes before euthanasia, the animals were injected with the thymidine analogue 5-bromo-2’-deoxyuridine (BrdU; 100 mgi kg; 20 mg/kg). Immediately after anesthesia (pentobarbital 30 mgikg IP) and exsanguination, the left proximal tibial growth plate was isolated, and the animal was euthanized with a n overdose of barbiturate. The growth plate was submerged in the primary fixation solution and further trimmed into 1 x 1 x 3 mm blocks with epiphyseal bone on one end and metaphyseal bone on the other end. The fixation protocol for these blocks consisted of a primary fixation in 2% glutaraldehyde-2% paraformaldehyde in 0.05 M cacodylate buffer (pH 7.3) with 0.7% ruthenium hexammine trichloride for 2 hours. The blocks then were rinsed in 0.05 M cacodylate buffer (pH 7.3), followed by secondary fixation in 0.2% OsO,-O.7% ruthenium hexammine trichloride in 0.05 M cacodylate buffer (pH 7.3) for 2 hours. After a second rinse in 0.05 M cacodylate buffer (pH 7.31, the blocks were rapidly dehydrated in graded alcohols, cleared in propylene oxyde, and embedded in epon-araldite. Growth plate tissue fixed according to this protocol contains hypertrophic chondrocytes preserved in their native state, which makes meaningful stereology possible (Farnum and Wilsman, 1987; Hunziker et al., 1982, 1983,1984). An additional advantage is that this protocol allows the immunocytochemical identification of cells in the S-phase of the cell cycle using BrdU. Following the collection of the left proximal tibial growth plate, the right proximal tibial growth plate was isolated and placed in airtight bags. The right proximal radial growth plates were stored at -20°C until used for the measurement of the rate of longitudinal bone growth. All collections were done between 9:OO and 11:OO A.M. The proximal radial growth plate of one 35-day-old male hooded rat was used for the reconstruction and measurements of individual growth plate chondrocytes. The tissue collection and tissue processing for this study were as described above, except that secondary fixation was omitted. One micrometer thick sections were cut, put on acid cleaned slides, and etched in 50% sodium ethoxide (pH 12.5) for 15 minutes. Etching resulted in both deplastification of the sections and denaturing of the nuclear DNA. Following etching, sections were passed through graded alcohols, washed in PBS (0.05 M phosphate buffered saline a t pH 7.3), and incubated with a n antiBrdU monoclonal antibody (Beckton Dickinson Immunocytometry Systems, Mountain View, CA 94039) a t a dilution of 25 plilml PBS (1:40 dilution of stock solution), for 12 hours at 4°C in a humidified slide chamber. The primary antibody was localized with a n avidinbiotin-peroxidase complex (biotinylated horse antimouse IgG and avidin-biotin-peroxidase complex; Vector Laboratories Inc, Burlingame, CA 94010). After incubation with primary antibody, the sections were washed in PBS and incubated with the biotinylated horse anti-mouse IgG (9 pl biotinylated horse antimouse IgG in 1 ml PBS). Following another wash, the sections were incubated with the avidin-biotin-peroxidase complex (9 p1 avidin stock solution and 9 pl biotinylated peroxidase stock solution per 1ml PBS). Both incubations were for 30 minutes in a humidified slide chamber a t 4°C. The BrdU incorporated was demonstrated by a final incubation for 30 minutes in a fresh solution of 0.05% 3,3‘-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) and 0.02% hydrogen peroxide in PBS. Chondrocytes that had incorporated BrdU (chondrocytes in the S-phase of the cell cycle) were demonstrated by a brown precipitate over the nucleus (Fig. 1). Method controls (deletion of the primary antibody, deletion of the secondary antibody, and staining of unlabelled growth plate tissue a s a negative control) resulted in blocking of immunolabeling. Two specificity controls were done. Incubation of the primary antibody (25 pl stock solution per 1 ml PBS) with BrdU (1,5,10, or 100 pgiml PBS) for 48 hours a t 4°C before the immunocytochemical procedure resulted in complete ablishment of labelling a t BrdU concentrations 2 10 pgiml PBS. In contrast, pre-incubation of the primary antibody (25 pl stock solution per 1 ml PBS) with thymidine (10, 50, 100, or 1000 pgiml PBS; Sigma, St. Louis, MO) did not affect the intensity of the immunolabel. 258 G.J. BREUR ET AL. Fig. 1. Localization of chondrocytes in the S-phase of the cell cycle using a monoclonal antibody against the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). a: BrdU containing cells, identified by their dark nuclei, are demonstrated in the proximal portion of the proliferative zone. b Immunolabelling is completely abolished if the primary antibody is incubated with BrdU (210pgiml PBS) before the immunocytochemical procedure. Bar = 100 pm. c: Pre-incubation of the primary antibody with thymidine (10,50,100, or 1000 pgiml PBS) does not affect the intensity of the immunolabel. Bar = 30 km. Stereological Analysis of Unbiasedly Determined Growth Plate Strata the micrographs ( x SOO), growth plates were divided proximodistally in 8 equal, narrow strata. In each stratum measurements for the following stereological parameters were made: mean cell volume (ik)) chondro, cytic volume fraction (VdVwri), the mean principal diameters of chondrocytic profiles (Tf(hor) and X v e r t i ) , and the percentage of cells in the S-phase of the cell cycle (Labelling Index). In defining zones of growth plates, investigators most often have used subjectively applied criteria, based on cell size or organization of chondrocytes (Brighton, 1978; Buckwalter et al., 1985, 1986; Cruz- Strata analysis Three blocks of each growth plate were randomly choosen for stereological analysis. One section cut vertically (parallel to the long axis of the bone) a t 1 pm was obtained from each block and stained with azure 11, basic fuchsine, and methylene blue (Humphrey and Pittman, 1974). Two unbiasedly choosen fields per section (including epiphyseal bone, the entire growth plate, and metaphyseal bone) were photographed. On 259 ENLARGEMENT O F GROWTH PLATE CHONDROCYTES Orive and Hunziker, 1986; Ham and Cormack, 1984; Holtrop, 1972a,b; Hunziker et al., 1987; Hunziker and Schenk, 1989; Scott and Pease, 1956). In this study we divided the growth plate in eight strata of equal height. With this approach the definition of growth plate zones was completely independent from cell size and chondrocytic organization, which ensured a n unbiased approach to the analysis of the process of chondrocytic enlargement. In preliminary studies we had determined that approximately eight strata were required to obtain enough sensitivity to detect minor changes in the parameters of interest. Reference volumes Labelling Index per unit area is equal to the Labelling Index per unit volume. Chondrocytic volume fraction. The chondrocytic volume fraction of a stratum V(c)/V(stri was estimated from micrographs generated for the stereological analysis using point counting techniques (Cruz-Orive and Hunziker, 1986). Vw is the total volume of chondrocytes in that stratum, and V(str)the total volume of the stratum. Mean cell volume. The mean cell volume i i c ) of a stratum was calculated using the following equation (CruzOrive and Hunziker, 1986; Gundersen and Jensen, 1985; Gundersen, 1986): vw = (11/3) x 31, In each animal, the volume of the proximal tibial where iic)is the volume weighted mean chondrocytic the mean cubed intercept length. The growth plate V(ppii, the volume of unbiasedly deter- volume, and mined strata V(stri (see strata analysis), and the volume point sampled intercept length was measured from the of newly produced bone per day V(bg/dayiwere measured. micrographs with the aid of a n image analysis system Reference volumes were calculated by multiplying the (Bioquant IV, R&M Biometrics Inc, Nashville, TN). Of growth plate area in a plane perpendicular to the long each growth plate stratum, at least 300 intercepts unaxis of the bone by respectively, the mean-growth plate der 16 randomly choosen angles were measured (Cruzheight tippi), the mean stratum height tiw, and the Orive and Hunziker, 1986). This technique is suitable length growth per day h g i d a y ) . The growth plate area for volume estimations of anisotropic objects, and can perpendicular to the long axis of the bone was esti- be used on vertical sections (Baddely et al., 1986; Cruzmated by measuring the principal diameters of the in- Orive and Hunziker, 1986; Gundersen and Jensen, tact growth plates with a sliding mechanical caliper 1985; Gundersen, 1986). Chondrocytic numerical density. Assuming minimal (accuracy 10 p.m). The estimate of the projected area cell size variability, the chondrocytic numerical denwas obtained by using the ellipse formula (Cruz-Orive sity Nic)/V(str), the number of chondrocytes per mm3 was and Hunziker, 1986; Howell et al., 1960): calculated using the relationship (Cruz-Orive and area = (d4)x longest diameter x shortest diameter Hunziker, 1986): Nici/V(str)= [V(c)/V(str)] / UICJ] Growth plate height was measured on 1 pm thick sections cut from each of 3 blocks per growth plate. On where Nw is the total number of chondrocytes and each section, measurements were made a t 2 unbiasedly the mean chondrocytic volume in that stratum. chosen locations along the growth plate from microNumber of chondrocytes per stratum. The relationship graphs used for the stereological analyses. MeasureN(c) = [N(ci/Vistri] X Vistri ments were made from the epiphyseal to the metaphyseal bone. The estimated mean height of each was used to calculate Nw, the number of chondrocytes individual growth plate was obtained as the mean of 6 in a stratum. measurements. The mean stratum height in the proxNumber of hypertrophic chondrocytes leaving the growth imal tibial growth plate was equal to the growth plate plate per day. The number of hypertrophic chondrocytes height divided by eight (number of strata). leaving the growth plate per day N(out/day) was estimated by Basic estimators N(out/day) = [N~ci/V(stri]~~,~ X V(bg/day) Labelling index. where [N~ci/V(str~]~~,~ is the numerical density of the terTo identify chondrocytes in the S-phase of the cell cycle, 3 serial 1 p.m thick sections were cut of each of 3 minal, juxta-metaphyseal stratum, and V(bgiday)the volrandomly selected blocks of each growth plate. The me- ume of newly formed bone per day. dian section was stained with azure 11, basic fuchsine, and methylene blue (Humphrey and Pittman, 19741, Chondrocytic morphology Principal cell profile diameters. while the two surrounding sections were used for the In each stratum, the mean principal profile diameidentification of S-phase cells. Photographs of the median section served as a reference section and for the ters X ( h o r l and X i v e r t i were measured. The X ( h o r l of a recording of cell profiles containing nuclei. The preced- chondrocytic profile was defined as the caliper diameing and the succeeding sections were used to determine ter of that profile measured perpendicular to the long whether nucleated cell profiles of the median section axis of the bone. The X v e r t i was defined a s the length of were BrdU-labelled or not. The Labelling Index was the intercept bisecting the cell profile measured paraldefined as the ratio of BrdU-labelled cell profiles and lel to the long axis of the bone. These definitions were nucleated cell profiles from the median section. At choosen under the assumption that cells have a n ellipleast 200 nucleated cell profiles per stratum were an- soid shape with principal axes perpendicular and paralyzed. In this study, we assumed that all cells, includ- allel to the long axis of the bone. In addition, such ing cells in the S-phase of the cell cycle, had the same measurements describe the principal cell profile diamnuclear diameter measured perpendicular to the long eters in relation to longitudinal bone growth (Kember, axis of the bone. Under these conditions, the obtained 1960, 1972, 1973, 1978; Cruz-Orive and Hunziker, 260 G.J. BREUR ET AL. 1986; Hunziker et al., 1987; Hunziker and Schenk, 1989; Thorngren and Hansson, 1973a,b, 1981; Sissons, 1955). Axial ratio of chondrocytic profiles. The axial ratio of chondrocytic profiles was calculated as the ratio X(vert): Xlhorl and describes how flat a cell profile is relative to the long axis of the bone (Buckwalter et al., 1985). An axial ratio smaller than 1represents a n oblate ellipsoid cell profile; a n axial ratio of 1, a circular profile; and a n axial ratio larger than 1, a prolate ellipsoid cell profile. Temporal relationships of the process of chondrocytic enlargement The spatial relationships of chondrocytes within a growth plate reflect the temporal changes that take place during the process of chondrocytic enlargement (Ham, 1952; Ham and Cormack, 1984; Hunziker et al., 1987). Assuming that each chondrocyte in a column represents a later stage of development than the preceding, more proximal chondrocyte, the strata not only represent different stages of the enlargement process, but also represent a certain time period. Because growth plates, over a short time period, can be considered a steady state system where the rate of cell gain in the proliferative zone is equal to the rate of cell loss at the metaphyseal junction (Hunziker and Schenk, 1989; Kember, 1960, 1972, 1973, 1978, 1983; Kember and Kirkwood, 1987; Thorngren and Hansson, 1973a,b, 1981), definitions of kinetic parameters in cell renewal systems can be applied (Cleaver, 1967). Turnover time. The turnover time of a stratum Tlturn) is the time required for the replacement of the total number of cells in that stratum. This time is equal to the mean amount of time each cell spends in this compartment if no cell divisions takes place. Thus, the cell turnover time of a stratum can be estimated by (Cleaver, 1967; Quastler, 1959, 1960; Quastler and Sherman 1959) T(turnlstr = N~c~,tJK~out),t, where K(out),tris the mean number of cells leaving the stratum per hour. This study was restricted to the process of cellular enlargement, and therefore only the most distal stratum containing proliferating cells (Stratum 3) and strata without proliferating cells (Strata 4 through 8) were included. Because of these restrictions and the fact that the growth plate is a steady state system, the efflux K(outIst, of each of the included strata is approximately the same and can be estimated as follows (Cleaver, 1967; Quastler, 1959, 1960; Quastler and Sherman 1959): Klout)str = K(outIterm= “out! X 11/24] where K(out)te,m is the chondrocytic efflux from the terminal, juxta-metaphyseal stratum, and N(nutithe total number of chondrocytes leaving the growth plate per day. Rate of gain or loss. The rate of gain or loss per hour of parameters related to cell morphology (Om, X(hor1 and Xlvert!) were calculated for the strata for which Tlturn) was estimated. The derivation of this parameter is illustrated in this example of the estimation of the rate of mean cell volume change Aih,,,/hr = [U (C)str~ ( c str] i / ~T(turn),t, ~ ~ ~ In this equation, ~ ( cst, )is the ~ ~mean ~ ~cell volume of the stratum preceding the stratum of interest. u ( ~and ) ~ ~ ~ T(turn),trare, respectively, the mean cell volume and the turnover time of the stratum of interest. Statistical analysis The arithmetic means and standard deviations of all parameters in identical strata of the 6 animals were calculated and compared with the means of adjacent strata using one-way analysis of variance and Fisher’s analysis for least significant differences (protected LSD) (Snedecor and Cochran, 1980). Serial Section Reconstruction and Measurement of Individual Chondrocytes One tissue block was randomly choosen for the analysis. One hundred vertical (parallel to the long axis of the bone) serial sections were cut. Sections were 1 pm thick, except every fourth section, which was cut a t 2 pm. The accuracy of the microtome was verified in a preliminary study in which the actual thickness of sections cut at 1 pm was measured using a Watson interference objective. In this study, the actual mean section thickness of 36 unbiasedly choosen sections cut at 1pm was 0.995 pm (20.06). One micron thick sections were stained with azure 11, basic fuchsine, and methylene blue (Humphrey and Pitmann, 1974). The 2 pm thick sections were used for the immunocytochemical identification of cells in the S-phase of the cell cycle. A growth plate region 80 pm wide and 100 pm deep (83sections), spanning the growth plate from the proximal end of the growth plate columns to metaphyseal bone, was analyzed. Columns within this region were unbiasedly selected for reconstruction using the forbidden plane, or “brick” rule (Howard et al., 1985). Camera lucida drawings of cell profiles of interest were made at ~ 7 4 8 and , the individual cell profiles were digitized. The columns were reconstructed and volumes of individual cells were measured using a Eutectics 3-dimensional image analysis system (Eutectics Inc., Raleigh, NC). With this system, the estimated volume of individual cells is equal to the cumulative total of the product of the section thickness and the cell profile area of all sections containing profiles of that cell. A correction factor for the 2 pm thick sections was included. The principal caliper cell diameters X(vert)and Xlhorl, the caliper cell diameters parallel and perpendicular to the long axis of the bone, respectively, were measured on reconstructions magnified at x 1,000. The caliper cell diameter in the third dimension was calculated a s the product of the section thickness and the number of sections containing profiles of that cell. Adjustments for 2 pm thick sections and incompletely filled sections (minus one section thickness) were made. The mean of this diameter and Xlhor) is the reported X(hori. Because the spatial arrangement of chondrocytes within columns reflects their development in a temporal fashion, and because growth plates can be considered a steady state system (Hunziker et al., 1987; Hunziker and Schenk, 1989; Kember 1960,1972,1973, 1978,1983; Kember and Kirkwood, 1987; Kember and Walker, 1971; Thorngren and Hansson, 1973a,b, 1981), the time that a cell remains in a certain position of the column (or remains in a certain stage of development) can be estimated. This time, T(celli,is equal to the time a hypertrophic cell remains in the most distal position ENLARGEMENT OF GROWTH PLATE CHONDROCYTES of the column, and was estimated by (Gray and Scholes, 1951; Hunziker et al., 1987; Hunziker and Schenk, 1989; Sissons, 1955) 261 Chondrocytic morphology During the process of cellular enlargement, the mean cell volume ixC) increased approximately 10 Tlcell) = 24 x IXlvert)term / tlbgiday)] times. The smallest mean chondrocytic volumes were where tibg/day) is the longitudinal bone growth per day, found in the strata with active cell proliferation, Strata and Xivewterm the mean vertical diameters of the eight 1,2, and 3 (1,050, 1,250, and 1,590 mm3, respectively). most distal hypertrophic chondrocytes in this recon- Mean cell volume slowly increased to 2,530 ~m~ in struction. The cell maturation time Tim), the time be- Stratum 5, and then increased with significant steps tween cell proliferation, and cell death was estimated towards its final volume (12,840 pm3) in Stratum 7 and by (Gray and Scholes, 1951; Hunziker and Schenk, Stratum 8. The mean vertical and horizontal chondrocytic profile diameters,Jhe profile diameters measjred, 1989; Kember, 1960; Sissons, 1955) respectively, parallel (Xlvert))and perpendicular (X[hor)) R m ) = n X T(c~II) to the long axis of the bone, followed a similar pattern. In strata 1, 2, and 3, was 5.4, 5.3, and 6.4 pm, where n is the number of cells between the last BrdU- respectively. In the more distal strata %ert) increased labelled cell of a column and metaphyseal bone. significantly, and the maximum value (25.5 pm) was Finally, the position of each cell in a column was obtained in Stratum 8. In the most proximal three determined by measuring the distance from each cell to strata W(hor) was 12.7, 14.0, and 13.9 pm, respectively, the chondro-osseous junction with the epiphysis. and i t increased to 21.0 pm in Stratum 8. The smallest axial ratio was found in Stratum 2 (0.42). It increased RESULTS with significant steps from 0.50 in Stratum 3 to 1.26 in Stereological Analysis Stratum 7, and obtained its final value 1.31 in Stratum 8. The axial ratio was approximately 1 (1.08) in Stratum 6. Significant differences (P < 0.01) related to ih, Reference volumes The mean growth plate height t(gpi), the mean Xvert)and X f h o r ) were found between Stratum 4 and 5, 5 stratum height j ( s t r ) , and the mean longitudinal bone and 6, and 6 and 7. In addition Xcvert) was significantly growth per day t(bg/day) were 577 pm (+30), 72 pm (54, larger in Stratum 4 than in Stratum 3 ( P < 0.01). and 331 pmiday (?28), respectively. The estimated Twenty percent of the final mean cell volume was obmean values of the corresponding 3-dimensional pa- tained from Stratum 2 to Stratum 4 ( 2 3 3 hours), which volume increase mainly resulted in a n increase in rameters growth plate volume V(gpi),stratum volume X(vert). The remaining 80%of the final mean cell volume Vlstr), and the volume of newly produced bone per day V(bgiday) were 13.66 mm3 (k1.231, 1.71 mm3 (+0.16), and was obtained from Stratum 5 to Stratum 7 (520 hours); this volume incre_ase resulted in significant increases 7.81 mm3/day (_t0.72), respectively. in both Xtvert) and Xlbor).During the last 5 hours of chondrocytic enlargement, the mean cell volume and the Basic estimators mean principal cell diameters did not change signifiThe mean Labelling Index, the fraction of cells in the cantly. All results, including the statistical analyses, S-phase of the cell cycle, was 0.15 in Stratum 1(epiph- are summarized in Table 1. The relationship between yseal end of the growth plate). 0.19 in Stratum 2, 0.08 OW,Wivert),and Xihor) in the different strata is depicted in in Stratum 3, and 0 in all other strata. These data Figure 2A. The pattern of these parameters after time normalsuggest that active cell proliferation only took place in the proximal 3 strata of the growth plate. The chondro- ization reflected the pattern before time normalization. cytic volume fraction V<c)/V(str), the fraction of a stratum A phase with moderate rate increases in Strata 3,4, and occupied by chondrocytes, increased from 0.21 in Stra- 5 is followed by a phase with major rate increases from tum 1 to 0.67 in Stratum 8 (metaphyseal end of the Stratum 5 to 6. No significant gains or losses were made growth plate). The highest number of cells per stratum in Stratum 7. The rate of ain in mean cell volume Nw and the highest number of chondrocytes per mm3 gradually rose from 18 pm5/hr in Stratum 3 to 230 (N<c)/Vlstr)) was found in Stratum 2 (474,200 cells/stra- pm3/hr in Stratum 5. Then, it abruptly increased to 807 tum and 277,500 cells/mm3) and this number decreased pm3/hr in Stratum 6 (P < 0.01). This rate of cell volume towards the metaphysis (Stratum 8: 90,200 cellsistra- increase was maintained in Stratum 7 (840 pm3/hr) and tum and 53,200 cells/mm3). The total number of hyper- then dropped to -11 km3/hr in Stratum 8 (P < 0.05). trophic chondrocytes leaving the growth plate per day The rate of increase of Xivert) in Stratum 3 was 0.06 N(outiday1and per hour K(out)was 414,600 (269,200) and pm/hr, which gradually increased to 0.39 pm/hr in Stra17,280 (?2,880), respectively. The turnover time Titurn), tum 5. The rate significantly increased to 1.14 pmihr in the time required for the replacement of the total num- stratum 6 (P < 0.01) and 1.18 pm/hr in stratum 7, and ber of cells, was the highest in Stratum 3 (19.7 hours) then significantly decreased to 0.16 ym/hr in Stratum and decreased towards Stratum 7 and 8. The turnover 8 ( P < 0.01). The rate of change OfX(h0r)followedthe rate time of Stratum 8 was 5.3 hours. The estimated matu- of change of the mean cell volume t v c ) and Xvert). A ration time, the time from the end of the last cell divi- significant increase was found between Stratum 5 and sion in the proliferative zone to cell death in the jux- 6 (0.15 pm/hr to 0.43 pmlhr; P < 0.05), and a significant tametaphyseal cartilage (the total of all turnover decrease between Stratum 7 and Stratum 8 (0.42 pm/hr times), was 57.8 hours (59.3). All results, including the to 0.13 pm/hr; P < 0.01). The relationship between these results of analyses for comparison of significant differ- 3 parameters is displayed in Figure 2B. All results are ences between adjacent strata, are listed in Table 1. summarized in Table 1. 262 G.J. BREUR ET AL. TABLE 1. Stereological estimators of the proximal tibia1 growth plate of the rat (standard deviations are given in parentheses) Strata Parameters 1 2 3 4 5 6 7 8 0.00 (0.00) 0.00 io.001 0.63 (0.05) 0.67 (0.031 Basic estimators LI 0.15 (0.071 VicjNistri 0.21 (0.031 Nid NiciNistri (mm 2 0.19 (0.031 2 0.08 (0.04) 0.34 (0.031 348,800 (71,480) 2 474,200 3l 206,700 __*I_ 277,500 (51,950) (37,980) 0.32 (0.02) 5 (80,360) Kiouti (cellsihrl 5 5 0.33 (0.031 1,250 (1801 (2101 - X i v d (pm) - 5.4 (0.3) Xihori (pml 12.7 (1.31 Axial ratio 0.49 (0.061 2 2 0.00 (0.001 0.04 (0.061 0.51 (0.05) 152,200 (21,9701 5 (17,2401 88,900 (14,840) 17,280 (2,8801 17,280 (2,8801 5 225,600 205,700 (23,1801 5 131,700 17,280 (2,8801 (38,870) 13.4 (2.81 1,590 (180) 0.00 (0.001 5 333,800 (57,5601 19.7 (4.01 Chondrocytic morphology oici (pm3i 1,050 0.00 (0.00) 2,530 (3801 5 5 8.9 (1.7) 4,550 6.4 (0.41 8.6 (0.71 5 12.2 (1.41 14.0 (1.21 13.9 (0.91 14.9 (1.41 2 16.2 (1.21 0.42 (0.031 0.50 (0.041 imihr (pm3ihri 18 (101 2 0.62 5 (0.061 75 (311 97,000 i10.7301 83,800 (8,6201 90,200 (9,5701 57,200 (7,8501 49,400 (6,5601 53,200 (7,960) 17,280 (2,8801 17,280 (2,880) 17,280 (2,8801 2 5 5 ( 1,0001 5.3 (0.31 0.80 (0.08) "" 5 0.18 (0.051 0.39 (0.09) -0.01 (0.04) 0.07 (0.051 0.15 (0.05) 5.7 (0.91 5 5.0 (1.01 9,040 f 1,560) 5.3 (0.51 12,840 (1,250) 12,840 (1,7401 18.5 (1.51 5 24.0 (2.2) 25.5 (2.21 18.4 (0.91 5 20.4 (1.0) 21.0 (0.3) 1.08 _T_ (0.11) 807 (2101 230 (75) 0.06 (0.031 5 1.26 1.31 (0.11) (0.101 840 (5211 5 -11 (272) 1.14 (0.261 1.19 (0.601 0.16 (0.231 0.43 (0.24) 0.42 _II_ (0.281 0.13 (0.211 *Significantly different a t the 95% confidence level. **Significantly different a t the 99% confidence level. yseal bone, chondrocytic volume rapidly increased. The rapid cell volume increase was accompanied by rapid Our reconstruction consisted of nine columns, a total increases in both X(verti and X(horl (caliper diameter perof 168 chondrocytes. Only two of those columns were pendicular to the long axis of the bone). Approximately complete columns (originating in the zone of actively 100 pm away from the metaphysis, the maximum valproliferating cells delineated by immunolocalization of ues of uw, X(vertJ, and X ( h o r ) were obtained. All spheroid BrdU and continuing uninterrupted to the chondro- cells (axial ratio 0.9-1.1) and prolate spheroid cells (axosseous junction with the metaphysis). Six columns ial ratio > 1.1) were found in the phase of rapid volume were incomplete distally (epiphyseal columns), and one increase (Fig. 3). The variability of the studied paramcolumn was incomplete proximally (metaphyseal col- eters a t any arbitrary level of the growth plate (for umn). The total reconstruction and the individual col- example 300 pm away from the epiphysis) indicated substantial cell morphological differences between umns are displayed in Figure 3. The results of the serial section reconstruction con- chondrocytes in adjacent columns of the growth plate. firmed the findings of the stereological analysis of nar- The relationship of uiCi, X(vert1, and X(hor1, and their porow strata. BrdU labelled cells (cells in the S-phase of sition in the columns is demonstrated in Figure 4. Within a column, the development of the mean cell the cell cycle) were confined to a narrow zone a t the proximal ends of the columns, approximately 28-1 19 volume and both principal cell diameters reflected the hm away from epiphyseal bone. Distally, from the zone pattern described in the stereological analysis of narcontaining proliferating cells, cells gradually doubled row strata. All three parameters increased towards the their volume towards a transition point approximately metaphysis regardless of whether columns were com215 hm away from the epiphysis. During this gradual plete or incomplete. Again, changes in X(verti most increase in cell volume cells maintained their oblate closely reflected changes in cell volume. Finally, all spheroid shape, and their caliper vertical chondrocytic cells in the proximal portion of a column were oblate diameter (X(vert1, the chondrocytic diameter parallel to spheroid, but more distally oblate and prolate cells ofthe long axis of the bone) increased only moderately. ten succeeded each other (Figs. 3, 5). The transition from small flattened chondrocytes to Beyond the transition point 215 pm away from epiphAnalysis of Individual Cells Using Serial Section Reconstruction 263 ENLARGEMENT OF GROWTH PLATE CHONDROCYTES 30 b 1200 --1.2 = Ai(c)lhour A Ax(vert) I hour 25 800- 20 1 Ax(hor)lhour 1 I -0.8 L 3 0 15 4000 -E, 10 4 { 3,' 1 2 3 4 5 -0.6 400- -0.4 . 3 T 0 5 - 0.2 oJ 0 t 600- 6 7 -2ooJ 0 8 1 STRATUM 2 3 4 5 6 7 8 1-0.2 STRATUM Fig. 2. Changes in chondrocytic morphology during the process of cellular enlargement as measured stereologically in eight unbiasedly defined, narrow growth plate strata. a: Mean cell volume G(c) (m), mean vertical cell diamet_er X(vert1 (A),and mean horizontal cell diameter X(hor1(0). b Rate of change per hour A u d h r (m), A X(vertdhr (A)and A X(hodhr (0).Standard deviations are listed in Table 1. B metaphyseal column epiphyseal columns complete columns Fig. 3. a: Serial section reconstruction of nine growth plate columns. Bar = 50 pm. b: Individual columns. Two columns were continuing uninterrupted from the epiphyseal end of the columns to the chondro-ossoeous junction with the metaphysis, six columns were incomplete distally (epiphyseal columns), and one column was incomplete proximally (metaphyseal column). more spheroid or prolate spheroid chondrocytes at a point approximately 250 pm away from epiphyseal bone was very rapid. In the six transitions analyzed, cells in this region nearly doubled their volume (1.82 0.66) within two sequential positions, and tripled their * volume (2.96 2 1.15)within three sequential positions. The rate of metaphyseal bone growth was 290 pmlday and the mean X(vert1 of the eight most distal hypertrophic chondrocytes was 33.2 pm. Thus, each chondrocytic position represented 2.8 hours (T(cel1)). Therefore, G.J. BREUR ET AL. -45 18000 -40 16000 -35 ~ 12000 f + 10000- m + t 0 + 8000- 8000 6000- 9 . 6000 40002000 0 0 50 100 150 200 250 300 30 . 350 - 20 if -10 2000 5 0 F 3 -15 4000 - , 400 ..... . -25 , 50 100 150 200 250 300 350 400 450 DISTANCE FROM EPIPHYSEAL BONE (prn) DISTANCE (prn) Fig. 5. Changes in chondrocytic morphology during the process of cellular enlargement as measured in individual cells within a column using serial section reconstruction. The position of each cell in a column was determined by measuring the distance from each cell to the chondro-osseousjunction with the epiphysis. Chondrocytic volume v(c) (El,vertical cell diameter Xlvert) (A),and horizontal cell diameter X(hor) (0). .40- 3530- and the metaphyseal bone was 17.2. Thus, the estimated mean maturation time T m was 47.3 hours. DISCUSSION 0 50 100 150 200 250 300 350 400 450 DISTANCE (prn) 40 , I 351 30 I l l 0 0 50 , , , , , , , , 100 150 200 250 300 350 400 1 450 DISTANCE (pm) Fig. 4. Changes in chondrocytic morphology during the process of cellular enlargement as measured in 168 individual cells using serial section reconstruction. The position of each cell in a column was determined by measuring the distance from each cell to the chondroosseous junction with the epiphysis. a: Chondrocytic volume VW. Oblate spheroid cells (O), spherical cells (01,and prolate spheroid cells ( + ) . b: Vertical cell diameter Xlvert). c: Horizontal cell diameter Xlhor). this rapid increase in volume took place within a time span of 5.5 to 8.5 hours. In addition, the mean number of cells in a column between the last BrdU-labelled cell In this study, using two different and independent techniques, and using the physiological criterion of loss of BrdU labelling as a n indicator of where all growth plate chondrocytes have left the cell cycle, we demonstrated that (a) cell volume increase starts immediately following cell division in the proximal portion of the proliferative zone, (b) the process of chondrocytic enlargement as i t relates to longitudinal bone growth consists of two morphologically distinguishable phases, (c) the rate of cell volume increase and the rate of cell shape modulation is significantly higher during the second than during the first phase, and (d) that cell volume increase during the first phase results mainly in an increase in vertical chondrocytic diameter, while cell volume increase during the second phase results in a large, significant increase of the vertical chondrocytic diameter, and a smaller but significant increase of both horizontal chondrocytic diameters. The transition point between the first and second phase of chondrocytic enlargement corresponded with the natural transition from flattened chondrocytes to more rounded chondrocytes, often described by other investigators as the junction between the proliferative zone and the hypertrophic zone (Brighton, 1978; Buckwalter et al., 1985,1986; Engfeldt, 196913; Ham and Cormack, 1984; Holtrop, 1972a,b; Moss-Salentijn, 1974; Scott and Pease, 1956). Stereological, Serial Section, and Kinetic Analysis In the present studies we used three new morphological approaches to study chondrocytic hypertrophy. First, in our stereological study, unlike previous stereological studies by other workers (Brighton et al., 1973, 1982; Buckwalter et al., 1985, 1986; Buckwalter and Mower, 1987; Cruz-Orive and Hunziker, 1986; ENLARGEMENT OF GROWTH PLATE CHONDROCYTES 265 Hunziker et al., 1987; Hunziker and Schenk, 19891, Therefore, they may challenge one of the main undergrowth plate regions were defined independently from lying assumptions of previously used kinetic models natural or physiological strata, thus ensuring a n unbi- describing the performance of growth plates (Hanson, ased approach to chondrocytic hypertrophy. Further- 1967; Hunziker and Schenk, 1989; Kember, 1960, more, the obtained stereological parameters were used 1972, 1973, 1978, 1983; Tayler et al., 1987a,b; Thornfor a newly developed kinetic analysis of growth plate gren and Hanson, 1973a,b, 1981). behavior. This kinetic analysis is independent from the Chondrocytic Enlargement columnar arrangement of chondrocytes, and indepenDuring the first phase of chondrocytic enlargement, dent from the parameters mean chondrocytic height and mean septum thickness, parameters that only can in the present study approximately 30 hours, and corbe estimated accurately using time consuming serial responding to Strata 3 and 4 or a zone 120 to 215 pm away from the epiphyseal bone, cell volume increased section reconstructions. As a second approach, we used computer-aided serial gradually, and 20% of the final cell volume was obsection reconstruction and measurement of chondro- tained. During the following 20 hours, corresponding to cytes within columns. With this technique sampling is Strata 5, 6, and 7 or a zone 215 to 350 pm away from also independent from the natural and physiological epiphyseal bone, the remaining 80% of the final cell growth plate strata. In addition, this technique com- volume was acquired. The change in chondrocytic morphology from a flatplements the stereological analysis; the data obtained with the stereological analysis reflect average tened shape in the first phase of chondrocytic enlargechanges, while the data obtained with the serial recon- ment to a rounded shape during the second phase of struction analysis provides very precise measure- chondrocytic enlargement is accompanied by changes ments, however, of a limited number of chondrocytes. in cellular activity. Alterations in chondrocytic activity Finally, this is the first study in which chondrocytic are reflected by changes in proliferative activity, inproliferation was studied on the same tissue as used for termediary metabolism, synthesis of matrix constituthe stereological analysis of chondrocytic enlargement. ents, and mechanisms of cellular enlargement. For exApplication of stereological techniques for the study of ample, active cell division has been demonstrated in growth plate performance requires fixation protocols the proliferative zone, but not in the maturation and that preserve chondrocytes in a fully expanded state hypertrophic zones (this study; Farnum and Wilsman, (Hunziker et al., 1982). Using these fixation protocols, 1993; Kember and Walker, 1971). Also, chondrocytes chondrocytes closely resemble the morphology of chon- change from a n aerobic metabolism in the proliferative drocytes fixed using rapid freezing and freeze substitu- zone to a n anaerobic metabolism in the hypertrophic tion, and living chondrocytes in explants (Hunziker et zone (Brighton and Heppenstall, 1971; Brighton, 1978). The synthesis of matrix constituents characteristic al., 1982, 1984; Farnum and Wilsman, 1987; Farnum et al., 1990). Although recently optimal chemical fixa- for the hypertrophic zone is further evidence that the tion in combination with BrdU labelling was used for change in cell morphology reflects changes in cell functhe study of chondrocytic proliferation (Farnum and tion. Carbonic anhydrase (Gay et al., 1982; KumpuWilsman, 1993), chondrocytic enlargement was not ad- lainen and Vaanaanen, 1982; Vaanaanen 1984), Type X collagen (Adams e t al., 1989; Gibson and Flint, 1985; dressed in that study. Grant et al., 1985; Schmidt and Linsenmayer, 1985), Incomplete Columns and Calpain I1 (Shimizu et al., 1991) have been idenIn addition to confirming the results of the stereolog- tified exclusively in the hypertrophic zone, both intraical study, our serial reconstruction study demon- cellularly and extracellularly. Other matrix constitustrated the existence of columns incomplete at the ents like collagenase (Blair et al., 1989; Brown et al., epiphyseal or metaphyseal end. This observation may 1989; Dean et al., 1985, 19891, chondrocalcin (Poole et suggest that incomplete columns, as observed in the al., 1984; van der Rest et al., 1986), osmium-ferrocyarabbit by other workers (Moss-Salentijn et al., 19871, nide positive electron dense pericellular substance are not species dependent, and that they may be rec- (Farnum and Wilsman, 19831, and a periodic acid reognized in other growth plates and in growth plates sistant lectin Ricinus Communis I binding pericellular from other species a s well. This may be further support glycoconjugate (Farnum and Wilsman, 1988) have for the concept of regular replacement of chondrocytic been localized in the hypertrophic zone, but not in the columns (Rigal, 1962) and the hypothesis that epiphy- proliferative zone. There is also evidence that the change in chondroseal columns represent young, newly developing columns, and metaphyseal columns the final segments of cytic morphology is accompanied by a change in the exhausted columns (Moss-Salentijn et al., 1987, 1991). mechanisms by which chondrocytes enlarge. Several Recently, i t was suggested that columns even may be workers have demonstrated that the main portion of further subdivided in smaller groups of 4 to 16 cells cellular enlargement is the result of cell swelling (ab(Wilsman et al., 1993). The combined observations of sorption of water) rather than a n increase of cell orthese studies are important because they imply a pre- ganelles (hypertrophy), as evidenced by a volume inviously unrecognized regulatory level in the growth crease in cytoplasmic space and nucleoplasms between plate, namely, the regulation of the transient column proliferative and hypertrophic chondrocytes (Brighton or isogenous group. Furthermore, the finding of incom- et al., 1973, 1982; Buckwalter et al., 1986; Hunziker et plete columns and the hypothesis of transient columns al., 1987). However, i t has been suggested that in the or isogenous groups appear to be incompatible with the costochondral junction of the rat, cell swelling a s a concept that the functional unit for longitudinal bone mechanism for chondrocytic enlargement is restricted growth within the growth plate is the complete column. to the hypertrophic zone (Brighton et al., 1973). Qual- 266 G.J. BREUR ET AL. itative ultrastructural studies of the growth plate support this hypothesis. In flattened chondrocytes of the proliferative zone, organelles are tightly packed together, while in rounded chondrocytes of the hypertrophic zone, cell organelles are spread and the cytoplasm has a more loose appearance (Holtrop, 1972a,b; Scott and Pease, 1956). Furthermore, re-analysis of data presented by Buckwalter (Buckwalter et al., 1986) confirms this hypothesis. In the proximal radial growth plate of 15-day-old mice, 65% of the cell volume increase between the upper and lower proliferative zone was the result of a n increase in the volume of the Golgi apparatus and the rough endoplasmic reticulum; the balance was mainly provided by a n increase in cytoplasmic ground substance. Eight-five percent of the chondrocytic volume increase between the distal portion of the proliferative zone and the distal portion of the hypertrophic zone was contributed by a n increase in cytoplasmic space and nucleoplasm, while the balance again was mainly provided by a n increase in cytoplasmic ground substance. These studies together would suggest that cellular enlargement, during what we have described as the first phase of cellular enlargement and corresponding to Strata 3 and 4, is due to true hypertrophy, while the cell volume increase in the second phase, Strata 5,6, and 7, is mainly the result of cell swelling. Consequently, the transition from flattened cell to a more rounded cell would have to be accompanied by synthesis or activation of membrane structures that facilitate active transport of water and electrolytes against the high osmotic pressure of the matrix (Hunziker et al., 1987; Rothstein, 1989). Recently, i t was hypothesized that potassium channels, responsible for a large outward-directed K' current in chondrocytes from the hypertrophic zone, may play a n important role during the swelling of growth plate chondrocytes (Walsh et al., 1992). In a previous study we demonstrated a positive linear relationship between the final volume of hypertrophic chondrocytes and the rate of growth of a growth plate (Breur et al., 1991). We saw this as evidence that the final volume of hypertrophic chondrocytes may be regulated independently from physiological processes associated with endochondral ossification. The data of this study indicated that the final volume of hypertrophic chondrocytes was obtained a t least 100 pm proximally from the metaphysis, thus a t least 5 hours before cell death. These two studies together would suggest that a control mechanism for the final volume of hypertrophic chondrocytes must act at, or proximally from, this point. The functional significance of a period without substantial cell morphologic changes at the end of chondrocytic enlargement is unclear. However, we speculate that it is related to preparations for the upcoming conversion of cartilage into bone and the imminent cell death. This study's contribution to the understanding of chondrocytic enlargement in the growth plate is twofold. It demonstrates that cell volume increase starts immediately following cell division in the proximal portion of the proliferative zone, and that chondrocytic enlargement consists of a phase of slow and a phase of rapid cellular enlargement. Although this study clarifies several aspects of chondrocytic enlargement, i t also raises new questions in relation to the kinetics of the growth plate. For instance, the finding that chondrocytic enlargement starts immediately following cell division in the proximal portion of the proliferative zone may imply cellular heterogeneity in this portion of the growth plate. Thus, in addition to actively proliferating chondrocytes, this stratum may contain cells that have left the cell cycle and have started the phase of slow chondrocytic enlargement. Circumstantial evidence for such a heterogeneity has been reported in epiphyseal cartilage (Wilsman and Vansickle, 1971) and in growth plate cartilage (Farnum and Wilsman, 1993; Rigal, 1962; Simon and Cooke, 19881, but definitive proof of functional heterogeneity has not been given. Further definition of the functional heterogeneity may permit a more accurate estimation of cell cycle time and enhance our understanding of the process of chondrocytic maturation. Another, more general question is related to the role of matrix synthesis in bone length growth. It is generally understood that bone length growth is a function of chondrocytic proliferation, chondrocytic enlargement, and matrix synthesis (Seinsheimer and Sledge, 1981; Buckwalter and Sjolund, 1989), while the rate of bone length growth is only determined by the number of newly produced chondrocytes per column per day and the mean height of terminal hypertrophic chondrocytes (Hunziker and Schenk, 1989; Kember, 1960, 1972, 1973, 1978; Thorgren and Hansson, 1973a,b, 1981; Sissons, 1955). Consequently, many recent studies related to bone length growth are restricted to chondrocytic proliferation and volume and shape changes during chondrocytic enlargement (e.g., Breur et al., 1991; Buckwalter et al., 1985, 1986; Farnum and Wilsman, 1993; Hunziker et al., 1987; Hunziker and Schenk, 19891, even though data of several studies have implied that the cartilage matrix contribution to longitudinal bone growth may vary from 30% to as much a s 60% (Buckwalter et al., 1986, Breur et al., 1992; Hunziker e t al., 1987; Hunziker and Schenk, 1989). It seems unlikely that the important contribution from cartilage matrix to bone length growth is completely inconsequential for the determination of the rate of bone length growth. Thus, revisiting the role of cartilage matrix formation in bone length growth may be justified. LITERATURE CITED Adams, S.L., K.M. Pallante, and M. Pacifici 1989 Effects of cell shape on type X collagen gene expression in hypertrophic chondrocytes. Connect. 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