Age and pattern of the onset of differential growth among growth plates in rats

Age and Pattern of the Onset of Differential Growth among GrowthPlates in Rats

Norman J. Wilsman,1 Elizabeth S. Bernardini,1 Ellen Leiferman,1 Ken Noonan,2 Cornelia E. Farnum3

1Department of Comparative Biosciences, University of Wisconsin–Madison, Madison, Wisconsin 53706, 2Department of Orthopedics andRehabilitation, University of Wisconsin–Madison, Madison, Wisconsin 53706, 3Department of Biomedical Sciences, Cornell University, Ithaca,New York 14853

Received 3 April 2007; accepted 7 September 2007

Published online 10 April 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20547

ABSTRACT: Differential growth is the phenomenon whereby growth plates in the same individual at the same time all have uniquelydifferent axial growth velocities. Differential growth is clearly present in the adolescent skeleton. In this study we ask two questions. Whenand by what pattern does the phenomenon of differential growth begin? Second, to what extent are the development of differential growthvelocities correlated with changes in hypertrophic chondrocyte volume and/or with changes in chondrocytic production/turnover? Fourgrowth plates (proximal and distal radial; proximal and distal tibial) were studied at 24 different time points in Long-Evans rats between the17th gestational day (when differential growth does not exist) and postnatal day 27 (when differential growth is well established). Growthvelocities were measured using fluorochrome labeling. Using stereological methodology, multiple chondrocytic kinetic parameters weremeasured for all growth plates. Elongation of the proximal radial growth plate decreases relative to elongation in the other three growthplates in the late fetal phase. Differential growth is fully expressed at postnatal day 13 when the other three growth plates start to decreasedaily elongation at different rates. Differential growth is primarily associated with differences in hypertrophic cell volume manifested whengrowth deceleration occurs. This study also illustrates that differential growth is superimposed on systemic regulators that affect all growthplates simultaneously. The most dramatic illustration of this is the sharp decline in growth velocity in all four growth plates that occursperinatally. � 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1457–1465, 2008

Keywords: growth plate; physis; growth; growth velocities; hypertrophic chondrocyte

Longitudinal bone growth is the product of discrete butlinked operations carried out through chondrocyticdivision and differentiation. The mechanics of this processoccur in all growth plates. Germinal chondrocytes passthrough a series of regulated gates, enter the cell cycle,divide, and leave the cell cycle.1–3 They differentiate,swell,4–7 mineralize the matrix, degrade the matrix, andfinally die by apoptosis at the chondro-osseous junction.This differentiation cascade is (probably) identical in allgrowth plates.8–10

It is curious, therefore, that when regulation of growthis considered at the level of the individual animal andwhen we alter the perspective from discrete events in thecellular maturation cascade to rates of cellular change,growth velocities are not uniform but vary from onegrowth plate to the next—growth plates in differentlocations in one individual at one moment in time: growthplates are all different. This phenomenon—differentialgrowth—is a hallmark of growth plate biology, and wasfirst documented 150 years ago.11,12 Differential growthis superimposed on the well-documented phenomenon ofage-related differences in growth velocity and growthplate activity where all growth plates in the body acce-lerate or decelerate together, and is superimposed ondifferences that are used to determine the ages ofindividuals.13–17 Differential growth is the phenomenonwhereby growth plates on opposite ends of the same bonein the same individual at the same moment in timeelongate at rates that may differ by a factor of 3 to 7.

Preliminary studies in our lab suggested that growthplates in the fetal radius and fetal tibia were all elongating

at similar rates; however, at 28 days of age in the rat,differential growth is fully expressed with proximal anddistal radial, and tibial growth plates are all elongatingat significantly different rates. Of all the chondrocytickinetic parameters measured, hypertrophic cell volumes(also all different) seem to be particularly significant tothis process.8 Pinpointing the timing and pattern of theemergence of differential growth (between the late fetalperiod when growth plates have similar growth velocitiesand 28 days of age when they all have different growthvelocities) should help identify those mechanisms andcontrols that are prime movers of the process.

The purpose of this study was to (1) document theemergence of differential growth by daily measurementof changes in growth velocities in four different growthplates (proximal and distal radial and proximal anddistal tibial) and (2), using stereologically measuredkinetic parameters of growth plate chondrocytes, deter-mine whether it is changes in the rate of cell proliferation(turnover) or changes in chondrocytic volume duringhypertrophy that are primarily responsible for effectingdifferential growth.

MATERIALS AND METHODSFour growth plates, the proximal and distal radial and theproximal and distal tibial growth plates from Long-Evans ratpups were studied. These four growth plates were sampleddaily starting at gestation day 17 (1 day before the earliestvisible signs of calcification in rat long bones) through the first2 weeks of age. Thereafter, we sampled pups every other dayfor the third week and every fourth day during the fourth week.This design focused most intensively around the peripartumperiod, which we believed to be critical for detecting theemergence of differential growth. This study was reviewedand approved by the Institutional Animal Care and UseCommittee.

JOURNAL OF ORTHOPAEDIC RESEARCH NOVEMBER 2008 1457

Correspondence to: Norman J. Wilsman (T: 608-263-4434; F: 608-263-3926; E-mail: [email protected])

� 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Growth Velocity by Fluorochrome LabelingElongation velocity was measured using calcein fluorochromelabeling. Calcein was prepared as a solution of 10 mg/mL of1.3% sodium bicarbonate. For each prenatal day sampled,calcein was delivered to fetal pups by injection to an anesthe-tized mother (30 mg/kg, i.v., subclavian vein). For each gesta-tional age, two mother rats were used and the number of fetusesin each of the 10 mothers was similar, varying from 11 to 14.Twenty-four hours after receiving i.v. calcein, the mothers wereeuthanized with pentobarbital (50 mg/kg, i.p.) and six ratfetuses were chosen for collection. After excluding the fetuses atthe ends of each uterine horn to avoid sampling those thatmight be atypically small for gestational age, the choice offetuses was done randomly before the uterus was opened.

For each postnatal day sampled, two pups were chosenrandomly from four litters. Twenty-four hours prior to euth-anasia, each rat pup was injected with calcein (5 mg/kg, i.p.) andwas euthanized 24 h later with pentobarbital (50 mg/kg, i.p.).

Fixation, Embedment, Sectioning, and Label DetectionFollowing euthanasia, the right radius and tibia were collectedand each growth plate (including epiphyseal cartilage/bone onone end and metaphyseal bone on the opposite end)was sectioned longitudinally into sections approximately250 mm thick. The sections were mounted on slides in glycerin,coverslipped, and the fluorochrome label was visualized underepi-fluorescence microscopy using a 405 nm excitation filter, a455-nm dichroic mirror, and a 470-nm barrier filter (Fig. 1).

Calcein18–20 is a member of a family of fluorescent markersthat include oxytetracycline, alizarone complexone, and xylenolorange. Calcein is incorporated into all active mineralizingmatrices, including a narrow band at the chondro-osseousjunction. To estimate elongation per 24 h, the distance betweenthe leading edge of the fluorescent calcein band and the

chondro-osseous junction was measured using an ocularreticle.7,21 Measurements were taken from each growth platefor each pup (two measurements per fetal pup growth plate,three per postnatal pup growth plate) and were averaged. Theseaverages were then averaged for each growth plate across allpups of the same age.

Left growth plates were trimmed into 1� 1� 2-mm blocksand fixed in 2% glutaraldehyde in 0.05 M cacodylate buffer(pH 7.35) with 0.7% ruthenium hexamine trichloride (RHT).7,22

Tissue blocks were oriented such that growth plates wouldbe sectioned vertically,23 and blocks were embedded in Epon-araldite. For each animal and each growth plate, two tissueblocks were selected randomly from all available blocks. Fromeach block two sets of sections 1.5 mm thick were cut. The setswere spaced 15 mm apart. Absolute volumes of individualgrowth plates are estmated from measures of the majorand minor axes and the height of the growth plate.23

Kinetics of Growth Plate Chondrocytes—Stereological FrameworkThe details of the mathematical derivation of the stereologicalframework for chondrocytic kinetics of growth plate chondro-cytes has been described previously.7 In brief, a growth plate ismodeled as an idealized unit cylinder with diameter¼ 1 mm(diameter is arbitrary). This method of modeling a growthplate as a unit cylinder builds on the pioneering modeling ofchondrocytic kinetics by Sisson and Kember,7,24,25 but differesfrom Kembers columnar model by using entirely three-dimensionally based parameters. As Kember pointed out whenthe questions of interest involve comparing different growthplates in one individual, one growth plate over time, andespecially when comparing growth plates from differentspecies where absolute volumes (in stereological terms, VT)vary extensively, for example rats and children, there needs tobe a procedure for normalization.24–26

Figure 1. Micrograph of the dis-tal radial growth plate viewed underepifluorescence microscopy (250 mmscale bar included). This micrographillustrates the technique of fluoro-chrome labeling to measure growthvelocity. At the time of injection thefluorochrome label is permanentlyincorporated and fixed in a narrowband at the mineralizing front at thechondro-osseous junction. With time(in this example 24 h) and continu-ed new endochondral ossification,the chondro-osseous junction sepa-rates from the fluorochrome labelby a distance equal to the growthvelocity divided by time interval. Thehalf-life of calcein in the bloodis estimated to be 7 min.20 Whenmeasuring the distance to the chon-dro-osseous junction we used theleading edge (edge furthest fromthe growth plate) of the fluoro-chrome label as a starting point.This basic technique can be modifiedto include double labels with dif-ferent fluorochromes such as oxy-tetracycline, alizarine complexone,or calcein using correspondinglyappropriate epi-fluorescent filters.Time intervals must be a multipleof 24 h to cancel out any circadianeffect. Other metaphyseal fluores-cence represents autoflourescence.

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Kember’s unit of normalization primarily was an idealizedchondrocytic column in two dimensions. In this study we use athree-dimensionally based unit cylinder model. Differentialgrowth is, by definition, differences in axial growth velocity. Bymodeling growth plates as unit cylinders located in the centralaxis of a bone, we maximize the study of axial elongation andwe minimize the confounding effect of lateral growth.26

In the unit cylinder the number of chondrocytes producedper day (NNEW) is estimated by:

NNEW ¼ ½Vvproliferative zone=vðcÞproliferative chondrocytes�� ½p � 0:52 � Htproliferative zone� � GF

� ½24hrs=Total Cell Cyclehrs�

where the growth fraction (GF) is the percent of chondrocytesin the proliferative zone that actually are in the cell cycle.Whenever GF has been measured,3 the result has been 100%,or GF¼ 1.

The number of chondrocytes lost per day at the chondro-osseous junction (NLOST) is estimated by:

NLOST ¼ ½Vvterminal hypertrophic zone=vðcÞhypertrophic chondrocytes�� ½p � 0:52� � ½Elongation velocity=24hrs�

In a 24-h time frame, measuring five independent param-eters to calculate NNEW and three different and independentparameters to calculate NLOST, we have shown that NNEW/24h¼NLOST/24 h, thus validating relatively steady-state kineticsover a 24-h period. Therefore, in this study we only measuredNLOST/day as an index of NNEW/day.7 If one is interested inabsolute numbers for a given growth plate (VT), all that isrequired is to multiply the unit cylinder value by the product ofthe axes of the growth plate measured at tissue collection(essentially multiply by the number of unit cyliners in the entiregrowth Plate23). However, data presented in this way (VT) lacka method of normalization among growth plates becausechanges in major and minor axes of the four growth platesand absolute total volume of a growth plate (VT) over time are afunction of lateral growth.26

Hypertrophic Chondrocyte Volumes and Chondrocytic DensitiesOf the 24 different time points at which we measured growthvelocities, we selected 14 time points for stereologicalanalysis of chondrocytic kinetics. At each of these timepoints, we measured hypertrophic chondrocytic volumes

(v(c) hypertrophic chondrocytes) using the point sampled mean linearintercept method, applying eight angles of intercept.7

We calculated numerical densities (Nvterminal hypertrophic zone)from volume fractions (Vv) and mean cellular volumes(v(c)hypertrophic chondrocytes). Volume fraction of the hypertrophiczone (Vvterminal hypertrophic zone) was determined using an imageanalysis system (NIH Image). Accurate thresholding of thesystem was tested against volume fractions determined usingpoint-counting techniques.27–29 Calculated numerical den-sities were tested against numerical densities determinedusing the disector technique.28 No significant differences werefound in comparing these two methods. For each of thesestereological parameters, a minimum of four independentmeasurements (two blocks per rat pup; two pups per timeperiod) were averaged to represent an independent estimateper growth plate per time period. These data were analyzedusing ANOVA with Scheffe’s method of multiple comparisons(StatView 4.5 for Macintosh). Correlation coefficients werecalculated directly. The basis for this analysis has beenpresented previously.3

RESULTSDifferential growth may be defined as the point at whichthe daily growth rate in one growth plate is significantlydifferent from that of other growth plates at the sameage. The data (Fig. 2) capture that portion of develop-ment when differential growth emerges: at gestationday 18 (day �4 in Fig. 2), all four growth plates wereelongating at approximately the same rate (200 mm/day), while at the end of the sequence (27 days of age),differential growth is evident as all six possible pairs ofgrowth plates demonstrate significantly different ratesfrom each other. By the last time point, the proximaltibia is growing almost nine times faster than theproximal radius. When does this pattern first emerge?The answer has two parts. First, differential growthclearly exists in the fetal rat. The proximal radiusestablishes its own particular growth trajectory veryearly in the process while the other three growth platesremain clustered together until well after birth. Exceptfor the first day of the study, the proximal radius has a

Figure 2. Pattern of changesin growth velocity in four dif-ferent growth plates. On the17th–18th day of gestation(day �4) all growth plates areelongating at 200 mm/day. Inthe late fetal period the prox-imal radial growth plate velo-city is significantly slower thanthe other three growth plates.By postnatal day 13 all fourgrowth plates have signifi-cantly different growth velo-cities. Significant decelerationin birth growth velocitiesoccurs in all four growth platesperinatally. Bars¼ standarderror.

DIFFERENTIAL GROWTH VELOCITIES IN GROWTH PLATES 1459


growth rate that is significantly slower from all othersfor the entire sequence.

The second part of the pattern shows that the dailygrowth rates for the distal radius and proximal and distaltibia remain in tandem throughout the fetal period andinto the second week of life. No new developments indifferential growth occur until day 13, at which time allfour growth plates differ significantly from each other.After day 13, all four growth plates maintain the sameorder of fastest to slowest and there is an overall gradualdecline in daily rates with increasingly divergent rates.Thus, the emergence of differential growth is seen in twophases: first the prenatal divergence of the proximalradius, and second the later divergence in the secondweek by the proximal and distal tibial and the distalradial growth plates.

Additionally, there are trends that affect all growthplates in the same manner. For example, toward the endof the sequence (days 13–24), there is an overall gradualdecline of growth rates across all growth plates (lesser inthe proximal radius because of its already slow growthvelocity), a trend that has been reported in severalstudies and across several species.30 Further, there is adramatic perturbation in growth rates in the peripartumperiod. The fetal period is marked by a starting point atwhich all four growth plates grow at about equal rates(200 mm/day), followed by a rapid acceleration to a peakthat represents the fastest rates observed over the entiresequence. This acceleration is abruptly halted over thenext few days, just prior to birth, but resumes during thefirst week of life. These changes are somewhat lesspronounced in the proximal radius, but are dramaticacross all four growth plates. This pattern of waxing andwaning of all growth plates simultaneously is wellknown and not the subject of this study; however, whatthe growth velocity curves from this study is the knownwaxing and waning superimposed demonstrate thepattern of the emergence of differential growth.

Is differential growth best explained by changes/differences in the rate of cell production (comparingFig. 2 with Fig. 4) or by changes/differences in hyper-trophic cell volume (Fig. 2 with Fig. 3)? Visual inspectionof these three graphs demonstrate that we need toconsider the prenatal period separately from the post-natal period.

Prenatal PeriodIn the prenatal period, volumes of hypertrophic chon-drocytes did not vary significantly among time periodsor among different growth plates at any one time period(Fig. 3). On average, regardless of growth plate orprenatal time period, fetal hypertrophic chondrocyticvolume was 11,400 mm3 2350. In the prenatal periodthere was no correlation (or even a slight negativecorrelation; r¼�0.01) between rate of growth andhypertrophic cell volume.

In this study NLOST was measured. However, in theshort time frame of 24 h, we previously demonstratedthat NLOST¼NNEW.7 In the prenatal period the numberof chondrocytes produced/lost varied day to day, andfluctuations of number of chondrocytes lost/day paral-leled each other in each of the four growth plates in apattern similar to changes in growth velocity. Forexample in the prenatal period, in the distal tibialgrowth plate, the number of chondrocytes turned overper day varied from a low of 8600 cells/day in the slowestgrowing 18th gestational day to a high of 23,000 cells/dayjust 2 days later on the 20th gestational day. In theprenatal period, number of chondrocytes lost/day corre-lated (r¼0.79, p<0.001) with growth velocity (Fig. 4).

Postnatal PeriodPostnatally, hypertrophic chondrocytic volume variedwith time period and with growth plate (p<0.01). Thefluctuations of hypertrophic cell volumes paralleledeach other in each of the four growth plates and fluc-tuated in a pattern similar to the fluctuations in rate of

Figure 3. Changes inhypertrophic chondro-cyte volumes in four dif-ferent growth plates overthe course of this study.The changing patternin cellular volumes inthe postnatal adolescentperiod parallels changesin growth velocity duringthe same period.

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growth. For example, the smallest hypertrophic chon-drocytes (4000 mm3) were found in the slowly growingproximal radial growth plate at 24 to 28 days of age. Thelargest hypertrophic chondrocytes (20,000 mm3) werefound in the rapidly growing proximal tibial growthplate at 7 to 13 days of age. In the postnatal period,hypertrophic cell volume correlated significantly(r¼ 0.70; 0.01>p>0.001) with rate of growth (Fig. 3).

Alternatively, in the postnatal period, the number ofchondrocytes lost/day remained relatively constant(Fig. 4). Within a given growth plate and even amonggrowth plates, the number of new cells producedper day varied but not significantly. For example atthe time of the emergence of differential growth at13 days of age the number of new chondrocytes producedper day was about 10,5001500 cells per day in all fourgrowth plates.

DISCUSSIONA complex array of factors, both genetic and epigenetic,are involved in the mechanisms by which growthplate cellular activity during endochondral ossificationresults in bone elongation. (recent reviews, refs. 8–10,31, 32). Although mechanisms regulating the differ-entiation cascade of growth plate chondrocytes can bestudied by the reductionist and transgenic approachesof molecular biology,31 stereological approaches tounderstanding the kinetics of chondrocytic performanceparameters in growth plates growing at different rateshave been a valuable approach for the analysis ofhow the chondrocytic differentiation cascade is con-verted quantitatively into incremental bone elongationthrough time.5,7,31 Interstitial growth contributes toelongation during both the proliferative phase (throughchanges in cellular numbers) and the hypertrophicphase (through changes in cellular size). Matrix syn-thesis contributes during both phases. Although therelative magnitude of contributions made during theproliferative and hypertrophic phases vary in growth

plates growing at different rates (Fig. 5), even over aneightfold range of growth from approximately 50 mma day to 400 mm days, it has been demonstrated that thesingle biggest contribution to differential growth resultsfrom cellular swelling and associated shape changeduring hypertrophy.4,5,7,9 Therefore, for a study inwhich multiple time points and multiple growth platesare analyzed, chondrocytic volume increase and elonga-tion velocity are the two variables most likely to yieldsignificant comparative information.

A particularly interesting aspect of growth platebiology is that each individual growth plate of the bodycan be considered to have a biological life span. For thepurposes of this study, the ontogeny of an individualgrowth plate can be considered to start with formation ofthe primary center of ossification of the bone prenatally.From this perspective, the proximal and distal growthplates of a given bone begin their formation simulta-neously. The timing of formation of the secondary centerof ossification postnatally is a second key biological timepoint, and this differs for the two ends of a given longbone. For the radius, the secondary center forms earlierdistally than proximally, and for the tibia it is thereverse—the proximal secondary center forms prior tothe distal one. A third critical time point when consid-ering biological age of a given growth plate is the timingof closure. The proximal radial growth plate closesearlier than the distal radial growth plate; the distaltibial growth plate closes earlier than the proximal one.Differential contributions to overall length of a bone fromthe proximal and distal growth plates results from theirrelative rates of growth while both growth plates areopen, as well as from the relative differences in overalllength of the growth period for the two growth plates.

The timing of formation of the primary center ofossification is different in the radius than in the tibia, andthus the growth plates associated with these two bonesare formed initially at different stages of development.The timing of formation of the secondary centers in the

Figure 4. Changes inchondrocyte production/turnover in four differentgrowth plates over thecourse of this study.The changing pattern inthe prenatal period paral-lels changes in growthvelocity during the sameperiod.



four growth plates studied also differs, as do their timesof closure. If changes in growth velocity are a function ofbiological age, one would predict to see the same form tothe curve of growth velocity for all four growth plates, butoffset from each other depending upon the biological ageof each specific growth plate. However, the data generat-ed in this study demonstrate a trajectory of changingvelocity that is essentially identical in form and in activeacceleration for three of the four growth plates (distalradial, and proximal and distal tibial) from birthuntil day 10–13 postnatally. Growth velocities in thesethree growth plates track each other up to this point as afunction of chronological age, rather than biological age.It is interesting to note that differential growth for thesethree growth plates emerges as differences in rate ofelongation when all growth plates begin to slow theirrates of elongation. Thus, these three growth platesaccelerated at the same velocity, but decelerated at

different velocities. Unlike the other three, the velocitycurve for the proximal radial growth plate essentially isone of decreasing velocity throughout the period ofgrowth plate activity, including the prenatal period.The degree of separation of growth velocities seen in thisstudy at 27 days contuniues through 35 and 42 days.33

One could hypothesize that epigenetic factors aresignificant for the timing of the start of differentialelongation in these three growth plates, but thathypothesis was not directly tested in this study. Onepossibility is the emergence of differential biomechanical(compressive, tensile, and strain) forces as significantcontributors to the timing of the onset of differentialgrowth in specific growth plates. These biomechanicalsignals in the fetal period could originate from in uteroskeletal muscle contractions.34 Prenatally ossification ofthe primary diaphyseal center could change the forcesseen by the growth plate chondrocytes. Postnatally,

Figure 5. The three micrographs are of 1.5 mm-thick plastic-embedded sections stained with methylene blue/azure II/basic fuchsin of thedistal radial growth plate (scale bar¼200 mm). Three time points for illustration where chosen by referring to Figure 2 and drawing animaginary line parallel with the x-axis at about 350 mm/day, and noting time points where the growth velocity for the distal radial growthplate crossed this line. Three of these time points (day 20 gestation, and days 13 and 18 postnatal) are illustrated in Figure 5. The significantpoint is that all three micrographs illustrate the same growth plate elongating at 350 mm/day but at three different time points in itsbiological life span. What differs at each time point is the primary variable by which this growth velocity is achieved. At the earliest time point(left, day 20 gestational), mean hypertrophic cell volume is low (9540 mm3); however, the number of chondrocytes being produced is high(19,070 cells/day). In contrast, at the latest illustrated time point (right, day 18 postnatal), mean hypertrophic cell volume is high (14,64 mm3)while the number of cells turned over is only 12,710 cells per day. The micrograph in the center at 13 days of age represents the chronologicalpoint of emergence of differential growth. Visual comparison on the micrographs on the right and left illustrate an increase in matrix volumeper hypertrophic chondrocyte (3520mm3 vs. 6240mm3).5,21,23 Raw data and calculations are not included in this manuscript. This is consistentwith what is expected if ‘‘matrix directed cellular swelling’’4,6 is the chief engine of growth.9 This hypothesis requires an adequate volume ofbiomechanically normal matrix to direct the chondrocytic shape change that accompanies the increase in cellular volume of chondrocytes asthey hypertrophy.37

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while the timingof theossificationofprimaryandsecond-ary centers of ossification is primarily a developmentalphenomenon (radius before tibia), the subsequent effectof differential growth velocity of the growth plate mightbe mediated through biomechanical signals.35–37 Suchsignals then would be accentuated through gravity,especially as pups begin rigorous ambulation at 2 weeksof age.

This leads to the generalization that regulation ofgrowth velocities is a function of (1) regulating thenumber of cells produced multiplied by (2) regulating theamount that each cell increases its volume, with normalmodulation of cell shape.5–7,38 Both functions exist in allgrowth plates. All functions can be regulated independ-ently. One function or the other may dominate over theother functions. Which of these fundamental processesaccounts for the emergence of differential growth? Theanswer appears to be differential regulation of hyper-trophy. Late gestation through birth and the earlyneonatal period are periods without fully expresseddifferential growth, even though growth velocities in allfour growth plates rise, fall, and rise again. Theprimarily factor associated with these changes in velo-city of all growth plates are changes in cell productionkinetics (compare Fig. 2 with Fig. 4; r¼0.79). Afterthe second week of age to adolescence, while growthvelocities all slowly decrease, all four growth plates haveunique and significantly different velocities from eachother. During this period significant differences ofhypertrophic cell volume emerge (compare Fig. 2 andFig. 3; r¼ 0.70). In the postnatal period while differencesin cell production kinetics have a positive correlationwith growth velocities (r¼ 0.35) this correlation is notstatistically significant. When we consider velocity ofgrowth as the variable of interest, this study demon-strates that at any one time in any one growth plate,regulation of the kinetics of proliferation and regulationof the kinetics of hypertrophy occur simultaneously.Regulation of either the kinetics of proliferation orhypertrophy or both determine final growth velocity.

One candidate for the dramatic suppression of growthin the peripartum period is the fetal cortisol surge,released by the maturing adrenal glands to initiate thebirth process. The growth retarding effects of cortico-steroid therapy on children are well known, and experi-mental animals receiving synthetic cortisone have beenshown to experience a dramatic deceleration or totalcessation of bone elongation at their growth plates.39,40

IGFs are widely accepted to be the primary modulators ofgrowth hormone (GH) on skeletal tissues.41 Fetal cortisolmay act directly to suppress local expression of IGF-1 bychondrocytes or may act indirectly by suppressing thesynthesis or release of GH. In an interesting parallel,human neonate IGF-1 levels are known to drop precip-itously the first day after birth and remain low for 3 days,returning to prebirth levels by the end of the first weekof life.42 Therefore, maternal fluctuations of estrogensor progesterones may be another contributor to the de-crease in growth at birth.

As we consider the array of locally produced moleculesknown to influence growth plate chondrocytes, it is usefulto think about their differential role in regulating cellproduction, hypertrophic chondrocyte volume, cell shape,and integrating these cellular activities across theseregulatory gates.43,44 This is what has made the discoveryof the hedgehog protein and its internal negative feedbackloop Ihh—PTHrP—(alternatively TGF-b45) such a fasci-nating discovery.46,47 If, as many studies suggest, Ihh isinducible by biomechanical signals,48–50 and if differentgrowth plates are exposed to differential biomechanicalenvironments,35–37 then the negative Ihh—PTHrP–TGF-b feedback loop could be applied differentially. Consistentwith this argument is the report that the lamb tibiaelongates only when the lamb is recumbent and by infer-ence relatively unloaded. During standing/ambulation thelamb tibias’ length is static. Although most regulatoryinputs are continuous regardless of whether the tibia isloaded or unloaded, at the level of the individual lamb thetibia only elongates when unloaded.51

Clinically, these concepts can be taken to the level ofthe whole animal for consideration of how all regulatoryinputs integrate to create the clinically relevant variable,which is velocity of elongation. Growth velocity is aclassical example of an emergent property of a complexsystem. It is analogous to the concept that behaviorcannot be studied at the level of the neuron. Only whenmultiple neurons are included in the system doesbehavior emerge as a property of the system. Regulatoryinputs to growth plate chondrocyte differentiation occurat all levels of biological organization. Growth velocity isa variable that plays out at the level of the individualanimal. The clinical relevance of understanding differ-ential growth is that sometimes corrections need to bemade that have the potential to affect all growth plates inthe body. In contrast, in other scenarios what is needed isfor the clinician to be able to influence growth velocity of asingle growth plate differentially or even differentiallyon the different sides of the same growth plate as inangular deformities.52

ACKNOWLEDGMENTSThis project was supported by the National Institute of Health(NIH), National Institute of Arthritis, Musculoskeletal & SkinDisease (NIAMS): AR 35155-18, and a Merck-Merial ResearchFellowship to Dr. Bernardini. We would also like to acknowl-edge and thank Dr. Ian Stokes for his leadership, and hiswillingness to share software code, as a part of our collabo-rative effort to develop Matlab compatible software to greatlyfacilitate point-sampled, mean cubic intercept measures ofmean cell volumes. None of the authors received financialsupport for this study.

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Age and pattern of the onset of differential growth among growth plates in rats