J . Zool., Lontl. (1993) 231, 543-562

Comparative quantitative histology of mammalian growth plates

J . K . K I R K W O O D

T Ltrrinary Scicnce Group, IristitutP of Zoology, Rtpvit's Ptirk. London nl N ' I 4R 1'

A N D N. F. KEMBER

Drpurtnierit of'Afcdica1 Electronics und Phjwics, St Burtholonieu.'s hledictrl C'ollrgc, Churtc~rhusr Squtirt>, Lotidon ECIAf 6 BQ

(With 7 plates and 1 figure in the text)

Variation i n the growth rate of long hones is a function o f the number o f dividing cclls in the columns of the prolireration (flat) cell zone of the growth plate, the frequency with which they divide, and the sire to which they grow prior to ossitication. In a previous study we found that the wide variation in hone growth rates seen among specie5 of hirds was largely associated with variation in thc numhers ofcells in thc flat cell zone. Ilere we have undertaken a similar study of the growth plates of mammals and have examined variation in the morphningy and cell kinetics of the tihial growth plates of a variety of spccies. The hone growth rates tended to he lower than those observed i n birds and were particularly low in the mthropoid primates. Although quite marked variation in flat cell numhcrs I S apparcnt, the results suggest that variation in cell division rate may play a relatively greater role in variation in honc growth rate among mammals than i t does in birds. and that thc very low hone growth rates seen in the primates are due, i n pnrt, to lower rates of cell division than in other species.

Contents

Pagc Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545

Collection and preparation o f specimens . . . . . . . . . . . . . . . . . . . . 515 Morphometric and histological measurements . . . . . . . . . . . . . . . . . . 545 Estimation of tibia growth rates . . . . . . . . . . . . . . . . . . . . . . . . 546

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547 Tibia length and body mass. . . . . . . . . . . . . . . . . . . . . . . . . . 547 Morphology of the growth plates . . . . . . . . . . . . . . . . . . . . . . 517 Growth kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556 Interspecies variation in growth plate morphology . . . . . . . . . . . . . . . . 556 Interspecies variation in growth plate kinetics . . . . . . . . . . . . . . . . . . 556

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561

Introduction

The growth rate of the advancing tip of a long bone is a function of the number of dividing cells in the growth plate. the frequency with which these cells divide. and the sire to which they

543 ( 1993 The Zoological Society of London

544 J . K . KIKKWOOD A N D N. F. K E M B E R

subsequently grow in the hypertrophic zone prior to ossification (Kember ef al., 1990; Kember & Kirkwood, 1991 ). In a recent study we examined the relationship of long bone growth rate to these aspects of growth plate micro-anatomy in a variety of species of birds (Kember et al., 1990), and found that variation between species in bone growth rate was largely associated with variation in the number of proliferating cells.

As far as we are aware, the cell kinetics of bone growth has been studied in only five species of mammals: rats (e.g. Walker & Kember, 1972), mice (including a number of mutants) (Thurston, Johnson & Kember, 1985a, h), rabbits (Kember, 1985). and pigs and man, for which the data are more limited (e.g. Thurston & Kember, 1985). Most of these studies of bone growth plates were based on the use of tritiated thymidine to label cells in the synthesis phase of the cell cycle. The data for these mammals reveal a similarity between species in the patterns of cell division in the growth plates. Briefly, within each column of cells in the cartilaginous growth plate, there is a reserve cell

Secondary epiphyseal centre of ossification

Inert cell zone

Reserve cell zone t

PLAT^ I . Section through the proximal epiphyseal plate of the tibia of a red-necked wallaby hlucroy~us ruf;~gri.tru.s illustrating the structure o f the growth plate. (H. & E. Photographed at ,X 16.) Thls scction shows the hl-oad incrt cell zonc and the highly organized columns of flat cells typical o f B Type I 1 growth plate (see text).

H I S T O L O G Y O F M A M M A L I A N G R O W T H P L A T E S 545

zone where the division rate is low, a clearly defined proliferation zone in which the cells are flat in shape (the flat cell zone), a zone in which cells undergo hypertrophy and maturation, and a zone o r mature hypertrophied cells with calcified inter-cellular matrix (see Plate I) . There is variation between species in the numbers of cells in the different zones, the percentages of dividing cells and the sizes of the hypertrophic cells. There are also variations in the histological structure of the plate with, for example, human bone showing a wide zone of inert cells between the active growth plate and the bony epiphysis (Kember & Sissons, 1976). The structure of avian growth plates is broadly similar to that of mammals (Kember & Kirkwood, 1987) although, as in mammals. there are considerable differences between species in the numbers of cells in each zone (Kember P I ol., 1990).

Mammals generally grow more slowly than birds of comparable adult size (Case, 1978; Kirkwood & Webster, 1984; Kirkwood et ul., 1 9 8 9 ~ ) although, as in birds, there is wide variation between species. In this study we set out to examine growth plate morphology and tunction among mammals in order to explore the basis of this variation in growth rates between mammals and birds and amongst mammals. Our aims were: ( 1 ) to examine variation in the structure of the growth plate among species of mammals; and (2) to undertake a preliminary study of the relationship of bone growth rate t o features of the micro-anatomy of the growth plate among these species and others for which data already exist.

Bone diseases associated with dysfunction of the growth plate may occur in some animals reared in captivity, and particularly in those that grow more rapidly than is usual for individuals of their species (see, e.g. Hedhammar et al., 1974; Riddell, 1981; Serafin, 1982; Yamasaki & Itakura, 1988). Part of the rationale for this study was to provide information on interspecies variation in form and function of the growth plate to assist in the investigation of the aetiology of these abnormalities.

Materials and methods

Collection und prepurution qf specinirns

Tibiae were collected at post-mortem from young animals that had died from various ciiuses at the Zoological Society of London's collections. While none of these animals was considered to be suffering from any form of bonc disease, the possibility cannot be excluded that poor nutrition associated with a terminal state would have had effects on some of the growth plates. The animals from which the specimens were collected are listed in Table I . In each case ;I tibia was removed and fixed in formol saline. The overall length (distance between articulating surfaces) was measured and recorded before histological processing. Small bones were processed whole but the larger ones were split longitudinally and small sections, including both epiphyseal and mctaphyseal bonc, were cut from the centre of the proximal and distal plates. These pieccs. about 5 x 5 mm and 7 mm thick, were decalcified in EDTA, as wcrc the entire smaller bones, and embedded in wax for histology. Sagittal sections were cut and stained with haciiiatovylin and cosin for cxamination.

Morpliornetric uiid histologicul t i i e m ~ i ~ r i ~ ~ ~ ~ e n t ~

The dimensions of various parts of thc proximal and distal growth cartilages were measured as follows:

1. A c;ilibi-ated eyepiece graticule was used to tind the dimensions ofthc inert zone and the flat cell zone in the direction parallel to the axis of the bone (Plate I ) .

7 . An estimate was made of the number ofcells in the flat cell zone ( N ) as the mean ofcounts orcells down at least 3 columns. Where columns were discontinuous, counts were made by moving laterally at the discontinuity and by continuing the count down an adjacent column.

546 J . K. KIRKWOOD A N D N F. KEMBEK

Animal

Marsupialia Brush-tailed bettong Red-necked wallaby

Primates Brown lemur ( 1 ) Bushbaby Squirrel monkey Common marmoset (2) Mandrill Orang utan

Rodenlia Springhaas Rock cavy Orange-rumped agouti Chinchilla

Hyracoidea Rock hyrax

Aruodactyla Vicuna Reindeer (2) Blackbuck Mouflon

Post-natal age (days) -

PE PE

2 1 1

I80 2

15

5

14 I

-

1

30 5 I

21

Adult tibia Icngth" Tibia (mm)

~ length - - (mm) Mean S.D. n

98 101 284 -~

I I S 33 55

80 46h 59 70 236 66 227

-~

-~

65 126 35 44h I00 29 59

32" 67

I96 I65 290 157 206 - 22x

5 7

4 5 3 3

I I 15

4

6 2

N/D

6

-

18 10 2

2 6

I 0 16 8 8 7 8

3

7 4

6

7 8 4

Adult weight"

(kg) ~-

I .3 14

3. I 0.25 0.925 0.341

18.2 57.5

3.5 8.5 2.7 0.75

3.6

5 0 4 181 35 40

-- ~

.' Adult wcight data from Macdoiicild (1984) and Walkcr (1968)

PE Approximately the time of pouch cxit Mean of two boncs

3. The calibrated graticule was used a t high magnification to mcasurc thc average hcights (the diamctcr parallel to the direction of longitudinal growth) of hypertrophic cclls (h,pm). Normally the hcight o f u sequence of 5-6 cells was measured and the mean dimension calculated but. wherc the hypertrophic zone was small, heights of individual cells were recorded. For each plate a t least 50 cells were measured in order to find the average height.

The overall mature tibia lengths (ATI,,mm) of the species studied were measured from spccimcns made available to us at the Natural History Museum, London.

Ektimnrion of tihio grotr.tJ? rrrtc.s

Estimates o f thc instantaneous growth rate o f the tibia (GR, pm/day), at ages corresponding to those of thc animals from which ou r spccimcns wcrc collcctcd. were dcrivcd in scvcral ways. a s dcscribcd below:

I . From data for the spccies published in thc litcraturc (common marmoset Cd/ i / /?r i .~ juc(~1iii.s- ~c Appendix).

1IISTOLOGY OF M A M M A L I A N GROWTH P L A T E S 549

epiphyses. In some species with a secondary centre, an inert zone was evident and the sizes of these inert zones are given in Table 11.

While the histology of the growth plates generally conformed to the expected pattern for endochondral ossification, in addition to variations in cell counts and sizes, there were some noteworthy differences between species in detailed structure. The plates could be classified into three general types. Type I was typified by the rodent pattern as seen in rats (Ruttus riorzyyicus) (Kember, 1985); Type I1 was seen in the artiodactyls and typified by the pig (S24.Y .smifu) (Thurston & Kember, 1985); Type I11 is the primate pattern of which the human growth plate is a typical example (Kember & Sissons, 1976). The essential differences between these types lie (a ) in the degree of organization of the cartilage columns, which is high in Types I and 11. and (b ) in the presence of an inert zone between the active proliferating cartilage and the epiphyseal growth plate in many of the sections of Types I1 and 111. Types I, I1 and I11 are best illustrated here by Plates V, VI and 11, respectively.

Brief descriptions of features of the morphology of the growth plates examined are given below. These apply to both proximal and distal growth plates since few obvious differences between them were observed.

h fizrsiipicrliu

Brush-tailed bettong ~~~c.rtoizgiupcnici(lufu. This Type I growth plate was from a n animal at the age of leaving the pouch. The hypertrophic cells had very thin walls.

Red-necked wallaby Macropus rufbgriseus. The epiphyseal secondary centre had developed. This was a Type I1 plate with well-defined columns in the cartilage growth plate below a wide inert zone (Plate I) .

Primates

Brown lemur Lrmirr ,fult~us. These bones from two-day-old animals showed no secondary centres in the epiphyses. The plates were of the Type I11 pattern. The columns in the growth plate were not well defined and a few blood vessels passed through the plates. The metaphyseal trabeculae were very short.

Bushbaby Gulago stwy&n.si.s. The bone from this one-day-old animal showed bipolar growth plates (Type H I ) with endochondral ossification in process directed both towards the epiphysis with its secondary centre and towards the metaphysis.

Squirrel monkey Sainziri sciureus. This specimen from a one-day-old animal had a secondary centre in an early stage of formation. In this Type 111 plate there were no clear cell columns but cells were arranged in short linear groups.

Common marmoset Ctrllithri.riacchus. Two specimens of these Type I11 plates were examined. The specimen from the neonate showed an immature growth plate with poorly defined columns and very short metaphyseal trabeculae. The other specimen from a six-month-old animal had a narrow plate that was nearly ‘sealed off’ by calcified tissue at the metaphyseal edge. In the mature plate there was still evidence of an inert zone between the columnar zone and the secondary centre of the epiphysis.

Mandrill Mandrillus .sphin.u. This specimen from a two-day-old animal showed a Type I I I plate (Plate IT). There was a secondary epiphyseal centre and a wide inert zone. The cells were not

550 .I. K . K I R K W O O D A N D N . F . K E M B E R

P I A i t ’ 11. Section through the proximal epiphysed plate of the tibia of a mandrill Ai’mdri//us .sphi!~.\-. ( H . & E. Photographed at I 16.) This IS a typical Type 111 growth plate ( x e text) in which there I S a wide incrt cell zone but which lacks a clear columnar arrangement o f cells in thc flat a n d hypertrophic cell mncs.

arranged in a clear columnar structure even in the hypertrophic zone. There was therefore an irregular edge to the metaphyseal border of the plate.

Orang utan P o n g o p ~ ~ ~ ~ i u r z r s . The specimen was from a 15-day-old animal. Tt showed a Type 111 growth plate with no secondary centre evident and a poorly defined proliferation zone.

Roikcw t ia

Springhaas Pedetes cupensis. The growth plate from this five-day-old animal was of the Type I pattern. There was no epiphyseal secondary centre but there was a very deep zone of flat cells

H I S T O L O G Y O F M A M M A L I A N G R O W T H PLATES 55 I

(Plate 111). A few blood vessels passed through the cartilage plate and there was an irregular metaphyseal margin to the growth plate.

Rock cavy Kcwith7 w p s t r i s . This showed a narrow Type I growth plate with a well-developed secondary centre.

Orange-rumped agouti Dtr.sj.proctu q u f i . This specimen from a two-week-old animal showed a typical Typc 1 plate (Plate IV).

Chinchilla Chinchillu Ilrniger. This showed a typical Type I growth plate (Plate V).

H.twc*oidcw

Rock hyrax P~-octrritr c u p c w s i s . There was ;I developing secondary centre in the epiphysis with a narrow inert zone. The columns were well defined i n this Type I plate.

532 _ I . h. h l K h W U U U A N L J N . P . hkiVIHLK

PLATE 1V. Section through the proximal epiphyseal plate of the tibia of a11 orange-rumped agouti f ) u . ~ ~ ~ / r o ~ ~ / o ugur;, 16.) This is ;I Typc I growth plate (see text) in which the cells are organiicd in clcnr columns. ( H . & E. Photographed at

but which I d s a n inert mne.

PLATF. V. Section through the proximal epiphyseal plate of the tibia of a chinchilla Ch~w/ii / /u / u ~ I ; , ~ w . (H. & E. Photographed at - 16.) This is a Type 1 growth plate (see text) in which the cells :ire organized in clear columns. hut which lachs an incrt zone.

IllSTOLOCiY 01.' M A M M A L I A N G R O W T H P L A T E S 553

.4 rtiodiicij-lu

Vicuna I'icx~riu ricwgriu. The proximal plate in this specimen was wedge-shaped with a 50"0 difference in thickness of the plate across the width of the section. The pattern conformed to Type TI (Plate VI).

Reindeer Run'qjjiv turmdus. The specimens examined were from two- and five-day-old animals. A wide inert zone lay above the zone of columns. The outstanding feature of these Type 11 growth plates was their irregular border with the metaphysis and the appearance of cartilage cells embedded i n the traheculae. There was evidence of invasion of the hypertrophic zone beforc the

P L A T E VI. Section through the proximal epiphyseal plate of the tibia of a vicuna L'iu1gm r i ~ ~ u p i u . (H. & E. I h . ) This is a typical Type 11 growth plate with a wide inert zone and a highly organized columnar Photographed a t

arrangement or cells in the flat and hypertrophic zones.

551 J . K . K I R K W O O D A N D N . F . K E M B E R

cells had swollen to expected size. The appearance was that of premature invasion by the metaphyseal blood vessels (Plate VII) .

Blackbuck Antilopt~ cewicupru. The Type I I growth plate from this one-day-old animal was narrow with few cells in the inert zone. The metaphyseal border was ragged but not with such marked irregularity as in the reindeer.

Mouflon Oris nzu.si?m~. In this species from a three-week-old animal the growth plate was of the Type 11 pattern. An inert zone was present and there were a few blood vessels traversing the growth plate.

Gloic,tli kiric~tics

There was a wide variation in the linear growth rate of the tibia among the young niammals

P I . A r t VIl . Section through the proximal cpiphyscal plate 01' the tibia of a reindccr Rm,q:t/c,r. rtrvuiltius. ( H . & E. 16.) I n this Type I I growth plate there is I wide inert zone and the cells arc arranged in clriir columns. Photographcd at

IIISTOLOGY OF M A M M A L I A N GROW.It1 PLATFS 555

T A I ~ L F I l l Estimates of tibia groit.th rute.v, l i ypc~trophic~ celldiametc~r.s, nurnbc~r.s ufflcrt ceNc atidof rlir main ci4IcIit'i.son ratc it7 f l l r / /a l w//

zonrs f;w the inrlit~i(luul,s of ruriet.~ of .spec.ic~.c ~ t i t c l i i v l l i iw

Age L 1; G R I1 PI. /

Squirrel monkey I - 80 330 22 15 76 0.20 Common marmoset (I) Neonate -- ~ 240 19 13 I27 0.10 Mandrill 2 166 270 22 12 96 0.13 Orang utan 15 161 ~ 80 20 4 I47 0.03 Agouti 14 55 74 5 1 0 18 28 31 0.76 Chinchilla I 30 35 600 24 25 58 0.43 Reindeer ( 1 and 2) 2 & 5 I25 70 1000 19 53 I98 0.27 Blackbuck I 49 150 220 21 10 55 0 . I9 MouRon 21 ~ 500 18 28 54 0.52

Key: L: Adult tibia length minus tibia length at the time the specimen was collected ( L e i - L , ) t i : The time on the fitted curve at which length is half the asymptotic value (scc Methods) GR: Growth rate / I : Mean height (diameter in the plane of growth) of hypertrophic cells Pr: Number o f cells produced per day by each column in the growth plate N : Number ofcells in the Rat cell zone (a proportion ofwhich arc proliferating). The figures indicate the sum of thc number of cells in the columns of both proximal and distal plates f i Cell division frequency (divisions per day) calculated /= P r / N

Animal (days) (mm) (days) (pm!day) (pin) (C'clls:day) N (Divisions:day) .. -~ ~~

examined, ranging from 80 pm/day in the orang utan to a n estimated 1000 pm/day in the reindeer (Table 111). For some of the species we were unable to obtain measurements or estimates of bone growth rate.

The relationships between longitudinal growth rate and thc ccll kinetics o f the growth platc are described by the expressions:

and

where,fis the frequency of cell divisions (divisions per day) and Pr is the cell production rate (cells per day): N , G R and h have been defined above.

From the results of the measurements of h (Table Ill), it is clear that it is not variation in the mature size of the cells that accounts for the large variation in tibia growth rates seen among the species examined here. Hypertrophic cell sizes varied within fairly narrow limits in the sections examined here, ranging from 15 ,urn in the proximal plate in the brown lemur to 30 pm in the distal plate of the red-necked wallaby. The mean values for h a t the proximal and distal growth plates were 20.8 f 3-75 ,urn in = 19) and 20.7 f 4.29 pni ( n = 20), respectively.

I n contrast, there was considerable variation in the depth of the flat cell zone and in N , the number ofcells in this zone (Table TI). I t is in this zone that cell division occurs and the count of flat cells gives an estimate of the number of proliferating cells per column. The deepest growth plates were seen in the springhaas in which the flat cell zones in both proximal and distal plates were about 3500 pm deep and their columns consisted of over 300 flat cells. At the other end of thc spectrum was the bushbaby in which the flat cell zones were 300--400 pm deep and consisted of columns of just 12- 17 cells.

556 J . K . K I R K W O O D AND N . F. K E M B E R

In birds, variation in the growth rate of the tarsonietatarsus bone between species is largely associated with variation in the numbers of cells in the proliferation zone (Kember et a/., 1990). However, in the mammals examined here, this relationship was not so apparent. I t was notable. for example, that although the growth rate of the orang utan tibia was very low, the number of cells in the proliferation zones was similar to that in species whose bones grew more rapidly (Tables 11, 111). This must reflect a difference in the frequency of division of the cells in the proliferation zone.

For the animals for which estimates of GR, Nand h were available (Table III), we calculated f'as described by Equations 4 and 5. The results (Table 111) indicate that there was quite a wide range in f between these species, with the frequency of division being particularly low (0.03 divisions per flat cell per day) in the orang utan. These appear to be robust findings but, in our attempts to draw conclusions from the data, it must be remembered that many of the values for growth rates are estimates.

Since a proportion of the cells in the proliferation zone do not divide (Kember, 1983). .f' underestimates the division frequency of the actively dividing cells. The proportion of cells in the proliferation zone that are actively dividing may vary between species and with stage of growth and, in the absence of information about this, the true division frequencies of the dividing cells cannot be deduced here. Studies in which the dividing cell population has been labelled suggest that the proportion of cells which actively divide tends to decrease as the number of flat cells increases (Kember et al., 1990).

Discussion

Interspecies rariation in grmvth plate morphologjx

We have already noted the general similarity in the structures of the growth plates between species. The basic difference between that of the rodents (and others with Type I plates) and those of the artiodactyls (Type I1 plates) and primates (Type 111 plates) is the presence of an inert zone between the active growth plate and the bony epiphysis in the latter orders. The primates showed cartilage columns that were in comparison poorly organized with the typical pattern for artiodactyls. It is possible that this may have been partly due to the immaturity of the animals (young rats do not develop well-defined columns until three weeks of age) but, alternatively, i t may be a feature of slower-growing bones.

There were two noteworthy findings in the histology. The first was the structure of the metaphyseal border in the reindeer where rapid invasion by blood vessels appeared to prevent full maturation of the hypertrophying cartilage cells prior to ossification. The second was the striking depth of the flat cell zone in the springhaas (Table TI). From this we infer that the growth rate of this bone at this stage of growth was probably very rapid.

For most of the species we had specimens of tibia1 growth plates from only one individual so the degree of intraspecies variation could not be assessed. It is possible that there is a sex effect in some cases and effects of age have been described in other species (e.g. by Walker & Kember, 1972).

Interspecies twiation in growth plate kinetics

Among both mammals and birds there is an association between time taken to grow Tand adult mass Ma. The larger the animal the longer it tends to take to grow (although a considerable amount of variation in Tremains after the influence of Ma has been taken into account) and, to be

HISTOLOGY O F M A M M A L I A N GROWTH P L A T E S 551

more specific, between species T tends to increase with M , raised to about the 1 / 3 power (Ricklefs. 1979; Zullinger c t a/., 1984). In birds, as we have pointed out elsewhere (Kirkwood et d.. 1989rr), since the length of the tarsometatarsus bone also tends to increase with the 1/3 power of M;, between species, i t follows that T tends to increase in almost direct proportion with adult tarsometatarsus length. Thus variation in the growth rate (pm/day) of the tarsometatarsus between species of birds is independent of mass (Kirkwood et a/., 1989~).

Likewise, for the mammal species studied here, we found that ATL scaled with mass raised to a power close to 1/3 (0.79+0.033) and this is consistent with previous studies (Alexander e t ul., 1979). Thus we would predict that limb bone growth rate would probably be independent of adult body mass among species of mammals also, and this is supported by our finding here that log GR was not significantly correlated with log Ma ( r = -0.034, IZ =9-data from Table TIT). This being so we might further expect that between-species variation N , h and,fwould also all be independent of mass. These predictions are supported by our findings, in which correlations of N , h and,f'with Ma (all log-transformed) were not significant ( r=0 .33 , n = 17; r = -0.30, n= 17; and r = -0.16, n = 9, respectively-data from Tables I, I1 and 111). As was previously found for birds (Kember et al., 1990),,f'appears thus to be independent of variation in metabolic rate.

Among mammals there is a wide variation in T that is not associated with M:,. The Anthropoidea tend to grow more slowly than individuals in other taxa and the apes are the slowest-growing of all in relation to M , (Case, 1978; Zullinger et a/., 1984; Kirkwood, 1985). In birds (Kember et a/.. 1990), variation in the growth rate of the tarsometatarsus was found to be largely related to N . In contrast, among the sample of mammals studied here there was no significant correlation between GR and N (r=O.26, 1z=9, data from Table 111).

To explore further the impact of variation in N on the dynamics of long bone growth across species, this was examined in relation to the cell production rates of the growth plates. In Fig. I the estimated rates of cell production Pr (calculated as GR/Iz) are plotted against N for all the species for which these data are available (Tables 111 and IV). Across all 18 species, there was a significant correlation and N explained 82':;) of the variation in Pu: for all species ( I Z = 18). Pr= -5.4+0.359 N (r=0.905, P<0.001).

C 0 0

-0

.- c 3 50- $2 a - - 8

1501 100

0 0 . c. A

A A A A

558 J . K K I R K W O O D A N D N . F. K E M B E R

The primates had low rates of P r in relation to N (Fig. I ) . Excluding these made rather little impact on the slope and intercept of the regression but the proportion of the variation then explained by N increased to 93%: for all species except primates ( n = 13), Pr= 5.8+0.339 N

There were also similar and significant relationships of P r and N amongst birds and amongst the non-primate mammals: for birds ( n = 6) , Pr = 18.3+0.306 N ( r =0.956, P < 0.01 ): for non-primate mammals ( n = 7), P r = 13.8 + 0,300 N ( r = 0.840, P < 0.05). However, Pr was not significantly correlated with N when all the mammals were lumped together (r=0.315, N.S., I Z = 13).

The mean lengths of the avian tarsometatarsus and the mammalian tibia are very similar in relation to body mass: 79 mm per kg'?' (this study) and 77 mm per kg' 3h (Kirkwood ef al., 1989c0, respectively. However, the times taken to achieve these lengths differ between the classes. The mean growth rate of the tarsometarsus in birds was found by Kirkwood et ul. ( 1 9 8 9 ~ ) to be 2000 pmlday, whereas the mean growth rate of the tibia found here for mammals was 430 pm/day. This difference is not attributable to a difference in hypertrophic cell diameters as these tend to be greater in mammals (mean 30.7 pm-this study) than in birds (mean 15 pm-Kember et ul., 1990), and there was no correlation between 11 and Pr among the mammals included in this study ( r = -0.35, tz =9). As shown above (Fig. I ) , the tendency for lower flat cell numbers partially explains the lower growth rate of long bones in mammals; however, our results also indicate some marked differences in mean cell division rates between species.

The estimates of f'derived here (Table 111) range from 0.4-0.8 divisions per day in the Type I plates, to 0.2-0.5 divisions per day in the Type I1 plates and 0.03-0.3 in the Type 111 plates. There was no significant correlation between fand PI- (calculated GR/h) for the mammals included in this study ( r = 0.5 I , n = 9) or among the larger dataset ( r = 0.35, n = 18). The mean values offfor birds, primates, and mammals other than primates, were, respectively: 0.39 (S.D. = 0.055, IZ = 6), 0.10 (S.D. = 0.065, n = 5), and 0.42 (S.D. = 0.190, n = 7). The difference in means between the birds and the non-primate mammals was not significant, but the mean for the primates was significantly lower than those for both other groups (cf. birds t=8.03, P<0.001: cf. non-primates t=3.57, P < 0.01 ).

( Y = 0.965, P < 0.00 I ).

Animal

G R PI ./' (pm/ h (cells/ (divisions/ day) (pm) day) N day) Source Bone

Aves Rheu uniericunu Ciconiu uhdimii Thre.skiornis aethiopicus Lophophoru.v impepnus Me1opsiiiucu.s utidulutus Gullus domrsticus

Mammalia Honw supicws Rut tus norregic,u.s Oryto/agtis cunicu1u.r

Tarsometatarsus Tarsometatarsus Tarsometatarsus Tarsometatarsus Tarsometatarsus Tarsometatarsus

Distal femur Tibia Tibia

1680 2000 1740 750

1070 1050

60 700 670

14 120 360 15 133 377 15 116 191 15 50 129 12 90 182 I9 55 146

21 3 50 30 23 70 26 26 55

0.33 0.35 0.40 0.39 0.49 0.38

0.06 0.33 0.47

Kember ct a/. (1990) Kem ber t't ul. ( 1990) Kember et ul. (1990) Kember ef a/ ( 1990) Kember rt ul. ( 1990) Kember et ul. (1990)

Kember (1985 and unpubl.) Kember (1985 and unpubl.) Kember (1985 and unpubl.)

~-

See Table 111 for key to parameters measured

H I S T O L O G Y O F M A M M A L I A N G R O W T H PLATES 5 59

Thus, it appears that the slow growth rates of the tibiae of the primates, especially of the orang utan and human, arid to a lesser extent of the other Anthropoidea, are consequences of low cell division rates rather than of particularly small flat cell populations. The cell division rates in the flat cell zones of man and orang utan appear to be about an order of magnitude lower than those estimated in the rabbit, rodents and artiodactyls and approach two orders of magnitude lower than the division rates observed in the long bones of the domestic fowl ( Kirkwood et ul., 1989h). The slow post-natal growth rate seen in anthropoid primates is one of the characteristic features of the suborder, and it has been suggested that it may have evolved because a prolonged learning period prior to maturity conferred advantage (Napier & Napier, 1967). Regardless ofwhether this slow growth is an adaptation or a constraint, it appears that i t is, in part, a consequence of a reduced cell division rate.

Some nutrient deficiencies (e.g. of vitamin D or phosphorus) can lead to thickening of thc growth plate as a result of failure of maturation and mineralization of cartilage in the hypertrophic zone, and this feature is important in the radiographic diagnosis of rickets (Jubb, Kennedy & Palmer, 1985). The results here indicate that considerable non-pathological variation in growth plate thickness may be expected to occur between species in relation to variation in bone growth rates.

We wish to thank Dr Juliet Jcwell and Miss Daphne Hills of the Natural History Museum for permitting examination of bones and Miss Kathy Thorpe for assistance in collection of the growth platc specimens a t post-mortem.

R E F E R E N C E S

Alexander, R . McN., Jayes. A. S., Maloiy, G. M. 0. & Wathuta, E. M. (1979). Allometry of the limb bones of mammals

Case, T. J . (1978). On the evolution and adaptive significance of postnatal growth rates in the terrestrial vertebrates.

Chang, T. K. (1939). Skeletal growth in Ancon sheep. Growth 1 3 111-267. Glassman, D. M. (1981). The relation of long hone diaphyseal length to chronological age in immature saddle-back

tamarins, Sopinus /i/scirollis. Priniutc.~ 25: 252-36 I , Hearn, J . P. ( 1982). The reproductive physiology ofthe Corninon marmoset C ~ l / / i / / ~ r i . x j u c ~ ~ / ~ u ~ in captivity. I n / . Zoo Yb. 22:

138-143. Hedhammar, A., Wu, F., Krook, L., Schryver, H. F., De La Hunta, A., Whalen, J . P.. Kallfelz. F., Nunez, E. A,. Hintz,

H. F., Sheffy, B. E. & Ryan, A. D. (1973). Overnutrition and skeletal disease: an experimental study in growing Great Dane dogs. Co,nr/l I’eft. 64 (Suppl. 5 ) : 1 1 160.

Jubb, K. V. F., Kennedy, P. C. &Palmer, N. (1985). Pafhu/ug.v Nf’Lio~~.sSti(.u~iiiiiu/.~ 1. (3rd edn). Orlando, San Diego etc.: Academic Press.

Kember, N . F. (1983). Cell kinetics of cartilage. In Cuvtilugr: 139-180. Hall. B. K. (Ed.) . New York: Academic Press. Kember, N. F. ( I 985). Comparative patterns of cell division in epiphyseal cartilage plates in the rabbit. J . Ancrr. 145: I85

190. Kember, N. F. & Kirkwood, J . K. (1987). Cell kinetics and hone growth in birds: a general theory. Cell Ti.s.su~~ Kine,,. 11:

41 7-48 5. Kember, N . F. & Kirkwood. J . K . (199 1 ). Cell kinetics and the study oflongitudinal bone growth. In Fundunientuls of’honc

grondic mt~rhorkhgy t in( io/?~l i~.ufi~n.s: 153- 162. Dixon, A. A., Sarnat, B. G. & Hoyte. D. A. N. (Eds). Boca Raton: CRC Press.

Kember, N . F., Rirkwood. J . K.. Duignan, P. J., Godfrey, D. & Spratt, D. J. (1990). Comparative cell kinetics of avian growth plates. R r s . L’ct. Sci. 4 9 283-288.

Kember, N. F. & Sissons, H. A. (1976). Quantitative histology of the human growth plate. J . Bone Join/ Surg. 58B: 126 435.

Kirkwood, J. K. (1985). Patterns of growth in primates. J . Zoo/., Lond. ( A ) 205 123-136.

from shrews (Sorex) to elephant (Lo.xodontu). J . Zool., Lond. 189: 305-314.

Q. Rer. Bid. 53: 143-28’7.

560 J . K . K I R K W O O D A N D N . F. KEMBER

Kirkwood, J. K., Duignan, P. J., Kember, N. F., Bennett, P. M. & Price, D. J. ( 1 9 8 9 ~ ) . The growth rate of the tarsometatarsus bone in birds. J . Zool., Lond. 217: 403-41 6.

Kirkwood, J. K,, Duignan, P., Spratt, D. M. & Kember, N. F. (19896). Patterns ofcell proliferation and growth rate in limb bones of the domestic fowl Galhts domesticus. Res. I h t . Sti. 47: 139-147.

Kirkwood, J . J . & Webster. A. J. F. (1984). Energy-budget strategies for growth in mammals and birds. .4nim. Prod. 38: 147 155.

Leopold, A. S., Riney, T., McCain, R. & Tevis, L. (1951). The Jawbone deer herd. Calif: Dep. Nut. Res. Dir. Fish Game. Camp Bull. 4 1-39.

Long, J. 0. & Cooper, R. W. (1968). Physical growth and dental eruption in captive-brcd squirrel monkeys. Soimiri sciureus(Leticia, Columbia). In Thesquirrelmonkey: 193-205. Rosenblum. L. A. & Cooper, R. W. (Eds). New York & London: Academic Press.

Macdonald, D. (Ed.) (1984). The encyclc~puedia yf mummuls. London: George Allen & Unwin. Masoud, I . , Shapiro, F., Kent, R. & Moses, A. (1986). A longitudinal study of growth of the New Zealand White Rabbit.

McEwan, E. H. & Wood, A. J . (1966). Growth and development of the barren ground caribou. 1, Heart girth, hind foot

Napier, J . R. & Napier, P. H . ( 1967). A handbouk ofliring primures. London & New York: Academic Press. Phillips, 1. R. (1976). Skeletal development in the foetal and neonatal marmoset (Callithri,~jacchu.s). Luh. ilnimuls 10: 3 17 ~

Ricklefs, R. E. (1979). Adaptation, constraint and compromise in avian postnatal development. Bid . Rer. 5 4 269-290. Riddell, C. (1981). Skeletal deformities in poultry. Adr:. wt. Sci. Comp. Med. 25: 377-310. Ringberg, T. M., White, R. G. , Holleman, D. F. & Luick, J. R. (1981). Body growth and carcass composition of lean

reindeer (Rangifer tarandus tarandus L.) from birth to sexual maturity. Can. J . Zoo/. 59: 10.10-1044. Serafin, J. A. (1982). The influence of diet composition upon growth and development of sandhill cranes. Condor 8 4 417-

434. Snow, C. C. (1 967). Some observations on the growth and development of the baboon. In The huboon in medicwl rrsrurch 2:

187-201. Vagtborg, H. (Ed.). Austin & London: University of Texas Press. Suttie, J. M. (1979). Influences of nutrition on growth and sexual maturation ofcaptive red deer stags. In Proceedings of’the

second international reindeerlcaribou symposium international 17-21 September 1979, Rnros, N o r u q : 341 349. Reimers, E., Gaare, E. & Skjenneberg, S. (Eds). Trondheim: Direktoratet for Vilt og Ferskvannsfisk.

Thurston, M. N., Johnson, D. R. & Kember, N. F. (1985~) . Cell kinetics of growth cartilage of achondroplastic (cn) mice. J . Anut. 140: 425-434.

Thurston, M. N. , Johnson, D. R. & Kember, N. F. (19856). Cell kinetics of growth cartilage in spondylo-metaphyseal chondroplasia (SMC) mice. J . Anut. 140: 435 -445.

Thurston, M. N. & Kember, N . F. (1985). In vitro thymidine labelling in human and porcine growth plates. Cell Ti.r.ruc Kinet. 18: 575-582.

Walker, E. P. (Ed.) (1968). Mammals ofthe norld. (2nd edn). Baltimore: The Johns Hopkins Press. Walker, K. V. R. & Kember, N. F. (1972). Cell kinetics of growth cartilage in the tibia. Cell Tissue Kinet. 5: 401-419. Yamasaki, K . & Itakura, C. (1988). Bone lesions in clinically normal and in lame pigs. J. comp. Puth. 98: 415-432. Zullinger, E. M., Ricklefs, R . E., Redford, K. H. &Mace, G. M. (1984). Fitting sigmoidal equations to mammalian growth

J . Orthop. Res. 4: 22 1-23 I .

length and body weight relationships. Can. J . Zool. 4 4 401-41 1.

333.

curves. J . Mammal. 65: 607-636.

HISTOLOGY OF M A M M A L I A N GROWTll P L A T E S 561

Appendix

Nnt t~s 011 the nietliod~s used to deriw estimutes qf'honr grou.th ratesfbr tlic .spc~cies csuriiirird

The data on growth rates given in Table 111 were derived as follows:

Sqttirrrl nionkcy

If we assume that the growth pattern is the same as that of the saddle-back tamarin (Glassman, 1984), the initial growth rate for the tibia is 330 ,um/day (the mean of males and females). However, the growth rate of the foot in the neonatal squirrel monkey is 330 ,um/day (Long & Cooper, 1968) and since the adult foot and tibia lengths are similar in the species the neonatal tibial growth rate may be about 430 pmiday. In Table 111 the mean of these values, 330 pn/day, was used for calculating cell division rates.

Conznzori nlurnioset

Data for growth for heel to knee are given by Hearn ( 1982). The initial growth rate is I3000 ,urn/ SO days = 340 pm/day and this is the estimate used in Table 111. From 100-1 SO days of age, growth is 7OOO/SO= 140 pm/day, and at 300 days about 40OO/SO or 80 ,um/day. Measurements on marmosets at the Zoological Society of London gave a growth rate for the tibia of 1 I 000 ,urn in 59 days=310 pm/day (days 14 to 66 after birth) and 6000 ,um in 59 days= 100 pmlday (days 165 to 224).

Data given by Phillips ( 1976) suggest an average growth rate of 180 pm/day for long bones over the first month after birth.

Mandrill

The growth of the mandrill tibia from 70 mm at birth to 236 mm in adults is similar to that in the baboon (Pupio spp.) given by Snow (1967). The initial growth rate for the baboon tibia is 370 ,um/day and this is the figure we have used for the mandrill.

Orang utun

Measurements on animals at the London Zoo indicated that the tibia grows about 3 cm in the first year, which indicates an average growth rate of 83 pm/day. The initial growth rate may be higher than this average.

Ch incliilla

The tibial growth rate of this species was estimated by using Equation 7 and taking t ; = 35 days, based on estimates from rat and rabbit data (Walker & Kember, 1973 and Masoud et a/., 1986).

Orange- runiprd agou t i

Equation 2 was used to estimate growth rate and a value of 74 days was assumed for t;. The

562 J. K. K I R K W O O D A N D N . F . K E M B E R

latter was estimated by scaling the t: value for the rat (see above), to take account of the 10-fold greater adult mass ofthe agouti (3 kg) assuming that t l increases with Af; (Kirkwood rt ul.. 1 9 8 9 ~ ) .

Reindeer

McEwan & Wood (1966) gave a growth curve for ?he hind foot of the caribou. Analysis of this curve gives a value of 90 days for the parameter t i . We used this value to calculate growth rate using Equation 7 . This yielded an estimate of 1000 pm/day for the neonatal growth rate. This estimate is supported by the measurements of Ringberg r t a/. (1981) of reindeer femurs, which indicated a growth rate of 1700 pm/day for neonates.

Blackbuck

Data for the hind food of mule deer Ocfocoiltws heniiotzu.v (Leopold ef ul., 195 1 ) give a f: value of I80 days while Suttie's ( 1979) data for the hind foot of red deer Cerrw elup/ius gives a t; of I50 days. We assumed f ; = 150 days for the blackbuck and, using Equation 2. estimated the tibial growth rate to be 2.b pm/day.

hf 0 z4fior1

Chang (1949) presented data on the growth of tibiae in domestic sheep. We have assumed that the tibial growth rate of the domestic sheep is a good estimate of that of the mouflon.