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Journal of the Anatomical Society of India

Effect of Alpha Tocopherol on the Growth Plate in Albino rats

Author(s): Abhaya, A., Khatri, K., Pradhan, S. and Prakash, R.

Vol. 52, No. 1 (2003-01 - 2003-12)

Department of Anatomy, University College of Medical Sciences & G.T.B. Hospital, Shahdra, Delhi. INDIA.

Abstract

Alpha tocopherol is the most biologically active stereoisomer of vitamin E and recent investigations suggest thatvitamin E is important for bone mineralization and normal endochondral ossification. Thirty Albino rats (8-20 days) included in the study weredivided into three groups. Experimental group C was given tocopherol (90mg/Kg body weight) daily, orally for 12 days. Group B was treated with equal amount of arachis oil (vehicle) daily for 12 days while group A animals were kept untreated. On 20th day animals were sacrificedand 6m thick longitudinal sections of decalcified radius were stained for light microscopy.

Growth plate was differentiated into resting, proliferative, hypertrophic zone I and hypertrophic zone II. Morphometric observations were recorded at the central and lateral regions of the growth plate. The height (vertical thickness) of growth plate showed a statisticallysignificant increase in the central region (P = 0.000). The zonal thickness of hypertrophic zone I also increased in central region (P = 0.004) while the zonal thickness of hypertrophic zone II showed a significant increase in the lateral region (P=0.040). Cell population of hypertrophic zone I increased significantly in central (P=0.000) and lateral region (P =0.002) while the cell population of hypertrophic zone II decreasedsignificantly in central (P=0.000) as well as lateral region (P=0.011). The number of cell columns decreased significantly (P= 0.028) inhypertrophic zone II. Intense matrix metachromasia was seen in vitamin E treated animals. These results suggest that vitamin E promotes mineral apposition, multiplication and maturation of chondrocytes and thereby may enhance longitudinal bone growth.

Key words: Alpha Tocopherol, Growth Plate, Radius, Rat

Introduction:

Vitamin E is the naturally occurring antioxidant biosynthesized in plants and found abundantly in vegetable oils, having eight different stereoisomers of which alpha tocopherol is known to have greatest biological activity (Meydani, 1995; Murray et al, 2000) Vitamin E and its vitaminers are the only lipid soluble antioxidants found in plasma and cell membrane where they function as free radical scavangers to inhibit lipid peroxidation of membrane lipids, thus serving an important role in maintaining cell integrity and function (Parker, 1989). Although vitamin E deficiency rarely occurs in children or adults but the premature and low birth weight infants are highly susceptible because placental transfer is poor and the infants have limited adipose tissue where much of the vitamin is normally stored (Winick, 1987).

The longitudinal bone growth results from a sequence of cellular events occurring in the growth plate and metaphysis beginning with proliferation of chondrocytes and progressing through chondrocytes maturation, matrix synthesis, chondrocytes hypertrophy, matrix mineralization and vascular invasion of hypertrophic zone II (Sissons, 1955; Kember, 1960; Rigal, 1962; Walker and Kember, 1972; Hert, 1972; Kember and Sissons, 1976; Seinsheimer and Slidge, 1981; Buckwalter et al, 1986; Hunziker et al, 1987). The effect of various dietary nutrients, trace elements, polyunsaturated fatty acids, lipids and hormones on cartilage, bone growth and mineralization have been studied extensively (Smith and Mclean, 1936; Becks et al, 1946; Simmons and Kunin, 1967; Mirsky and Silbermann, 1985, Deardan et al, 1986,

Leboy et al, 1989, Pacifici et al, 1991). Kinetic data on bone mineralization and formation and the thickness of mineralized zone in growth cartilage suggests that vitamin E is important for bone formation, mineralization and endochondral ossification (Ebina et al, 1991, Gallop et al, 1993, Xu et al, 1995). However there is a paucity of literature regarding the effect of vitamin E on the different zones of growth plate, therefore the present study was undertaken to evaluate the effect of vitamin E in central and lateral regions of various zones of the growth plate at the distal end of radius in 20 day old albino rat.

Material And Methods:

The inbred newborn Albino rats of Wistar strain procured from the animal house of University College of Medical Sciences, Shahdra, Delhi were kept undisturbed with their mothers, provided with food and water ad-libitum till 7th day after birth. The temperature and light and dark cycle of twelve hours was maintained. Thirty newborn rats were selected at random on 8th day and divided into control and experimental groups. The control group was further divided into normal or untreated control (Group A) and vehicle treated control (Group B). The experimental (group C) animals were given 90mg/kg body weight of alpha tocopherol (vitamin E) daily, orally for twelve days; the vehicle treated animals were given equal amount of arachis oil while the normal animals were kept untreated. On 20th day, animals were anaesthetized with ether and sacrificed by decapitation. Radius was dissected out after removal of all soft tissues, fixed in 10% formal saline and decalcified using neutral EDTA. The end point of decalcification was assessed by chemical method using ammonia solution. 6m thick longitudinal paraffin sections were stained with Haematoxylin and Eosin, Masson's trichrome and Toludine blue.

The growth plate was divided into several zones clearly demarcated on the basis of their cell morphology and arrangement of cells. From epiphysis to diaphysis these successively merging zones were distinguished as the resting, proliferative and hypertrophic zone (Fig.1). The hypertrophic zone was further divided into hypertrophic zone 1 (HT-1) and hypertrophic zone II (HT-II) on the basis of their varied cell morphology (Fig.2) as described by Brighten (1984). The central region was in the middle of the growth plate limited by secondary ossification centre of the epiphysis and metaphysis while the lateral region was towards the margin of growth plate near the perichondrial ring and contiguous to the marginal germinative zone as distinguished by Flores and Baeza (1990).

The following parameters were recorded.

  1. Width (horizontal thickness) of growth plate-at three different levels (proximal, middle, distal) excluding periosteum.
  2. Height (vertical thickness) of growth plate- was the sum of heights of proliferative, HT-I and HT-II zones, measured in central and lateral region.
  3. Individual zonal thickness of proliferative, HT-1 and HT-II zones, in both central and lateral region.
  4. Mean cell population of proliferative, HT-I and HT-II zones in both central and lateral region.
  5. Number of cell columns in HT-I and HT-II zones in both central and lateral region. All the linear measurements were recorded in the longitudinal direction of cell columns by Abercrombie method (Abercrombie, 1946) and the results obtained were statistically evaluated.

Observations:

All the rats included in the study survived well and gained weight steadily ranging from 1.5 - 2.5gm daily. The average total length of radius from its proximal end to the lowest margin of styloid process at the distal end was 13.33mm, 13.33mm and 13.53mm in group A,B and C respectively.

The growth plate at the distal end of radius was seen as a continuous plate surrounded by a dense cell layer of perichondrium, exhibiting all cell layers maintaining the columnar arrangement of chondrocytes in control as well as vitamin E treated animals. The chondrocytes in the resting zone were small, rounded or spindle shaped, seen both within and without lacuna and distributed singly, double or in small groups. In the proliferative zone the discoidal or cuneiform chondrocytes lying in lacunae were slightly increased in size and arranged in longitudinal columns. In the central region, the longitudinal columns of cells were more straight lying close to each other and comprised of more number of cells in each column as compared to lateral region where the columns were disposed obliquely. The margins of lacunae were demarcated by slightly deeper staining of matrix. In HT-I there was a gradual increase in the size of lacunae around the chondrocytes arranged in columns. The cell outlines of the chondrocytes were slightly distorted and nuclei became swollen or compressed at places. The HT-I was followed by HT-II where the lacunae had greatly increased in size with irregular margins and acquired a round to polygonal shape enclosing a chondrocytes, which had reduced in size, cytoplasm became vacuolated and nuclei became pyknotic. Some lacunae showed complete degeneration of chondrocytes. The lacunae were separated from each other by a thin septa and from other columns of hypertrophic cells by a thin bar of matrix. The matrix appeared homogenous throughout the growth plate except near the epiphyseal end and perichondrium where it stained more deeply. Vitamin E treated animals showed intense matrix metachromasia with Toludine blue.

The width (horizontal thickness) of the growth plate was greater than the height (vertical thickness) and was narrower towards the diaphysis and increased steadily distally up to its epiphyseal end. The width at its proximal, middle and distal end was decreased at all levels in group C animals in comparison to group A and B but it was not found to be statistically significant (Table 1)

Table No. I: Showing width of growth plate at distal, middle and proximal ends.

Group Width (horizontal thickness) of growth plate (m)
Distal Middle Proximal
A 1791.53 ± 87.11 1621.47 + 43.55 1581.00 ± 54.75
B 1761.33 ± 167.13 1615.13 + 134.81 1565.33 ± 127.01
C 1731.55 ± 137.37 1574.19 + 103.17 1545.55 ± 115.07

In group C animals a statistically significant increase in the height of growth plate in the central region (P = 0.000) and an insignificant marginal decrease in the height of the lateral region was observed but it was noticed that the lateral region was always higher than the central region in control as well as experimental animals (Table. II).

Table No. II: Showing height of growth plate in the central and lateral region

Group Height (vertical thickness) of growth plate (m)
Central Lateral
A 474.67 ± 52.29 523.73 ± 30.14
B 477.07 ± 35.17 508.27 ± 29.94
C 492.37 ± 53.70* 515.23 ± 49.21

*Statistically significant

The individual zonal thickness of proliferative zone in central region was increased marginally while in the lateral region it showed a marginal decrease in group C animals (Table III). The zonal thickness of HT-I in the central region was increased (P =0.004) in group C animals while it decreased in lateral region (Table III). The zonal thickness of HTII was increased in both central and lateral regions in group C animals but it was found significant (P =0.040) only in the lateral region (Table III).

A statistically insignificant increase was observed in the cell population of proliferative zone in central as well as lateral region of the growth plate in group C animals (Table IV). The number of cell columns in this zone was not recorded due to oblique course of longitudinal columns of chondrocytes. In group C animals, the cell population of HT-I was increased significantly in central (P= 0.000) as well as lateral region (P = 0.002) while the number of cell columns decreased both in central and lateral regions (Table V). In HT-II the cell population had reduced significantly in central (P = 0.000) and lateral region (P = 0.011) of the growth plate in group C in comparison to group A & B animals. Although the number of cell columns in HT-II of group C animals were reduced in the central as well as lateral region but it was found to be statistically significant only in the central (P =0.028) region (Table VI).

Table No. III: Showing individual zonal thickness of proliferative, hypertrophic Zone I (HT-1) & hypertrophic Zone II (HT-II) in central & lateral regions of the growth plate

Group Individual zonal thickness (m)
Central Lateral
Proliferative HT-I HT-II Proliferative HT-I HT-II
A 199.27 93.00 182.20 265.07 105.60 153.00
  ± 42.22 ±13.52 ±23.54 ± 41.61 ± 26.56 ± 24.42
B 190.73 94.33 192.07 242.60 99.67 165.93
  ± 52.98 ±15.52 ± 23.71 ± 41.93 ± 13.35 ± 22.46
C 203.64 96.99 191.29 245.72 101.11 167.40
  ± 46.68 ± 22.32* ± 0.43 ± 0.69 ±2.42 ± 29.48*

*Statistically significant

Table No. IV: Showing the cell population in proliferative zone in central and lateral region of growth plate.

Group Cell Population (Proliferative Zone)
Number of cells (central) Number of cells (lateral)
A 59.00 ±3.58 61.17 ± 2.99
B 61.50 ± 3.78 63.50 ± 3.62
C 62.93 ± 4.21 65.90 ± 4.54

Table No.-V Showing the cell population and number of cell columns in hypertrophic zone I (HT-I) in central and lateral region of growth plate.

Group Cell Population and Number of cell columns (HT-I)
Central Lateral
  No. of cells No. of columns No. of cells No. of columns
A 34.83 ± 4.12 6.50 ± 1.05 39.33 ± 5.13 7.00 ± 1.10
B 37.00 ± 2.83 5.67 ± 0.52 42.50 ± 3.45 5.83 ± 0.75
C 44.17 ± 3.63* 6.30 ± 0.65 48.60 ± 4.99* 6.97 ± 0.67

*Statistically significant

Table No. VI: Showing the cell population and number of cell columns in hypertrophic zone II (HT-II)in central and lateral region of growth plate.

Cell Population and Number of cell columns (HT-II)
Group Central Lateral
  No. of cells No. of columns No. of cells No. of columns
A 23.67 ± 3.39 5.17 ± 1.17 23.83 ± 3.06 5.00 ± 1.26
B 22.50 ± 1.38 4.33 ± 0.52 21.50 ± 1.52 4.33 ± 0.52
C 18.37 ± 1.35* 3.97 ± 0.41* 19.93 ± 1.55* 3.90 ± 0.31

*Statistically significant

Discussion:

The growth plate at the distal end of radius in albino rat was seen as a continuous plate maintaining the columnar arrangement of chondrocytes and surrounded by a perichondrial ring as described earlier by Dawson (1925) and Dodds (1930).

The central and lateral regions of growth plate are quantitatively different as reported by Flores and Baeza (1990) therefore it is important to consider these two regions separately as has been done in the present study. In the central region all the three zones of growth plate have contributed to the significant increase in the height (vertical thickness) but the contribution of HT-I was maximum (Table II,III) while in the lateral region, a marginal decrease in the total height of growth plate and a significant increase in HT-II may be due to decreased cartilage resorption and phagocytic activity at the metaphyseal side (Table II, III). These observations support the views of Matsumoto et al. (1991) and Xu et al. (1995) regarding the increase in the thickness of growth plate and HT II in chicks treated with vitamin E but in their study the regional differences of zones of growth plate were not taken into consideration, probably the mean combination of both regions may co-relate with their observations.

During the process of endochondral bone formation the proliferating chondrocytes give rise to hypertrophic chondrocytes, which then deposit a mineralized matrix to form calcified cartilage. A statistically significant increase in the cell population of HT-I in both central as well as lateral region of growth plate (Table V) clearly indicates that vitamin E had promoted the multiplication of chondrocytes and laying down of matrix rich in proteoglycans evident by the intense matrix metachromasia. The decrease in cell population of HT-II and decrease in number of cell columns of HT-I and HT-II in both regions of growth plate signifies that the size of lacuna enclosing the degenerating chondrocyte had increased tremendously with a simultaneous decrease in proportion of matrix per cellular profile (Table V, VI) promoting their rapid metabolic turnover (Dingle and Dingle, 1980; Handley et al, 1980; Thompson and Robinson, 1981).

As the longitudinal bone growth is equal to the rate of production of new cells per cartilage columns multiplied by the average size of hypertrophic cell (Sissons 1955) it appeared from the present study that vitamin E had promoted mineral apposition, maturation and multiplication of chondrocytes, chondrocytes hypertrophy, their rapid metabolic turnover and thereby enhancing longitudinal bone growth by increasing the trabecular bone volume and osteoid volume (Ebina et al. 1991). However the mechanism by which it mediates its effect on bone formation remains unclear.

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Figure 1: The photomicrograph of growth plate divided into resting (R), proliferative (P), and hypertrophic zone (HT). H and E stain, X 100.

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Figure 2: The photomicrograph of growth plate showing hypertrophic zone-1 (HT-I) in which the chondrocytes (CH) lying in lacuna (LC) increased horizontally, cell outlines slightly distorted, nuclei swollen and compressed and hypertrophic zone-II (HT-II) in which the greatly enlarged lacuna with irregular margins acquired round to polygonal shape enclosing the chondrocyte which had reduced in size, cytoplasm vacuolated and nuclei pyknotic. Some lacuna showed complete degeneration of chondrocyte. H and E stain, X400.

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