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The response of 0steoblasts to parathyroid hormone (PTH 1–34) in vitro
Metab.
Bone
Dis. & Rel. Res. 2, 335-338
Metabolic Bone D&ease h Related Reeeamh @ by S.N.P.M.D. (Park
of Osteeblasts to Pa
The S.J. JONES, Department Address
(1981)
A. BOYDE
of Anatomy,
and
University
for correspondence
H
I.M. SHAPIRO College
and reprints
Street, London WClE 6BT, England..
London,
Gower
Street,
London
: Dr. S.J. Jones Department
WClE
6BT, England.
of Anatomy,
University
College
London,
Gower
Results and Discussion
Abstract
Morphological study of osteoblasts of the endocranial aspect of the rat cahrartum showed that they responded to the l-34 fragment of parathyroid hormone in exactly the same way as to parathyroid extract : thus, the ooteoblasts become elongated and aligned parallel to one another and to the collagen in the most superflclal layer of the bone matrix. Fluctuations in PTH levels may conceivably relate to the lamellar texture of mature bone. Key Words : Osteoblasts tation - 1-34 PTH.
- Bone - Collagen - Orien-
Introduction We have previously
reported
the effect of parathyroid
(PTE, Lilly) on rat calvarial osteoblasts maintained in vitro as an exposed monolayer on their original bone surface (Jones and Boyde, 1976a ; 1976b ; Jones and Ness, 1977) or after migration on other substrates such as glass (Jones and Boyde, 1977a) or dental tissues (Jones and Boyde, 1979). In all cases, the osteoblasts elongated and aligned parallel to one another in domains under its influence. It was assumed that the effect of the PTE on the osteoblasts was due to parathyroid hormone, PTH, but this was not proven (Jones and Boyde, 1977b). We have repeated these experiments using the l-34 peptide fragment of parathyroid hormone, since this has been found to contain the structural requirements for biological activity (Parsons et al., 1973 ; Tregear et al., 1973) and does affect the shape of osteoblasts (Gaillard et al., 1979). hormone
1981)
extract
Osteoblasts cultured on bone in the control medium exhibited the changes previously reported, with dorsal ruffling, loss of elongation and order (Figure 1). The osteoblasts in the medium enriched with 0.5U/ ml PTH l-34 were elongated, and aligned parallel to one another in domains over all the bone surface (Figure 2). The cultures looked identical to ones where PTE 0.5U/ml had been used (Figure 3). It is likely, therefore, that the effect of PTE on osteoblasts in culture is due to the parathyroid hormone it contains, rather than some unknown component. In some areas, shrinkage of the osteoblasts had occurred to a greater extent than shrinkage of their collagenous substrate, leaving gaps between the cells. This enabled us to see that the alignment of the elongated PTH-treated cells matched that of the collagen fibres in the osteoid surface. This observation was confirmed where occasional cells had been lost during the preparation of the specimens. It has been recognised for many years that very large doses of parathyroid extract change the shape of osteoblasts in vitro and in viva (Heller et al., 1950 ; Gaillard, 1954), and it has recently been confirmed that parathyroid hormone is the responsible agent in producing spindle-shaped osteoblasts (Gaillard et al., 1979), p_erhaps through causing an influx of calcium into the cells. The parallel alignment of these cells in domains (Jones and Ness, 1977 and this study) which initially match the organization of the underlying collagen (Jones et al., 1975) may have some relevance to the unsolved problem of how the texture of lamellar bone is formed. Small fluctuations in the level of bone hormones in the serum may cause a reorganization the cells in domains with
Material and Methods Half calvaria were dissected from five 3-day old Albino Wistar rats and the overlying tissues dissected away so that a single layer of cells remained on the endocranial aspect of the bone. They were cultured in Dulbecco’s 1889 medium with loo/o added fetal calf serum either with, or without, added parathyroid hormone, at a concentration of O.!%J/ml, in the form of its active region PTH l-34. After 24 hours all organ cultures were fixed in 3% glutaraldehyde in 0.15M cacodylate buffer, washed in water, quenched in liquid CCIzF2 at its freezing temperature (--155X), transferred to liquid nitrogen and freeze-dried at -65°C. Specimens were mounted with the endocranial aspect of the bone uppermost, coated in vacua with gold and examined in a Stereoscan S4-10 scanning electron microscope operated at IOkV.
of the
arrangement
of
a movement across the formative surface and a consequent change in collagen orientation of the bone matrix produced. This would only produce a textural change when the rate of apposition was slow and fluctuated but not when fast and more constant. There are cyclical patterns in osteoblastic activity and the appositional rate of bone matrix which seem to coincide with osteoclastic intensity (Simmons and Nichols, 1966). Simmons (1979) presented a circadian timeframe for calcium metabolism based on known daily fluctuations in the secretion of parathyroid hormone in adult humans (Jubiz et al., 1972) and in rats. We would expect that such a fluctuation in physiological PTH levels would have its greatest
S.J. Jones
et al. : Osteoblasts and PTH in vitro
Figs 1, 2, 3. For explanation see text.
FIGURE 1 A
b
FIGURE2
morphological effect on the cells and matrix when the appositional rate was relatively low and the cells were in domains, as in lamellar bone formation, and to have the least, or a negligible, effect on orientation and order when the incremental growth rate of the bone was high and the cell height at its greatest, as in the formation of woven bone.
Acknowledgements : We are indebted to Dr. John Parsons, N.I.M.R. London, for kindly supplying us with the PTH l-34. Professor Irving Shapiro held a Fogarty international Fellowship for the duration of this work at University College London.
References Gaillard P.J. : In vitro experiments on the influence of parathyroid gland tissue on desmal bone. Excerpta Med. 8 : 401-410, 1954. Gaillard P.J., Herrmann-Erlee M.P.M., Hekkelman J.W., Burger E.H. and Nijweide P.J. : Skeletal tissue In culture. Cl/n. Orthop. Rel. Res. 142 : 198-214, 1979. Heller M., McLean F.C. and Bloom W. : Cellular transformations in mammalian bones induced by parathyroid extract. Am. J. Anat. 87 : 315339, 1950. Jones S.J. and Boyde A. : Morphological changes of osteoblasts in vitro. Cell Tiss. Res. 188 : 101-107. 1976a. Jones S.J. and Boyde A. : Experimental study of changes in osteoblastic shape induced by caloitonin and parathyroid extract in an organ culture system. Cell Tiss. Res. 169 : 449-465, 1976b.
S.J. Jones
et al.
: Osteoblasts
and PTH in vitro
337
4
of osteoblasts. Cell S.J. and Boyde A. : The migration Res. 184 : 179-193, 1977a. Jones S.J. and Boyde A. : Scanning electron microscopy Of bone cells in culture. Excerpta Med. Int. Congress Series No. 421. Endocrinology of calcium metabolism. Proc. Sixth Parathyroid Conf. Vancouver. pp. 97-104, 1977b. Jones S.J. and Bovde A. : Colonization of various natural substrates by osteoblasts in vitro. Scanning Electron Microscopv:l979/11 SEM Inc. AMF O’Hare, Ill. USA Eds. 0. Johari and R.P. Becker pp. 529538, 1979. and oollaJones S.J., Boyde A. and Pawley J.B. : Osteoblasts gen orientation. Cell Tiss. Res. 159 : 73-80, 1975. Jones S.J. and Ness A.R. : A study of the arrangement Of osteoblasts of rat calvarium cultured in medium with, or without, added parathyroid extract. J. Cell. Sci. 25 : 247283, 1977. J.M., Reiss E. and Tyler F.H. : Circadian Jubiz W., Canterbury rhythm in serum parathyroid hormone concentration in : correlation with serum calcium, phoshuman subjects phate, albumin and growth hormone levels. J. C/in. Invest. 51 : 2040-2046, 1972. Jones
Tiss.
FIGURE
3
Parsons J.A., Reit B. and Robinson C.J. : A bioassay for paraEndocrinology 92 : 454-462, thyroid hormone using chicks 1973. design and the implicaion of Simmons D.J. : Experimental research : an circadian skeletal rhythmicity. In : Skeletal experimental approach. Eds. D.J. Simmons and A.S. Kunin. Academic Press, New York, pp. 567-585. 1979. Simmons D.J. and Nichols G. : Diurnal periodicity in the
metabolic activity of bone tissue. Am. J. fbysiol. 210 : 410-418, 1986. Tregear G.W., van Rietschoten J., Greene E., Keutmann H.T., Niall H.D., Reit B., Parsons J.A. and Potts Jr. J.T. : Bovine parathyroid hormone : minimum chain length of synthetic peptide required for biological actfvity. Endocrinology S3 : 1349-1353,
1973.
Received Revised Accepted
: March 7, 1980. : May 14, 7980. : May 27, 1980.
RESUME L’Otude morphologique de I’endocrine du calvarium de rat a montr6 une Gponse au fragment 1-34 de la parathormone semblable ii celle obtewe avec I’extrait parathyroidien : les ostkoblastes s’allongent paralltiement I’un in I’autre et au collag6ne de la couche la plus superficielle de la matrice osseuse. Des fluctuations du niveau de la PTH peuvent done Cventuellemeut Otre en rapport avec I’organisatiun lamellaire de 1’0s mature.
Bone
Dis. & Rel. Res. 2, 335-338
Metabolic Bone D&ease h Related Reeeamh @ by S.N.P.M.D. (Park
of Osteeblasts to Pa
The S.J. JONES, Department Address
(1981)
A. BOYDE
of Anatomy,
and
University
for correspondence
H
I.M. SHAPIRO College
and reprints
Street, London WClE 6BT, England..
London,
Gower
Street,
London
: Dr. S.J. Jones Department
WClE
6BT, England.
of Anatomy,
University
College
London,
Gower
Results and Discussion
Abstract
Morphological study of osteoblasts of the endocranial aspect of the rat cahrartum showed that they responded to the l-34 fragment of parathyroid hormone in exactly the same way as to parathyroid extract : thus, the ooteoblasts become elongated and aligned parallel to one another and to the collagen in the most superflclal layer of the bone matrix. Fluctuations in PTH levels may conceivably relate to the lamellar texture of mature bone. Key Words : Osteoblasts tation - 1-34 PTH.
- Bone - Collagen - Orien-
Introduction We have previously
reported
the effect of parathyroid
(PTE, Lilly) on rat calvarial osteoblasts maintained in vitro as an exposed monolayer on their original bone surface (Jones and Boyde, 1976a ; 1976b ; Jones and Ness, 1977) or after migration on other substrates such as glass (Jones and Boyde, 1977a) or dental tissues (Jones and Boyde, 1979). In all cases, the osteoblasts elongated and aligned parallel to one another in domains under its influence. It was assumed that the effect of the PTE on the osteoblasts was due to parathyroid hormone, PTH, but this was not proven (Jones and Boyde, 1977b). We have repeated these experiments using the l-34 peptide fragment of parathyroid hormone, since this has been found to contain the structural requirements for biological activity (Parsons et al., 1973 ; Tregear et al., 1973) and does affect the shape of osteoblasts (Gaillard et al., 1979). hormone
1981)
extract
Osteoblasts cultured on bone in the control medium exhibited the changes previously reported, with dorsal ruffling, loss of elongation and order (Figure 1). The osteoblasts in the medium enriched with 0.5U/ ml PTH l-34 were elongated, and aligned parallel to one another in domains over all the bone surface (Figure 2). The cultures looked identical to ones where PTE 0.5U/ml had been used (Figure 3). It is likely, therefore, that the effect of PTE on osteoblasts in culture is due to the parathyroid hormone it contains, rather than some unknown component. In some areas, shrinkage of the osteoblasts had occurred to a greater extent than shrinkage of their collagenous substrate, leaving gaps between the cells. This enabled us to see that the alignment of the elongated PTH-treated cells matched that of the collagen fibres in the osteoid surface. This observation was confirmed where occasional cells had been lost during the preparation of the specimens. It has been recognised for many years that very large doses of parathyroid extract change the shape of osteoblasts in vitro and in viva (Heller et al., 1950 ; Gaillard, 1954), and it has recently been confirmed that parathyroid hormone is the responsible agent in producing spindle-shaped osteoblasts (Gaillard et al., 1979), p_erhaps through causing an influx of calcium into the cells. The parallel alignment of these cells in domains (Jones and Ness, 1977 and this study) which initially match the organization of the underlying collagen (Jones et al., 1975) may have some relevance to the unsolved problem of how the texture of lamellar bone is formed. Small fluctuations in the level of bone hormones in the serum may cause a reorganization the cells in domains with
Material and Methods Half calvaria were dissected from five 3-day old Albino Wistar rats and the overlying tissues dissected away so that a single layer of cells remained on the endocranial aspect of the bone. They were cultured in Dulbecco’s 1889 medium with loo/o added fetal calf serum either with, or without, added parathyroid hormone, at a concentration of O.!%J/ml, in the form of its active region PTH l-34. After 24 hours all organ cultures were fixed in 3% glutaraldehyde in 0.15M cacodylate buffer, washed in water, quenched in liquid CCIzF2 at its freezing temperature (--155X), transferred to liquid nitrogen and freeze-dried at -65°C. Specimens were mounted with the endocranial aspect of the bone uppermost, coated in vacua with gold and examined in a Stereoscan S4-10 scanning electron microscope operated at IOkV.
of the
arrangement
of
a movement across the formative surface and a consequent change in collagen orientation of the bone matrix produced. This would only produce a textural change when the rate of apposition was slow and fluctuated but not when fast and more constant. There are cyclical patterns in osteoblastic activity and the appositional rate of bone matrix which seem to coincide with osteoclastic intensity (Simmons and Nichols, 1966). Simmons (1979) presented a circadian timeframe for calcium metabolism based on known daily fluctuations in the secretion of parathyroid hormone in adult humans (Jubiz et al., 1972) and in rats. We would expect that such a fluctuation in physiological PTH levels would have its greatest
S.J. Jones
et al. : Osteoblasts and PTH in vitro
Figs 1, 2, 3. For explanation see text.
FIGURE 1 A
b
FIGURE2
morphological effect on the cells and matrix when the appositional rate was relatively low and the cells were in domains, as in lamellar bone formation, and to have the least, or a negligible, effect on orientation and order when the incremental growth rate of the bone was high and the cell height at its greatest, as in the formation of woven bone.
Acknowledgements : We are indebted to Dr. John Parsons, N.I.M.R. London, for kindly supplying us with the PTH l-34. Professor Irving Shapiro held a Fogarty international Fellowship for the duration of this work at University College London.
References Gaillard P.J. : In vitro experiments on the influence of parathyroid gland tissue on desmal bone. Excerpta Med. 8 : 401-410, 1954. Gaillard P.J., Herrmann-Erlee M.P.M., Hekkelman J.W., Burger E.H. and Nijweide P.J. : Skeletal tissue In culture. Cl/n. Orthop. Rel. Res. 142 : 198-214, 1979. Heller M., McLean F.C. and Bloom W. : Cellular transformations in mammalian bones induced by parathyroid extract. Am. J. Anat. 87 : 315339, 1950. Jones S.J. and Boyde A. : Morphological changes of osteoblasts in vitro. Cell Tiss. Res. 188 : 101-107. 1976a. Jones S.J. and Boyde A. : Experimental study of changes in osteoblastic shape induced by caloitonin and parathyroid extract in an organ culture system. Cell Tiss. Res. 169 : 449-465, 1976b.
S.J. Jones
et al.
: Osteoblasts
and PTH in vitro
337
4
of osteoblasts. Cell S.J. and Boyde A. : The migration Res. 184 : 179-193, 1977a. Jones S.J. and Boyde A. : Scanning electron microscopy Of bone cells in culture. Excerpta Med. Int. Congress Series No. 421. Endocrinology of calcium metabolism. Proc. Sixth Parathyroid Conf. Vancouver. pp. 97-104, 1977b. Jones S.J. and Bovde A. : Colonization of various natural substrates by osteoblasts in vitro. Scanning Electron Microscopv:l979/11 SEM Inc. AMF O’Hare, Ill. USA Eds. 0. Johari and R.P. Becker pp. 529538, 1979. and oollaJones S.J., Boyde A. and Pawley J.B. : Osteoblasts gen orientation. Cell Tiss. Res. 159 : 73-80, 1975. Jones S.J. and Ness A.R. : A study of the arrangement Of osteoblasts of rat calvarium cultured in medium with, or without, added parathyroid extract. J. Cell. Sci. 25 : 247283, 1977. J.M., Reiss E. and Tyler F.H. : Circadian Jubiz W., Canterbury rhythm in serum parathyroid hormone concentration in : correlation with serum calcium, phoshuman subjects phate, albumin and growth hormone levels. J. C/in. Invest. 51 : 2040-2046, 1972. Jones
Tiss.
FIGURE
3
Parsons J.A., Reit B. and Robinson C.J. : A bioassay for paraEndocrinology 92 : 454-462, thyroid hormone using chicks 1973. design and the implicaion of Simmons D.J. : Experimental research : an circadian skeletal rhythmicity. In : Skeletal experimental approach. Eds. D.J. Simmons and A.S. Kunin. Academic Press, New York, pp. 567-585. 1979. Simmons D.J. and Nichols G. : Diurnal periodicity in the
metabolic activity of bone tissue. Am. J. fbysiol. 210 : 410-418, 1986. Tregear G.W., van Rietschoten J., Greene E., Keutmann H.T., Niall H.D., Reit B., Parsons J.A. and Potts Jr. J.T. : Bovine parathyroid hormone : minimum chain length of synthetic peptide required for biological actfvity. Endocrinology S3 : 1349-1353,
1973.
Received Revised Accepted
: March 7, 1980. : May 14, 7980. : May 27, 1980.
RESUME L’Otude morphologique de I’endocrine du calvarium de rat a montr6 une Gponse au fragment 1-34 de la parathormone semblable ii celle obtewe avec I’extrait parathyroidien : les ostkoblastes s’allongent paralltiement I’un in I’autre et au collag6ne de la couche la plus superficielle de la matrice osseuse. Des fluctuations du niveau de la PTH peuvent done Cventuellemeut Otre en rapport avec I’organisatiun lamellaire de 1’0s mature.