Loading-related increases in prostaglandin production in cores of adult canine cancellous bone in vitro: A role for prostacyclin in adaptive bone remodeling?

JOURNAL OF BONE AND MINERAL RESEARCH Volume 6, Number 12, 1991 Mary Ann Liebert. Inc.. Publishers

Loading-Related Increases in Prostaglandin Production in Cores of Adult Canine Cancellous Bone In Vitro:

A Role for Prostacyclin in Adaptive Bone Remodeling?

SIMON C.F. RAWLINSON,' ALICIA J. EL-HAJ,' SARAH L. MINTER,' IGNATIUS A. TAVARES,' ALAN BENNETT,3 and LANCE E. LANYON'

ABSTRACT

Cyclic mechanical loading sufficient to engender strains of physiologic magnitude applied to recently excised canine cancellous bone cores in vitro increased the release of prostaglandin E (PGE) and prostacyclin (PGI,, measured as its breakdown product 6-keto-PGF1cr), during a 15 minute loading period in which PG levels were measured in perfusing medium at 5 minute intervals. Peak production occurred in the 0-5 minute sample. Mean levels preload compared to during load were PGE, 2.66 and 3.67 ng/ml (p < 0.002); and 6-keto- PGF,a, 543 and 868 pg/ml (p < 0.007). The elevated levels then declined to preload levels during the loading period. However, the 5-10 minute but not the 10-15 minute samples still contained levels greater than preload values. A second 15 minute period of load, 1 h following the end of the first, produced smaller increases in the levels of release that were statistically significant only for the first 0-5 minute sample during load (preload compared to load mean values, PGE, 1.09-1.66 ng/ml, p < 0.02; 6-keto-PGF1cr, 401-558 pg/ml, p < 0.04). Immunolocalization revealed PGE and 6-keto-PGF1a in lining cells and 6-keto-PGFla but not PGE in osteocytes. Addition to the medium of 1 pM PGE,, approximating the concentration produced by loading, had no significant effect on the specific activity of the extractable RNA fraction labeled with [3H]uridine, whereas 1 pM PGI, produced an increase similar to that seen previously with loading. These results suggest that both PGE and PGI, may be involved in loading-related adaptive bone modeling and remodeling. The presence of the PGI, breakdown product in osteocytes and the RNA response to PGI, suggest that this prostanoid may be an early factor in the cascade of events between strain in the bone matrix and subsequent osteoregulatory responses performed by bone cell populations.

INTRODUCTION

HE MECHANISM WHEREBY FUNCTIONAL LOADING influ- T ences bone modeling and remodeling behavior to ad- just bone architecture and maintain structural competence is not fully understood. The experiments reported here are part of a series to examine the biochemical responses in bone cells resulting from the loading of adult canine bone tissue.

Our long-term experiments in vivo using an avian ulna model have demonstrated that bone mass can be substan-

tially increased by exposure to short daily periods of applied dynamic load.".2) There is a dose-response relation- ship between peak strain magnitude of the dynamic strains engendered by loading and the adaptive changes in bone mass.'31 A single period of dynamic loading is sufficient to convert a quiescent bone surface to one actively producing new bone 5 days later.(4) This osteogenic response was sub- stantially modified by a high dose of indomethacin at the time of loading,") suggesting a prostaglandin (PG)-dependent step within the first few hours of the adaptive pro- cess. Strain-related responses in vivo occurring before his-

IDepartment of Veterinary Basic Sciences, Royal Veterinary College, London, England. 2School of Biological Sciences, University of Birmingham, England. 'Department of Surgery, King's College School of Medicine and Dentistry, Rayne Institute, London, England.

1345

1346 RAWLINSON ET AL.

tologic evidence of osteogenesis include an increase in the number of osteocytes incorporating [3H]uridine 24 h after loadingI6) and a local strain magnitude-related increase in osteocytic glucose-6-phosphate dehydrogenase (G6PD) activity within 5 minutes.'')

Using cores of cancellous bone removed from the distal end of dog femora and perfused with medium while being loaded to produce strains of physiologic magnitude, we re- produced two effects of loading demosntrated to be associ- ated with adaptive bone remodeling in vivo: ( I ) a rapid (within 5 minutes) increase in G6PD activity in resident bone cells, and (2) increased specific activity of extractable RNA 6 h after loading in the presence of medium supplemented with [3H]uridine.(8) Both increases were inhibited by indomethacin present in the medium during the period of loading.

The studies reported here used this in vitro cancellous bone core model to determine whether ( I ) there is a loading-related increase in PG production, (2) PG production sites can be identified in the resident bone cells by immunolocalization, and (3) exogenous PGs, similar in type and concentration to those produced by loading, can mimic the loading-induced increases in RNA production in resident bone cells reported previously.'")

MATERIALS AND METHODS

Cancellous bone core samples were removed with a trephine from the distal femora of adult dogs (mixed breeds and sex) immediately after a lethal injection of bar- biturate. The cores were then cleared of marrow by flush- ing with sterile phosphate-buffered saline, pH 7.4 (PBS, GIBCO) at 37°C. Cores were extracted from left and right femora to provide contralateral experimental and control samples, which were maintained in recirculating culture medium (0.3 ml/minute, peristaltic pump) in the perfusion chambers at 37°C as described previously.(8) The medium used was minimum essential medium (MEM) + Hank's salts and 25 mM HEPES (GIBCO) supplemented with 2.0 mM L-glutamine (GIBCO), 0.1070 bovine serum albumin (Sigma Chemical Co.), 100 IU/ml of penicillin, and 100 pg/ml of streptomycin (GIBCO). All other chemicals were purchased from Sigma unless otherwise stated. Mechanical load, calibrated in separate experiments to produce a peak bulk strain of 0.005, was applied intermittently at a fre- quency of I Hz for a period of 15 minutes (900 cycles).

Loading-related PG concentrations in the perfusate

The cores were preincubated in the loading apparatus for 4 h with recirculating medium ( 5 ml), which was re- placed hourly with fresh medium. In the fifth hour of pre- incubation and thereafter, fresh medium was allowed to flow through. Medium was sampled by collection of the single-passage perfusate for 0-5, 5-10, and 10-15 minutes (each sample 1.5 ml) to give three preload samples. The cores were subjected to a 15 minute period of load, followed 1 h later by a second 15 minute loading period. Three samples of single-passage perfusate were collected

during each loading period as before and stored frozen at -20°C for 5-7 days. Samples were also collected from control cores at the same time. PGE and 6-keto-PGF,c~ (the hydrolysis product of PGI,) concentrations were measured in duplicate by radioimmunoassay [method based on Jaffe and B e h ~ m a n , ' ~ ) with suitable dilutions of tritiated standards (Amersham International) and antisera]. Assay sensitivities were 10 pg, and the intra- and interassay coef- ficients of variation were 2-5 and 5-8%, respectively, de- pending on the prostanoid measured.

Cross-reactions of the antisera used were as follows: PGE antiserum (Wellcome), PGE,, 100%; PGE, , 33%; PGF,a , <0.01 To; PGF,a, <O.OI%; 6-keto-PGFIa, <0.05%; and thromboxane B, (TXB,), <0.01 To; 6-keto- PGF,a antiserum (Wellcome), PCF,a , 100%; PGF,a, 0.84%; PGE,, 0.1%; and TXB,, 0.02%. Because the PGE antibody does not distinguish between PGE, and PGE,, the results are expressed as PGE.

Localization of PC production

Cores were pretreated as in experiment I , loaded for 5 minutes, removed, blotted, immersed in 10% polyvinyl al- cohol, chilled in hexane (BDH, -70°C). and stored at -70°C. On the following day sections (10 pm) were cut from the middle of the core using a Bright's heavy-duty bone cryostat, air dried for 5 minutes, and fixed in 3.7% formaldehyde (BDH) in PBS for I h. After three 5 minute washes with PBS to remove excess formaldehyde the sections were background blocked using 5% heat-inactivated normal goat serum ( 1 h). This was aspirated and the sections incubated for 1 h at room temperature with primary antibodies against either PGE or 6-keto-PGFIcr (ICN Bio- medicals). To test for antibody specificity, sections were incubated with PBS alone. Three washes with PBS ( 5 minutes each) were followed by the addition of secondary antibody (goat antirabbit IgG-FITC conjugate) to all sections for 1 h at room temperature. Sections were washed with PBS as before, mounted in PBS-glycerol (BDH. 1.9) containing 5% n-propylgallate, and inspected using an Olym- pus BHS microscope with filters for epifluorescence.

Effect of exogenous PCs on RNA production

Cores were pretreated as in experiment I , except that the period of flow through was omitted and substituted with another 1 h incubation with recirculating (0.3 ml/minute) fresh medium. After the fifth hour of pretreatment the res- ervoir of medium was allowed to drain and all channels were refilled with fresh medium containing [3H]uridine ( 5 pCi/ml, Amersham International), unlabeled uridine (0.1 mM), and 1 pM of either PGE, or PGI, for the treated cores or vehicle (see later) for the controls. This PG concentration approximates to the mean difference in the medium PGE concentration (load minus control) determined from earlier experiments when PGs were allowed to accu- mulate in recirculating medium after a loading period of 15 minutes; the PGE level reached (mean + standard error of the mean, SEM) was 19.4 f 1 1 ng per 100 pl (n = 9), which is about 0.6 pM.

PROSTAGLANDIN PRODUCTION IN BONE REMODELING 1347

After recirculation perfusion for 6 h the cores were removed and stored at -70°C for 3-4 weeks until extraction and quantification of the RNA content as described previously.(*'

Preparation and use of PG solutions

PGE, (Sigma) was dissolved in absolute ethanol, filter sterilized, aliquoted, and stored frozen at -20°C. PGI, (Sigma) was dissolved in 100 mM n-glycyl glycine, pH 9.0, filter sterilized. aliquoted, and stored frozen at -20°C. Fresh media containing labeled and unlabeled uridine was prepared and, immediately after addition of either of the recently thawed PGs or vehicle, was added and allowed to recirculate.

Statistics

The results are presented as means * SEM and tested for significance using Student's /-test (two-tailed) for paired data.

RESULTS

Loading-related PG concentrations in the perfusare

Cancellous bone cores perfused with culture medium released PGE and PGI, (measured as 6-keto-PGF1a) into the medium as determined by radioimmunoassay (Fig. I ) . There were always greater amounts of PGE released into the perfusing medium than 6-keto-PGF,a. From the controls i t is apparent that the PG release had not decreased to their lowest levels at the time of the first loading period but did so 2 h after starting the tlow-through perfusion. Bone

_-

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LOAD

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.OAD

30 60 P O I 2 0

TIME (minutes)

organ cultures are normally kept for 24 h to allow PG release to stabilize. However, preliminary experiments led us to the conclusion that in this system the cores should be maintained for only 12 h. Measuring levels ai. this compar- atively early time point may account for tht: falling baseline seen, especially in the profile of PG release from control cores.

Immediate preload data and the three data points during each load are given in Table 1 and shown diagrammatically in Fig. 1 . PGE production was elevated in the 0-5 and 5-10 minute samples for the first loading period compared to their immediate preload production (p < 0.002 and p < 0.03, respectively). In the second period of loading, only the 0-5 minute PGE sample showed an increase (p < 0.02) from preload. Levels of 6-keto-PGFla also showed increases in the 0-5 and 5-10 minute samples (p < 0.007 and p < 0.03, respectively) for the first loading period and only the 0-5 minute sample in the second load (p < 0.04). There were no significant changes in either PGE or 6-keto- PGF,a in the other samples.

The absolute levels of increase of PG production in the second loading period are reduced compared with those in the first period. However, the proportional increase in the second loading period was similar to that of the first (PGE, first loading period 74% increase, second loading period 75% increase; 6-keto-PGF,a, 72 and 60%, respectively.

Localization of PGE and 6-keto-PGFIa

Immunolocalization, used only as a qualitative tech- nique, revealed that PGE was present in the lining cells of the trabecular surfaces but not the osteocytes, whereas 6-

I

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w

.... P... LOAD -ccNrFa

0 30 6 0

TIME (minutes)

$ 1 , . . . .

LOAD - 120

FIG. 1. (A) PGE release into perfusion medium with time. The control release decreased with time to a basal level after approximately the second hour of medium flow through. The first loading period elevated PGE production for the 0-5 and 5-10 minute samples (p < 0.002 and p < 0.03, respectively) compared to preload PGE production. The second period of loading elevated PGE production in the 0-5 minute sample (p < 0.02) but to a lesser extent than in the first loading response (p < 0.001). (B) 6-Keto-PGFla release into perfusion medium with time. As with A, release from controls decreases with time to a basal level after 60 minutes. The first loading period resulted in a higher mean 6-keto-PGFla release in the 0-5 and 5-10 minute samples (p < 0.007 and 0.03, respectively) compared to preload 6-keto-PGFIa production. The second period of loading elevated the production of 6-keto-PGFla only in the 0-5 minute sample ( p < 0.04) by an amount lower than in the first loading period (p < 0.05). Periods of load are shown as stipped bars. Data points are means f SEM ( n = 8).


2

TABLE 1. AMOUNTS OF PGE (NG/ML) AND ~ - K E T o - P G F , ~ (PG/ML)

LOADING AND THE 0-5, 5-10, AND 10-15 MINUTE SAMPLES DURING LOAD^

RELEASED INTO PERFUSION MEDIUM IN THE 5 MINUTE SAMPLE BEFORE

Load PCE (ng/ml) 6-Keto-PCFIa (pg/rnl) Sample

1 2.66 f 0.37 543 f 120 Preload 3.67 f 0.33 868 f 130 0-5 minutes 2.86 f 0.41 727 f 148 5-10 minutes 2.45 f 0.38 600 f 124 10-15 minutes 1.09 f 0.18 401 f 76 Preload 1.66 f 0.23 558 f 114 0-5 minutes 0.97 f 0.24 428 f 91 5- 10 minutes 1 . 1 1 f 0.22 395 f 89 10-15 minutes

aSee text for relevant levels of significance. Data points are means f SEM, n = 8.

keto-PGF,a was located in both lining cells and osteocytes (Fig. 2). This distribution was the same whether the sections were from loaded or control cores ( n = 6). Blank sections incubated without primary antibody showed no staining.

Effect of exogenous PGs on RNA metabolism

The mean specific activity of extractable RNA labeled with ['Hluridine increased with exogenous PGI, (1 pM) by 87% from 8.3 f 1.6 to 13.9 f 3.3 dpm/pg RNA x lo3 (p < 0.03; n = 10). PGE, at the same concentration (1 pM) had no effect (14.5 f 1.2 and 14.0 f 0.7 dpm/pg RNA x lo3, n = 4; Fig. 3).

DISCUSSION

A period of cyclic loading that engendered physiologic strain levels in cores of recently excised canine cancellous bone resulted in increased levels of PGE and PGI, (measured as 6-keto-PGF,a) in the perfusate compared to preload levels during the initial period of the 15 minute load. Repeat loading 1 h later produced smaller absolute increases of PG levels but similar percentage increases. Since the bulk strain levels were similar to those induced in vivo, the load-related PG production is not considered to be caused by cell damage. Immunolocalization demonstrated the presence of PGE in the lining cells immediately adja- cent to the endosteal bone surface, whereas 6-keto-PGFIa was demonstrated in both the lining cells and osteocytes. In both cases this immunolocalization most likely indicates the sites of production; however, the possibility cannot be ignored that they are instead the sites of action (which may be different).

The specific activity of extracted ['Hluridine-labeled RNA was increased by exogenous PGI,, but not by PGE,, which is consistent with PGI, production being an early step in the sequence of events; strain change-PG production - RNA production - influence on adaptive modeling and remodeling. However, although PGs were added in

the concentration (1 pM) that approximated the amount of the PGE produced by loading in a recirculating system using the same core model no such comparable data were available for PGI,. The concentration we added may therefore have been much greater than that produced by loading and the actual response to physiologic levels of PGI, be different from those demonstrated in this study. However, this model is probably not the best to investigate this possibility. More accurate evaluations could be obtain- able with dose-response studies using cells in culture.

Demonstration of the effects of PGs in relation to bone modeling and remodeling is not new. In vivo, Jee et al.'lO1 reported increased bone mass in the tibiae of rapidly growing rats injected daily with PGE, (3 and 6 mg/kg SC, sub- cutaneously) over a period of 21 days. They attributed this to a decreased number of osteoclasts, increased osteoblast numbers, and stimulation of osteoblastic precursor cell replication. Mori et al.'ll' demonstrated increased bone turnover in favor of formation in the vertebrae of rats injected with PGE, (3 and 6 mg/kg SC) daily for 30 days. In- creased endosteal and periosteal thickening with oral administration of PGE, was reported by Shih and Norrdin('" at fracture sites in dog tibiae. Long-term administration of PGE,'") or PGE,'I4) to human infants to treat congenital heart disease produced cortical hyperostosis as revealed by radiography.

The effects of PGs in vitro on extracted bone cell populations, cell lines, and organ culture have been extrapo- lated to suggest their role in osteoblastic cell replication, matrix mineralization, and collagen synthesis in vivo. Stretch-induced PGE, release increased DNA synthesis in bone-derived cells, an effect that could be blocked by in- d o r n e ~ h a c i n , ( ~ ~ ) and 0.1 pM PGE, increased the DNA synthesis in cultured fetal rat calvariae.'16) Within 30 minutes of application, PGI, (2.3 pM) doubled calcium uptake into calvarial disks removed from newborn rats'"'; 6-keto- PGF,a (10 pM), but not PGE, (25 pM), also caused a significant increase. Collagen biosynthesis was elevated by 0.1 pM PGE, in fetal rat calvariae(16) and in osteoblast-en- riched cell populations.(18)

Inhibition of bone resorption was also investigated in


FIG. 2. Photomicrographs of undermineralized cryostat sections of trabecular bone stained immunofluorescently for (A) PGE and (B) 6-keto-PGF,cr. Fluorescence is localized to the lining cells in A, whereas osteocytes are also fluorescent in B. Magnification x 310. Olympus BHS microscope, SPlan AP060 PL oil-immersion objective, EY 455 excitation filter.


0 : 20 z a

M

2

0 1

h s c >

1 0 0 a L! Y 0 W a ln a z C

PG12

TREATED

0-

1

PGE2

FIG. 3. Histogram showing the effect of exogenous PGs ( 1 pM) on the specific activity of extractable RNA labeled with ['Hluridine (dpmlpg RNA x lo', means f SEM). PGI, ( n = 10) increased the specific activity by 87% (p < 0.03), whereas PGE, had no significant effect.

vitro. Chambers and Ah1") found that a range of PGs di- rectly and transiently inhibited the motility and hence the activity of osteoclasts. PGI, was about 100 times more po- tent than PGE,. Fuller and Chambers120) suggested that the major role of PGs in bone physiology is their direct inhibition of osteoclastic resorption. Short-term incubations with PGE, (10 pM) showed decreased calcium release in the first and second hours of incubation,"" and PGE, (1 pM) reduced matrix degradation'zz); both these initial responses were followed by increased resorption during the following 24 h.

Early studies in vitro suggested that PGs cause bone resorption. Tashjian et al.12'L found that [40Caz+] release was elevated by 25 ng/ml of PGE, after 48 h, and [45Caz+] was elevated at 48 h by PGE, at concentrations ranging from 10 nMIz4) to 10 pM.Iz5) However, it is now thought that these findings may present an inaccurate picture, Cham- bers and Ali'"' suggested that in prolonged cultures the initial direct PG-mediated osteoclastic inhibition may be followed by increased osteoclastic activity via PG-mediated cAMP production by osteoblasts.

PG production by tissue and cultured cells subjected to mechanical strain is also not a new concept. It was first demonstrated in the rat gastric fundus,Iz6' and cells derived from fetal rat calvariae grown on collagen ribbons show enhanced PGE synthesis when subjected to tensile forces.'"' In mouse calvaria-derived osteoblasts, PGE, release depended on the strain magnitude and was indepen- dent of cycle time"8); PGI, was not investigated. Loading experiments using bone-derived cells from rat embryos seeded onto the bottom of irreversibly deformable plastic dishes showed increases in PGE,-mediated cAMP production that could be blocked by antiphospholipase antibodies or gentamycin added to the culture m e d i ~ m . ~ " ) The au- thors concluded that phospholipase A, activation is an initial step in the transduction of mechanical stimuli into biochemical events.

Pead et al.14' described the transformation of a quiescent periosteum into one actively forming new bone as "re- newed modeling." This osteogenic response to a single, brief period of cyclic loading was modulated by a high dose of indomethacin administered 1 h before loading,I5) indicating a PG-dependent step. Pead et al.'') also demonstrated autoradiographically, in the same model, increased uptake of ["Hluridine by osteocytes 24 h after loading. EI- Haj et al.,I8' using the cancellous bone core model, reported that indomethacin prevented the loading-induced increase in the specific activity of extractable ['Hluridine- labeled RNA.

The present study shows that both PGE and PGI, are produced by bone cores in vitro in response to cyclic loading. Their rapid production and the effects of PGI, on RNA synthesis are consistent with the notion of an early PG-dependent step in the cascade of events following loading and preceding adaptive modeling and remodeling. In this respect, maintenance of the architectural status quo (and also the inhibition of hormonally modulated resorption that accompanies loss of functional loading) must be considered adaptive. The return of both PGE and PGI, toward baseline levels during loading implies that con- tinued periods of dynamic loading lasting more than 5 minutes do not produce a greater osteogenic (antiresorp- tive) response. This is consistent with the finding of Rubin and Lanyon") that the osteogenic response to dynamic loading is saturated after 36 cycles (occupying only 72 s) a day and coincides well with the peak of PG production, which may be even earlier than the 5 minutes (300 cycles) in the experiments reported here.

PGI, has a short half-life in neutral aqueous solutions, and therefore only a short exposure to it seems necessary to increase RNA synthesis. 6-Keto-PGF,ol generally has low biologic activity (note Refs. 17 and 30), but possible involvement of this degradation product or the 6,15-diketo metabolite cannot be excluded as stimulators of RNA production.

Most studies on the involvement of PGs on bone remodeling in vivo, effects on bone cell populations in vitro, and PG production in response to load have concentrated on PGE. As far as we are aware our findings of loading-related PGI, release, differences in the sites of PGE and PGI, production, and the PGI, effect on RNA metabolism in bone organ culture are entirely novel.,

Our study suggests that elevated PGI, levels, apparently produced in both the lining cells and osteocytes, may have a different effect that PGE in the early responses to mechanical activation of bone cells in situ. Exogenous PGI, and loading, but not PGE,, elicit quantitatively similar increases in RNA production in bone cells. Osteocytes are the cells best placed to perceive strain changes in the matrix, and these cells have previously been shown to increase G6PD activity and RNA production in response to loading in vivo. The importance of the loadingrelated (and possible PGI, mediated) RNA increase in osteocytes depends on the RNA species produced. Osteocytes are sur- rounded by an unexpandable matrix, and RNAs coding for matrix proteins would not seem to be useful to them. It is possible that in response to increased strains in the sur- rounding matrix, osteocytes produce mRNA that is coded


for some cytokine or growth factor that is subsequently translocated to cells on the bone matrix surface to initiate new bone formation and/or influence resorption.

ACKNOWLEDGMENTS

We thank Mr. G.N. Hagger for his assistance with core sample preparation. This work was supported by the Well- come Trust. UK.

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41 1-420.

Address reprint requests to: L.E. Lanyon

Department of Veterinary Basic Sciences Royal Veterinary College

Royal College Street London NWI OTU, England

Received for publication February 19, 1991; in revked form June 5, 1991; accepted July 12, 1991.

Documents

Loading-related increases in prostaglandin production in cores of adult canine cancellous bone in vitro: A role for prostacyclin in adaptive bone remodeling?