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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

A number of early biochemical responses of bone cells to mechanical loading have been identified, but the full sequence of events from the sensing of strain to the formation of new bone is poorly characterized. Extracellular matrix proteins can modulate cell behavior and would be ideal molecules to amplify the early response to loading. The extracellular matrix protein, tenascin-C, supports differentiation of cultured osteoblast-like cells. The current study was carried out to investigate whether expression patterns of tenascin-C in loaded bones support a role for this protein as a mediator of the osteoregulatory response to loading. Tenascin-C expression was investigated by Northern blot analysis in rat ulnae subjected to an established noninvasive loading regimen engendering physiological strain levels. RNA extracted from loaded compared with contralateral control bones 6 h after loading showed a significant increase in tenascin-C transcript expression. The presence of tenascin-C was investigated by immunohistochemistry in bones of animals killed 3, 5, or 15 days after the initiation of daily loading. In animals killed at 3 or 5 days, periosteal surfaces undergoing load-induced reversal from resorption to formation showed enhanced tenascin-C staining. In animals killed at 15 days, the bone formed in response to loading was clearly demarcated from old bone by strong tenascin-C staining of reversal lines. Within this new bone, tenascin-C staining was seen in the lacunae of older but not more recently embedded osteocytes. The results presented here indicate that tenascin-C expression by bone cells is enhanced in the early osteogenic response to loading. This may indicate that tenascin-C acts as a mediator of the mechanically adaptiveresponse.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The components of the extracellular matrix are important in regulating cell differentiation and proliferation in a variety of tissues, through specific receptor-mediated interactions with cells. In bone, where the extracellular matrix plays a crucial structural role, its ability to influence cell behavior has not yet been studied extensively. The extracellular matrix protein, tenascin-C (hereafter referred to as tenascin), is expressed in association with tissue remodeling during the development and pathogenesis in a number of connective tissues including bone (reviewed in Ref. 1). Cells of the osteoblast lineage express tenascin from the onset of osteogenesis (whether intramembranous or endochondral), and continue to do so in growing bone.2,3 Although it is present on bone surfaces, tenascin is generally undetectable in mineralized bone. These observations have led us to investigate the possibility that tenascin is involved in the regulation of bone cell behavior. In recent experiments employing osteosarcoma-derived osteoblast-like cell lines, tenascin was found to stimulate alkaline phosphatase activity, and antitenascin caused a reduction in both alkaline phosphatase activity and collagen synthesis.4 This suggests that tenascin supports bone cells' differentiated activity.

To understand the potential role of tenascin in supporting osteoblast differentiation in vivo, it is important to investigate the regulation of tenascin expression in bone. No information is currently available on the regulation of tenascin expression in osteoblasts, although a number of relevant studies have been carried out in other cell types (reviewed by Chiquet-Ehrismann5). For example, tenascin expression in fibroblasts is stimulated by a number of factors, including transforming growth factor-β,6 basic fibroblast growth factor,7 tumor necrosis factor-α, interleukin-1, and interleukin-4.8 Of particular interest is the observation that the expression of tenascin by cultured fibroblasts is also stimulated by mechanical strain.9

Mechanical loading is important in the regulation of skeletal mass and architecture. It is well established from both exercise and artificial loading studies in vivo that increases in bone loading are associated with increases in bone mass.10 A number of early cellular responses to loading have been identified in osteoblasts and osteocytes, both in vivo and in vitro. These include an almost immediate strain magnitude-related increase in glucose 6-phosphate dehydrogenase (G6PD) activity,11,12 the release of prostaglandins, which appears to be essential for the adaptive response,13,14 and nitric oxide release, which occurs in bones in organ culture and osteoblasts in monolayer culture in response to load.15 A change in the profile of insulin-like growth factor-II transcripts is detectable 8 h after loading,16 and an increase in collagen mRNA synthesis, representative of new bone formation, is detectable 18 h after loading.17 The full cascade of responses leading to new bone formation, however, has yet to be elucidated. We have recently formulated the hypothesis that tenascin acts as an insoluble, medium-term mediator of the osteogenic response to mechanical loading. The studies presented here were carried out to: (1) determine whether expression patterns of tenascin in bones subjected to mechanical loading support this hypothesis and (2) investigate distribution patterns of tenascin in lamellar bone forming in response to mechanical stimulation. A well characterized in vivo model of bone loading, which utilizes noninvasive axial loading of the rat ulna, has been used in the studies.18

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Loading procedure

Female Sprague-Dawley rats (Charles River, Margate, Kent, U.K.) were anesthetized with xylasine (10 mg/kg) and ketamine (50 mg/kg). The left ulnae were subjected to axial compression using a loading device as described.18 Briefly, the olecranon and flexed carpus were held in padded cups of the loading device, then 1200 cycles of dynamic loading at 10 Hz was applied using a sine waveform. With this loading procedure, the medial face of the ulna at the midshaft experienced a strain of −2000 με (110 g rats) or −4000 με (240 g rats). Right ulnae were used as nonloaded controls. Animals (110 g) used in experiments to investigate tenascin transcript expression were subjected to a single period of loading, then sacrificed 6 h later. For immunohistochemistry, rats (240 g) were subjected to three different experimental regimens (Table 1).

Table Table 1. Experimental Procedure
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RNA extraction

The diaphyseal region of control and loaded ulnae was stripped of all attendant soft tissue, taking care not to damage the periosteum, and the bone marrow was flushed out with Dulbecco's phosphate-buffered saline (PBS). After three washes in PBS, the ulnae were chilled and ground to a fine powder in liquid nitrogen using a baked pestle and mortar. Total RNA was isolated using Ultraspec (Biogenesis, Cambridge, U.K.), an adaptation of the method of Chomczynski and Saachi.19 The RNA was quantified by measurement of its absorbance at 260 nm, and equal samples (10–20 μg) were electrophoresed on a 1.3% agarose gel containing 2.2 M formaldehyde. The gel was stained with ethidium bromide to visualize ribosomal RNA (28S and 18S), then photographed under UV illumination for scanning densitometry (Biorad Imaging Densitometer with Molecular Analyst Software, Richmond, CA, U.S.A.). The RNA was transferred and fixed onto Hybond-N nylon membrane (Amersham, Buckinghamshire, U.K.).

Northern blot hybridization

A mouse tenascin cDNA, mTN2S, which corresponds to a region close to the N terminus, including the first four epidermal growth factor–like repeats, was used to detect tenascin transcripts on Northern blots. mTN2S is a 577bp SacI restriction fragment of mTN2, the cloning of which has been described.7 The region recognized by mTN2S is present in all tenascin splice variants. cDNA probes for tenascin and β-actin were purified on agarose gels and labeled to a high specific activity with [α-32P] deoxyadenine triphosphate (3000 Ci/mmol; Amersham) by random hexanucleotide-primed second strand synthesis. The blot was first hybridized with labeled tenascin probe using the Quickhyb hybridization buffer (Stratagene, Cambridge, U.K.) at 68°C and washed under conditions of progressively increasing stringency. The final wash was with 0.1× SSC (20× SSC contains 3 M sodium chloride and 0.3 M sodium citrate, pH 7.0) and 0.1% SDS for 60 minutes at 60°C. Autoradiography was carried out at −70°C using intensifying screens and Hyperfilm MP (Amersham). The intensity of the autoradiogram bands was estimated by scanning densitometry. The levels of mRNA for tenascin were normalized against the combined levels of 28S and 18S RNA in the same sample. Results were analyzed by unpaired t-test and were considered significantly different at p < 0.05.

Immunohistochemistry

Left and right ulnae were excised and dissected free of most soft tissue, taking care not to damage the periosteum. The length was measured, then bones were halved longitudinally using an annular diamond saw. Bones were processed for immunohistochemical detection of tenascin as described.20 Briefly, bones were fixed in 4% paraformaldehyde for 2 h, demineralized in disodium EDTA at 4°C, infiltrated with sucrose, and embedded in optimal cryo-temperature (OCT) compound by rapid freezing on ethanol/dry ice. Transverse sections (10 μm) were cut in a freezing microtome, placed on 3-aminopropyltriethoxysilane (TESPA)-coated slides (Sigma Chemical Co., St. Louis, MO, U.S.A.), and stored at −70°C. For the present study, sections were cut from the region of the distal half of the ulna that corresponds to the functional midpoint, and that is known to be most responsive to load in terms of periosteal new bone formation.18

Sections were stained by indirect immunofluorescence, using rabbit antirat tenascin21 as described.20 Sections were pretreated with hyaluronidase, which enhances staining of tenascin in bone. They were washed and incubated in rabbit antirat tenascin or normal rabbit serum (1:200), then washed and incubated in rhodamine-conjugated swine antirabbit immunoglobulin (1:500 Dako Corp., Glostrup, Denmark). After a final wash, sections were mounted in 50% glycerol in PBS, then viewed and photographed with an Olympus photomicroscope.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Detection of tenascin transcripts in mRNA extracted from loaded bones

The mouse tenascin cDNA used in the current study detected a major transcript of about 7.3 kb in RNA extracted from rat ulnae. This is in agreement with previously published estimates for rat tenascin transcripts.22 RNA extracted 6 h after loading showed a significantly higher level of tenascin transcript expression in loaded ulnae than in paired contralateral control ulnae (Fig. 1). When densitometric values for tenascin expression were normalized for combined 28S and 18S RNA content, the level of tenascin expression in loaded bones was found to be almost 2-fold that seen in control bones. The mean value for control bones was 2.28 ± 0.38 and for loaded bones 4.15 ± 0.57 (mean ± SE). The values were significantly different (p < 0.05).

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Figure FIG. 1. Expression of tenascin transcripts in loaded bones. (A) Autoradiograph of Northern blot of RNA extracted from loaded or control ulnae and hybridized with tenascin cDNA. Each lane represents pooled RNA from four control (cont) or loaded ulnae prepared as described in Materials and Methods. Arrowheads indicate the position of 28S and 18S RNA bands. (B) The Northern blot shown in (A), visualized under UV light for ethidium bromide staining of ribosomal RNA (28S and 18S bands; arrowheads).

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Immunohistochemical detection of tenascin in the early response to loading

The growing ulnae investigated in the current study are undergoing modeling drift, with resorption occurring medially and formation laterally; this modeling drift is responsible for introducing and maintaining longitudinal curvature during growth. The loading regimen used in the current study results in reversal and new bone formation on the medial surface, as well as enhanced bone formation on the lateral surface.18 Transverse sections of control bones through the region known to be most responsive to load showed moderate continuous tenascin staining on lateral (forming) periosteal surfaces, but weak, discontinuous tenascin staining on medial (resorbing) surfaces (Figs. 2B and 2D). In rats killed 2 days after the initiation of loading (Group A), there was enhanced tenascin staining on the medial periosteal surface of loaded bones, concomitantly with the increase in osteoblastic proliferation and differentiation that has been shown to occur on such surfaces.23 The increase in tenascin staining on the medial surfaces of loaded bones above that seen in nonloaded bones was more obvious in rats loaded for 3 days and killed 2 days later (Group B; Figs. 2A, 2B, 2C, 2D). In loaded bones at this stage, multiple layers of cells on the medial periosteal surface were surrounded by a tenascin-containing matrix, whereas the corresponding surface of contralateral control bones showed only a fine discontinuous line of tenascin staining. Since lateral periosteal surfaces were undergoing formation even in control bones, they showed tenascin staining several cell layers deep; tenascin staining of the corresponding surface of loaded bones showed a similar distribution (data not shown). Since tenascin staining was abundant on this surface in control bones, it was not possible to determine whether staining was enhanced in loaded bones.

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Figure FIG. 2. Immunohistochemical detection of tenascin in ulnae of rats from Group B (4 days after initiation of loading). (A–D) Transverse cryosections of the medial region of the mid-diaphysis of loaded (A, C) or control (cont; B, D) ulnae, stained by indirect immunofluorescence for the presence of tenascin. Paired ulnae (A and B, or C and D) from the same rat. Large arrows indicate periosteal surface; small arrows indicate osteocytes stained positively for the presence of tenascin; m, marrow cavity; bv, blood vessel. Tenascin staining of the medial periosteal surface of control bones is seen in a discontinuous line along the bone surface (B, D), whereas in loaded bones the staining spans several cell layers (A, C). The magnification is the same for (A) and (B). Bar = 200 μm. The magnification is the same for (C) and (D). Bar = 100 μm.

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Immunohistochemical detection of tenascin in bone formed in response to load

In rats subjected to loading over a 12-day period and then sacrificed on day 15, the loaded ulnae showed measurable new bone formation on the medial surface, by comparison with the nonloaded ulnae, which had continued to undergo resorption medially, as described.18 The distribution of tenascin in the bone formed in response to loading was investigated. In tenascin-stained sections, new bone on the medial face was clearly demarcated from the older bone by strong staining of the line of reversal (Fig. 3). An additional observation of note was that within the new bone, only the osteocytes close to the reversal line showed strong staining for the presence of tenascin. The osteocytes that had become embedded more recently, that is, those closer to the periosteal surface, were unstained (Fig. 3).

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Figure FIG. 3. Immunohistochemical detection of tenascin in ulnae of rats from Group C (14 days after initiation of loading). (A,B) Transverse cryosections through the medial region of the mid-diaphysis of loaded (A) or control (cont; B) ulnae from the same rat, stained by indirect immunofluorescence for the presence of tenascin. m, marrow cavity. In (A), bone formed in response to load lies between the periosteal surface (closed arrow) and the reversal line (open arrow). The reversal line is strongly stained for the presence of tenascin. The magnification is the same in (A) and (B). Bar = 200 μm. (C,D) Adjacent transverse cryosections through the medial region of the mid-diaphysis of a loaded ulna stained by indirect immunofluorescence with antitenascin (Tn; C) or normal rabbit serum to demonstrate background fluorescence (NRS; D). Arrow indicates periosteal surface; open arrow indicates tenascin-stained reversal line. Within the bone formed in response to loading, only the deeper osteocytes (small arrows) show positive staining for tenascin. Bar = 100 μm.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The current study was undertaken as part of an investigation into the regulation of tenascin expression in bone cells. Endogenous tenascin supports the basal differentiation of osteoblast-like cells in culture,4 suggesting that it may be important in regulating osteoblast differentiation in vivo. If tenascin plays such a role, it can be expected that known osteogenic stimuli, such as increased mechanical loading, would induce its expression. These observations have led us to the hypothesis that tenascin helps to mediate the osteogenic response to loading. The results presented here support this hypothesis. We have detected an increase in tenascin transcript expression in loaded bones, and we have observed an increase in tenascin protein expression on periosteal surfaces known to respond to load.

The variety of cellular responses in bone that occur in response to mechanical loading and that lead to an osteogenic response are only just beginning to be identified.24 It seems likely that these responses form a cascade of events, each of which is required for new bone formation to occur. The results presented here suggest that an increase in tenascin expression may be one of the events in such a cascade. Tenascin expression in other tissues is induced by growth factors,6–8 an example of which is transforming growth factor-β, which is known to be present in bone and important in regulating osteoblast function. Thus, one possible explanation for the induction of tenascin after loading is that load-induced growth factors stimulate tenascin expression. The rapidity with which tenascin mRNA is increased in loaded bones (6 h after loading) suggests that the response is not dependent on the synthesis of other proteins, such as growth factors. It may, however, be dependent on other stimuli that are part of bone's early responses to loading. Such signals are known to include prostanoid and nitric oxide release and increased cellular G6PD activity.12–14

Tenascin, as an insoluble protein of the extracellular matrix, is likely to have a longer half-life in tissue than do any of the other early load-responsive factors so far identified. Prostanoids and nitric oxide are present only transiently, and growth factors are also likely to lose their activity or be removed from the site of synthesis much sooner than is tenascin. Indeed, the presence of tenascin in the line of reversal suggests that it is retained at this site for at least 10 days. Thus, if tenascin potentiates the response to loading, it remains in the environment of the cells that produce it long enough to support their continued differentiation over days.

The bone surface on which enhancement of tenascin expression was detectable immunohistochemically in loaded bones was the medial periosteal surface, which was undergoing resorption in control bones. No change in tenascin expression was detectable on the lateral periosteal surface, which in control bones was undergoing formation and therefore stained strongly for tenascin. One possible conclusion from these results is that tenascin expression is regulated by compressive loading (as experienced by the medial face), but not tensional loading (as experienced by the lateral face). A more likely explanation is that the immunofluorescence method used was simply unable to detect an increase in tenascin expression on surfaces already showing strong tenascin staining. The increase in tenascin expression on medial surfaces of loaded bones is likely to have been greater than the 2-fold increase detected in Northern blots of RNA extracted from whole diaphyses. This apparent discrepancy is probably due to the fact that the periosteal RNA would only represent a proportion of the total bone RNA, and tenascin expression on other bone surfaces, such as lining Haversian canals, is unlikely to have been affected by loading.

Since the distribution of strains sensed by loaded bones is not necessarily correlated with the distribution of the osteogenic response, it has been argued that cells other than osteoblasts must be primarily responsible for sensing strain. It has been suggested that osteocytes, which form a network throughout the bone substance and communicate with each other through gap junctions, are ideally suited to carrying out this function.24 Indeed, osteocytes are clearly load-responsive, since like osteoblasts they have been shown to release nitric oxide and to exhibit elevated G6PD activity and insulin-like growth factor I mRNA expression after loading.11,15,25 In the current study, we attempted to determine whether osteocytic tenascin expression was elevated in response to loading. This was not possible to assess in the immunohistochemical studies, since most osteocytes in nonloaded bones were positive for tenascin expression. This tends to suggest that increased osteocytic tenascin expression is unlikely to be important in the early signaling between osteocytes and osteoblasts. Any potential role for tenascin in transducing mechanical strain into an osteogenic response is thus more likely to be in supporting osteoblast differentiation and function.

The bone formed in response to loading provided a defined region of rapidly forming lamellar bone, with a known history, in which to investigate patterns of tenascin expression. We have previously observed that most, but not all osteocytes in growing rat bones express tenascin.20 In 4-week-old rats, strong tenascin staining was seen in osteocytes throughout the trabecular bone of the secondary center of ossification but was absent from osteocytes in the primary spongiosa of the metaphysis. In the current study, it was observed that in bone formed in the 2 weeks after the initiation of loading, the older osteocytes stained positively for tenascin, whereas the more recently embedded osteocytes closer to the periosteal surface were negative. Thus, it appears that osteoblasts lose their ability to express tenascin as they are embedded in bone matrix, but regain it with progressive maturation as osteocytes. Preliminary confocal microscopy studies on the distribution of tenascin in bone suggest that osteocyte-associated tenascin is present extracellularly, within the lacuna. What role this tenascin may be playing is unclear, but one possibility is that it prevents excessive adhesion of osteocytes to bone surfaces, allowing space for extracellular fluid. Such a role is supported by the observation that isolated osteocytes do not adhere to tenascin in vitro.26

The fact that antitenascin stained the reversal (or cement) line allowed us to distinguish easily between old bone and bone formed as a result of loading. The presence of tenascin in these reversal lines may not be specific for bones responding to load, since we have observed tenascin staining of reversal lines in normal adult rat bone (unpublished observations). Reversal lines are known to contain a distinctive, collagen-free globular matrix which is laid down by flattened (probably preosteoblastic) cells in the wake of resorbing osteoclasts.27 Osteopontin, osteocalcin, and bone sialoprotein have previously been shown to be present in reversal lines.28 Thus, the matrix of reversal lines appears to be made up of a complex mixture of noncollagenous proteins. Scanning electron microscopy studies carried out by Zhou et al.27 indicate that this globular matrix is secreted by flattened cells to coat the surface of demineralized collagen fibers left behind by osteoclasts; these secretory cells are found in an anatomical continuum with differentiated osteoblasts and are probably their precursors. Thus, it appears that in the orderly process of lamellar bone formation, bone-forming cells undergo a program of at least two phases of secretion of distinctive sets of matrix components: those found within the reversal line and osteoid. The role of the specialized matrix of reversal lines is likely to be the stimulation of further differentiation of the cells that secrete it, which would be an essential stage in the coupling of resorption with formation. The finding that tenascin, which stimulates differentiation of cultured osteoblast-like cells,4 is present in reversal lines, is in keeping with such a role.

The presence of tenascin in reversal lines may help to resolve the puzzle of how an osteoblast-secreted protein can be absent from the mineralized matrix of lamellar bone. The possibility that tenascin is secreted as one component of a distinctive globular matrix before, but not after, the final stages of osteoblast differentiation would explain its absence from lamellar bone matrix. Tenascin is, in fact, detectable in the woven bone matrix of human fetal bone, as well as dysplastic bone.29 Perhaps the osteoblasts of woven bone are forming bone so rapidly that they are unable to separate the phase of early globular matrix secretion from later collagenous matrix (osteoid) secretion, resulting in the poor organization of woven bone.

In conclusion, expression of tenascin is induced during the early osteogenic response to mechanical loading in bone and thus may act as a medium-term mediator of the adaptive response. Tenascin deposited soon after loading on previously resorbing surfaces may be of particular importance in the transition from resorption to formation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

This work was funded by a grant from the Arthritis and Rheumatism Council of Great Britain to Dr. Mackie.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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