Bone Formation Immunocal En

Endocrinology. First published October 2, 2003 as doi:10.1210/en.2003-1239

Title: Diabetes Causes Decreased Osteoclastogenesis, Reduced Bone Formation and Enhanced Apoptosis of Osteoblastic Cells in Bacteria Stimulated Bone Loss

Brief Title: Diabetes Modulates Bone Formation and Apoptosis

Authors: Hongbing He^, Rongkun Liu^, Tesfahun Desta^, Cataldo Leone^, Louis C. Gerstenfeld* and Dana T. Graves^

Affiliations: ^Department of Periodontology and Oral Biology, Boston University School of Dental Medicine, Boston, MA 02118 *Department of Orthopedics, School of Medicine, 700 Albany Street, Boston Medical Center, Boston, MA 02118

Corresponding Author: Dana T. Graves Boston University School of Dental Medicine, W-202D 700 Albany Street Boston, MA 02118 (617) 638-8547 (Phone) (617) 638-4924 (FAX) [email protected] Key Words:

apoptosis, bone formation, bone resorption, diabetes, inflammation,

osteoclast

Copyright (C) 2003 by The Endocrine Society

ABSTRACT

The most common cause of inflammatory bone loss is periodontal disease. Following bacterial insult inflammation induces bone resorption which is followed by new reparative bone formation. Since diabetics have a higher incidence and more severe periodontitis, we examined mechanisms by which diabetes alters the response of bone to bacterial challenge. This was accomplished with db/db mice, which naturally develop type 2 diabetes. Following inoculation of bacteria osteoclastogenesis and bone resorption was measured. Both parameters were decreased in the diabetic group. Diabetes also suppressed reparative bone formation measured histologically and by the expression of osteocalcin. The impact of diabetes on new bone formation coincided with the effect of diabetes on apoptosis of bone lining cells. Within 5 days of bacterial challenge, apoptosis declined in the wild type animals yet remained significantly higher in the diabetic group. Thus, diabetes may cause a net loss of bone because the suppression of bone formation is greater than the suppression of bone resorption. The diminished formation may be due in part to prolonged apoptosis of bone lining cells.

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INTRODUCTION

Bone is a tissue that undergoes frequent remodeling and has a large capacity for regeneration. In the adult remodeling occurs so that the skeleton is replaced approximately every 10 to 11 years (1). This physiologic remodeling is initiated by osteoclasts that resorb bone and is followed by the formation of an equivalent amount of new bone by osteoblasts (1), (2). Bone loss is noted when the amount of bone resorption exceeds the amount of new bone formation. This occurs in the aging skeleton, especially during menopause related osteoporosis (3). Bone loss also occurs as a result of metastasis to bone and during inflammation associated with arthritis and periodontal disease (4-7). Diabetes has also been associated with a net loss of bone. A number of studies have reported that type 1 diabetes alters bone remodeling by reducing the formation of new bone, leading to osteopenia. This has been shown by a decrease in bone mineral density in humans and alterations in the formation of new bone in animal studies (8-11). The impact of type 1 diabetes on bone is reflected by a significant delay in fracture healing (12, 13). In contrast, the presence of bone loss in type 2 diabetes is less clear and current understanding suggests that this form of diabetes is not typically associated with osteopenia (10, 14-16). The pathologic remodeling of bone seen in inflammatory diseases results from inadequate bone formation following resorption. One of the most common causes of bone loss in humans is periodontal disease (7). Periodontal disease is induced by bacterial plaque, which accumulates on the tooth surface and stimulates a host response in the adjacent gingiva that can lead to the destruction of connective tissue and bone

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surrounding the tooth (5). It has been suggested that periodontal disease is one of the significant and characteristic complications of diabetes (17). Both type 1 and type II diabetes increase the risk of periodontal disease three to four fold (18-20). Several mechanisms have been proposed to explain the greater incidence and severity of periodontal disease in diabetics. These include enhanced susceptibility to infection due to diminished neutrophil recruitment and function (21), (22), a more severe inflammatory response that leads to greater tissue destruction (23) and the effect of advanced glycation end products on formation (24). That latter can include enhanced formation of inflammatory cytokines (25, 26) and delayed wound healing(27). In general, these mechanisms would be expected to lead to enhanced formation of osteoclasts and increased bone loss. We report here that diabetes actually reduces the formation of osteoclasts following bacterial challenge. Rather the net loss of bone is due to a pronounced effect of diabetes on inhibiting new bone formation. The latter may be explained in part, by an increase in apoptosis of bone lining cells.

MATERIALS AND METHODS

Animals Db/db mice on a C57BL/KSJ background and normoglycemic db/+ heterozygous littermates were purchased from the Jackson Laboratory (Bar Harbor, ME). These mice develop diabetes at 6 to 8 weeks of age. They were 10-11 weeks old at the start of the experiments and had been diabetic for a minimum of 20 days. The degree of hyperglycemia during the experimental period was similar amongst mice in the diabetic

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group, typically having glucose levels of 400-450 mg/dl. Glycosylated hemoglobin levels in these mice were typically 10-15% for db/db mice and under 5% for normoglycemic littermates. All animal procedure protocols were approved by the Institutional Animal Care and Use Committee, Boston University Medical Center.

Inoculation of P. gingivalis Broth grown P. gingivalis at log phase growth were lightly fixed with 1% paraformaldehyde. P. gingivalis (5 x 108) in 50 ul of sterile PBS was inoculated at the midline of the scalp between the ears. This causes a reproducible inflammatory response that is located histologically between the coronal and occipital sutures. Mice were killed at 0, 1, 3, 5, 8 and 12 days following inoculation. For each data point there were six mice, n=6. The tissue was either fixed in paraformaldehyde for histological analysis as described below, or the soft tissue was removed from the calvaria and the calvarial bone was dissected and immediately frozen in liquid nitrogen, pulverized and total RNA extracted with Trizol reagent (Life Technologies Inc., Rockville, MD) according to the manufacturer’s instructions.

Preparation of Specimens Each calvaria was split along the midline and half was prepared for histologic sections by fixation in 4% paraformaldehyde at 4°C for 2 days. After fixation specimens were decalcified with Immunocal (Decal Chemical Corporation, Congers, NY ) for 12 days and washed with Cal–arrest (Decal Chemical Corporation). Specimens were embedded in a low melting paraffin and sectioned at 5 microns.

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Bone Histomorphometry Van Gieson stained sections prepared as described in (28) were used to measure newly formed bone. Newly formed bone collagen was stained blue while previously formed bone was stained red. New bone formation area and bone length was measured by image analysis software with the results expressed as new bone area per bone length of calvarium (mm2/mm).

TRAP Tartrate –resistant acid phosphates (TRAP) assay on tissue sections was performed as described in (29). Osteoclasts were counted as TRAP-positive multinucleated cells lining bone. The total number of osteoclasts was counted and expressed per mm length of bone.

TUNEL Assay Apoptotic bone lining cells were detected by an in situ terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate (TdT)-biotin nick end labeling (TUNEL) assay using a TACS 2 TdT kit purchased from Trevigen following the manufacturer’s instructions ( Trevigen, Gaithersburg, MD, USA). Slides were counterstained with nuclear fast red. TUNEL positive cells in the two to three cell layers adjacent to bone were counted at 1000x magnification and expressed as the number per bone length (number/mm). The area examined lay between the occipital and coronal sutures.

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Osteocalcin Expression Total RNA (4 µg) was incubated with a 32-P(UTP) labeled RNA probe specific for murine osteocalcin. Samples were subjected to RNase digestion using a kit from Pharmingen (BD Bioscience, Franklin Lakes, NJ) according to the manufacturer’s instructions. Following electrophoresis on 6% polyacrylamide gels, radiolabeled bands were visualized with a PhosphoImager (Biorad Laboratories, Hercules, CA). The optical density of the protected bands was measured with Image ProPlus software (Media Cybernetics, Silver Spring, MD), which was then normalized by the value of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the same lane. The experiment was performed twice with similar results.

Histologic Analysis All cells counts and histomorphometirc data were obtained by one examiner and confirmed by a second independent examiner with similar results. Van Gieson stained sections were used for histomorphometric analysis of bone. Previously formed bone and newly formed bone was measured between the coronal and occipital sutures. From our experience newly formed bone identified by van Gieson stain represents bone that has formed within four days of sacrifice. For example, newly formed bone measured on day 12 appears to have been formed after day 8, and between days 9 and 13. Bone loss was calculated by measuring the area of previously formed bone present on days 5 or 8 and subtracting it from the value measured on day 0. The net amount of bone present on day 12 consisted of the sum of the previously and newly formed bone area. Statistical

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significance was determined by one-way analysis of variance with significance set at the p< 0.05 level.

RESULTS

Bone Resorption Histomorphometric analysis was performed to quantify osteoclastogenesis and the amount of bone resorbed. Without stimulation few osteoclasts could be detected in either the diabetic or control groups (Figure 1A). At days three and five following inoculation, the increased level of bacteria-induced osteoclastogenesis was at least 50 % higher in the control versus diabetic mice, which was statistically significant (p<0.05). At later time points there was no difference between the two groups. At no time point were there significantly more osteoclasts induced in the diabetic mice than in the corresponding control mice. The amount of bone loss evident on days 5 and 8 was measured (Figure 1B). This was calculated by measuring the area of “old” bone present in Van Gieson stained sections. The amount of bone loss was significantly higher in the control group than the diabetic (p<0.05).

New bone formation Osteocalcin is produced by osteoblasts and its expression is directly proportional to the amount of new bone formed. Osteocalcin expression was measured by RNase protection assay. At the 0 time point, control and diabetic mice expressed a similar level of osteocalcin (Figure 2). On days 5 and 8 the values of both groups increased. However,

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at day 8 osteocalcin expression in control mice had increased further while levels in the diabetic animals were reduced. Recently formed collagen in connective tissue and bone can be identified in van Gieson stained histologic sections by its characteristic blue color (Figure 3). Bone formation was first observed in control mice on day 5, while none could be detected in the diabetic group at this time point. At day 8 new bone formation appeared in both groups, but the amount in control mice was considerably more than that in diabetic mice. Image analysis was used to quantify the amount of new bone formation. A small amount of new bone formation was observed on day 5 in the normoglycemic mice, while none was detected in the diabetic group(Figure 4 A). On day 8 (peak time point) there was four fold more new bone in the control than the diabetic group. On day 12 the absolute amount of newly formed bone had decreased. Nonetheless, the control group still had approximately eight times the amount of new bone formation than the diabetic at this time point. The difference between the diabetic and control groups on days 5, 8 and 12 was statistically significant (p<0.05). The total amount of bone present on day 12 was also measured (Figure 4 B). This value represents the net bone present, which reflects both old and newly formed new bone as well as the amount of bone that had been lost due to resorption. The total amount of bone present was greater in the control group than in the diabetic group(p<0.01).

Apoptosis The TUNEL assay was used to detect apoptotic cells adjacent to bone (Figure 5). At baseline virtually no apoptotic cells were detected. Relatively high numbers of

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apoptotic cells were found in both the diabetic and control groups on days 1 and 3. Starting on day 5 there was a continual decline in apoptotic cells over subsequent time points in the normoglycemic mice, while the number remained higher in the diabetic animals. On both days 5 and 8 following bacterial insult, apoptosis in the diabetic mice was 3 fold higher than controls and was 5 fold higher on day 12. The difference at these time points was statistically significant (p<0.05).

DISCUSSION Previous investigators have reported that there is an enhanced expression of cytokines in vitro that are capable of stimulating bone resorption in diabetics (23, 30, 31). These include proinflammatory mediators such as IL-1, TNF, IL-6 and PGE2. It has been proposed that the higher level of cytokine expression associated with diabetes is due, in part, to the formation of advanced glycation end products (AGE). Blockade of AGEs reduces the over-expression of these cytokines and inhibits alveolar bone loss stimulated by P. gingivalis in diabetic mice (24). Because diabetes is associated with higher levels of cytokines, particularly in response to bacterial products, it has been suggested that the increased risk and greater severity of periodontal disease in diabetics may be due to enhanced inflammation and bone resorption. However, the results presented here clearly demonstrate that, in comparison to control animals, the greater net bone loss in the diabetic group following bacteria-induced inflammation is not due to enhanced osteoclastogenesis. Thus, the reduced resorption is less important than the significantly diminished new bone formation. This is most likely a result of reduced coupling of bone

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resorption and formation in the diabetic group, which is based upon comparing the amount of new bone area divided by the number of osteoclasts. On day 8 the ratio is 9.6 x -4

-4

10 for normoglycemic mice and 3.0 x 10 for diabetics; on day 12 the ratio is 4.4 x 10 -4

for normal and 0.4 x 10

-4

for diabetic mice. Thus, the amount of new bone formed per

measure of osteoclastogenesis is three to ten times higher in the normoglycemic mice, suggesting that the degree of coupling is reduced in the diabetics. It has previously been noted that there is reduced fracture healing or osseous repair after marrow ablation in diabetics compared to normals (32-35). Several mechanisms have been proposed including diminished production of growth factors and expression of transcription factors that regulate osteoblast differentiation. We found that following bacterial insult apoptosis of bone-lining cells was induced. Diabetes had a profound effect on apoptosis of bone-lining cells by considerably lengthening the time during which there were high levels of apoptosis. This may explain findings that bone surfaces in diabetic mice are lined by fewer cells than bone in their normal counterparts (36). Moreover, it has been shown that increased apoptosis of bone lining cells decreases the amount of bone formed (37, 38). Studies of soft tissue wounds also indicate that diabetic mice have increased levels of apoptosis, which may interfere with the capacity for wound healing (39). Thus, diabetes may have a general effect on increasing apoptosis of matrix producing cells, which limits the repair of injured tissue. Although there are few reports describing the impact of diabetes on the pathological processes seen in inflammatory bone loss, there have been many reports on the impact of diabetes on bone mineral density, which reflects physiologic bone remodeling. It is generally accepted that type 1 diabetes causes diminished bone mineral

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density (8, 40-43). In contrast, adult type 2 diabetics have normal or even higher bone mineral density then non diabetics (11, 14-16). The reasons for the lower bone mineral density in type 1 diabetes are not known. Parfitt and colleagues have speculated that it is due to reduced formation during the period when peak bone mass is attained (11). The opposite conclusion was reached by Tuominen and colleagues, who examined patients that developed type 1 or type 2 diabetes after 30 years of age (10). Because the age of diabetic onset was after peak bone mass had been attained, they suggested that the reduced bone mass in type 1 diabetics was due to a higher rate of bone loss. Whatever the cause, it appears that type 1 diabetes has a more profound effect on physiologic bone resorption than does type 2 diabetes. In the present study, rather than examining physiologic bone remodeling, we examined pathologic remodeling initiated by an inflammatory stimulus in an animal model of type 2 diabetes. Under these conditions, we observed not only the inhibition of osteoclastogenesis and resorption in the diabetic animals but also the reduction of new bone formation to an even greater extent. The net effect of diabetes was to produce a loss of bone despite the finding that bone resorption was reduced compared to controls. Thus, type 2 diabetes may exert different influences on physiologic versus pathologic bone remodeling. One potential interpretation is that there is reduced coupling of bone resorption and formation in the diabetic group following bacterial inflammation, compared to the robust coupling that occurs in normoglycemic littermates. This is particularly evident on day 12 when there is very little new bone formation in the diabetic group, suggesting that the reduced amount of bone present will exist over a long period of time. The lack of consensus regarding the mechanism for diminished bone mass in type 1

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diabetic individuals and the absence of an equivalent effect in type 2 diabetics, along with results presented here point to the need for further investigation into the impact of diabetes on bone.

ACKNOWLEDGEMENTS This work was supported by grants from the National Institute of Dental and Craniofacial Research, DE11254 and DE13191.

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Figure Legend

Figure 1 A. Quantitative analysis of osteoclast numbers. TRAP positive osteoclasts were counted along the length of the calvaria as described in Materials and Methods. The number is expressed per mm of bone length in diabetic (db/db) (

) and normoglycemic littermate

control ( ) mice. B. Quantitative analysis of the amount of bone loss. The amount of bone loss was determined in histologic sections as described in Materials and Methods. The means ± standard error is based on six specimens for each data point. * indicates statistical significance between the two groups at a given time point, p< 0.05.

Figure 2 Osteocalcin mRNA expression is increased during repair of bacteria induced injury of bone. Osteocalcin and GAPDH expression was measured by RNase protection assay from total RNA obtained from calvaria following bacterial inoculation at the indicated time points. A. Autoradiograms of osteocalcin and GAPDH expression in control (–) and diabetic db/db (+) mice. B. Densitometric analysis of osteocalcin expression normalized based by GAPDH levels per lane. Diabetic (db/db) ( littermate control ( ) mice.

Figure 3

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) and normoglycemic

New bone formation is less in diabetic compared to normoglycemic mice following bacteria induced destruction. New bone formation was measured by van Gieson staining; new bone is blue while mature bone is red. Arrows point to newly formed bone area. Original magnification 400 x. Each photomicrograph is representative of six specimens for a given group.

Figure 4 A. Quantitative analysis of new bone formation. New bone formation was measured with image analysis assisted histomorphometry of van Gieson stand sections as described in Materials and Methods. The new bone formation is expressed as the area per bone length (mm2/mm). Diabetic (db/db) (

) and normal littermate control (

) mice. B. Net bone

area present on day 12. The area of old and new bone was measured in van Gieson stained sections from day 12 specimens. The means ± standard error is based on six specimens. * indicates statistical significance, p< 0.05.

Figure 5 Apoptosis of bone lining cells A. The TUNEL assay was performed on histologic sections following inoculation with bacteria at the indicated time points. TUNEL positive cells are in blue, while non apoptotic cells are counterstained with fast red. Shown are control and db/db mice at day 8. Arrows point to apoptotic cells. Original magnification 1000 x. B. Quantitative analysis of apoptotic bone lining following bacterial inoculation. The number of TUNEL-positive bone lining cells were counted at 1000 x magnification and the values are expressed per bone length. Diabetic (db/db)

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(

) and normal littermate control ( ) mice. The means ± standard error are based on

six specimens for each data point * indicates statistical significance, p< 0.05.

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A

*

num ber/m m

6 5 *

4 3 2 1 0

B

0d

Area Bone Loss (mm2)

Fig. 1

3d

5d

8d

12d

0.6

*

0.5 0.4

*

0.3 0.2 0.1 0 5d

8d

A 0day

5day

8day

12day

-

-

-

-

+

+

+

+ Osteocalcin

GAPDH

B 3 2.5 Relative intensity

Fig. 2

2 1.5 1 0.5 0 0

5

8

12

Fig. 3 5 day control

5 day diabetes

8 day control

8 day diabetes

New Bone Area (mm2/mm x 100)

A

5

*

4.5 4 3.5 3 2.5 2 1.5

*

1 0.5 0

* 5d

8d

12d

B 1.4

Total Bone Area (mm2)

Fig. 4

1.2

*

1 0.8 0.6 0.4 0.2 0 Control

Diabetic

A

Control

Diabetic

B Apoptotic cells per mm

8 *

7 6 5

*

4

*

3 2 1 0 0d

1d

3d

5d

8d 12d