WO1995012124A1

WO1995012124A1 - Osteocalcin as a marker of periodontal and peri-implant disease activity

Info

Publication number: WO1995012124A1
Application number: PCT/US1994/011680
Authority: WO
Inventors: William V. Giannobile; Ray C. Williams; Samuel E. Lynch
Original assignee: Institute Of Molecular Biology, Inc.; President And Fellows Of Harvard College
Priority date: 1993-10-29
Filing date: 1994-10-17
Publication date: 1995-05-04
Also published as: AU8017594A

Abstract

A method of diagnosing severity of periodontal disease or peri-implant disease in a human patient, said method comprising a) obtaining a sample of tissue or gingival crevicular or other fluid from the mouth of said patient, and b) measuring osteocalcin or fragments thereof in said sample as a measure of severity of disease.

Description

TITLE OF THE INVENTION OSTEOCALC N AS A MARKER OF PERIODONTAL AND PERI-IMPLANT

DISEASE ACTIVITY Background of the Invention

This invention relates to measuring severity of periodontal and peri-implant disease.

Periodontal disease and peri-implant activity presently are diagnosed by clinical parameters such as pocket depth, bleeding on probing, and radiographs. These parameters have limitations in that they lack ability to predict future attachment loss, and provide information only on the existence of past disease. The need for diagnostics in periodontology evaluating predictive markers of periodontitis is a focus of present research.

Periodontal disease is a general term used to describe specific diseases that affect the gingiva, as well as the supporting connective tissues and alveolar bone which anchor the teeth in the jaws. The periodontal diseases are among the most common infectious diseases in humans. In the last fifteen years, with the decline of dental caries in children aged 6-18, and better prevention programs for the general population, periodontal disease leading to tooth loss has assumed even greater importance. As more teeth are retained due to reduced caries, more teeth are at risk to be affected by periodontal disease (Shaw, J.H., N.Eng.J.Med. (1987) 117:996; Williams, R.C, N.Eng.J.Med. (1990) 332:3731. Thus, the recognition and diagnosis of periodontal disease has become even more important.

The use of clinical parameters for the diagnosis of periodontal disease has numerous limitations. For example, Haffajee and co-workers (Haffajee, A.D., et al., J.Periodontal (1991) 18.:117) have demonstrated that no clinical parameters have been shown to be predictive for periodontal disease activity. Thus there have been intensive research efforts to develop diagnostic tests for periodontal disease evaluation. Over 40 different tests for gingival crevicular fluid components have been studied, e.g., collagenase (Villela, B. et al., J.Periodont.Res. (1987) j22_.:264), alkaline phosphatase (Ishikawa, I. et al., Arch.Oral.Biol. , (1970) 15:1401; Binder, T.A. et al. , J.Periodont.Res. (1987) 22:14) . cathepsin-like activities (Kunimatsu, K. et al., J.Periodont.Res. (1990) 2J5:69; Cox, S.W. et al. , J.Periodont.Res. (1989) 4:353; Cox, S.W. et al., J.Periodont.Res. (1989) 2 :41; Beighton, D. and Life, J.S.C., Arch Oral.Biol. (1989) 3.4:843), and β- glucuronidase (Lamster I.B. et al., J.Periodontol. (1985) 5_6:139) .

Despite the plethora of such components, there are at present no diagnostic tests available which have been demonstrated to be highly predictive for future bone and attachment loss in periodontal disease. As the breakdown of these components is the ultimate concern of the practitioner, this destruction should be evaluated. Connective tissue-associated proteins such as glycosaminoglycans (Giannobile, W.V. et al., J.Periodontol. (1993) j54.:186) and osteonectin (Bowers,

M.R. et al., J.Periodontol (1989) _50:448) have been found in GCF from patients exhibiting clinical signs of periodontitis. However, no longitudinal studies have been performed which relate these components to future bone or attachment loss.

Osteocalcin (Bone Gla protein or BGP) is a vitamin K-dependent, calcium binding protein of bone and the most abundant noncollagenous bone protein. This extracellular protein has a molecular weight of 5.7 kD and contains three residues of a calcium-binding amino acid, gamma- carboxyglutamic acid (Gla) (Hauschka, P.V. et al., Proc.Natl.Acad.Sci. USA (1975) 72:3925; Lian, J.B. and Friedman, P.A. , J.Biol.Chem. (1978) 253:6623? Price, P.A. et al., Proc.Natl.Acad.Sci, USA (1976) 71:1447; Price, P.A. et al., Proc.Natl.Acad.Sci. USA (1976) 73:3374) . Osteocalcin is synthesized by osteoblasts as a 10 kD precursor. In the presence of calcium, the Gla residues allow specific conformational changes and promote osteocalcin binding to hydroxyapatite and subsequent accumulation in bone matrix (Hauschka, P.V. and Carr, S.A., Biochemistry (1982) 21:638). A fraction of the newly synthesized osteocalcin is released into the circulation and can be measured by radioimmunoassay (RIA) (Price, P.A. et al., J.Clin.Invest. (1980) 66:878; Delmas, P.D., Bone (1992) H:S17-S21).

Nanomolar concentrations of osteocalcin circulate in the blood, and it has been shown that it is a measure of bone turnover (Price, P.A. et al., J.Clin.Invest. (1980) _56_:878). The concentration of osteocalcin in bone is in a 1:1 molar ratio with collagen and in a constant proportion to hydroxyapatite (Hauschka, P.V., Haemostasis (1986) .16:258; Lian, J.B. et al. , Calcif.Tissue Int., (1982) 345:84) . The molecular and related regulatory properties of osteocalcin are that it has a Gla-dependent association to the hydroxyapatite crystal surface, and it is hypothesized that alterations in its hydroxyapatite binding may contribute to regulation of calcium homeostasis (Hauschka, P.V. and Carr, S.A. , Biochemistry (1982) H:638 and Hauschka, P.V. , Haemostasis (1986) 16:258). Furthermore, it exhibits chemoattractant activity for osteoclast progenitor cells (Glowacki, J. and Lian, J.B., Cell Diff. (1987) 21:247), human peripheral monocytes, and macrophages. Moreover, osteocalcin's synthesis in vitro is stimulated by 1,25 (OH)₂ Vit D₃ at concentrations that inhibit collagen synthesis in osteoblasts, promote bone resorption, and promote differentiation of progenitor cells capable of bone resorption (Lian, J.B. and Gundberg, C ., In Clinical Orthopaedics (1988) 226:267) . Finally, serum osteocalcin levels have been used as markers of several metabolic bone diseases such as paget's disease, hyperparathyroidism, and osteogenesis imperfecta.

Summary of the Invention The invention features a method of diagnosing severity of periodontal disease or peri-implant disease in a human patient, involving obtaining a sample of tissue or gingival crevicular or other fluid from the mouth of the patient, and measuring osteocalcin or fragments thereof in the sample as a measure of severity of disease.

In preferred embodiments, intact osteocalcin is measured, and a level about 8 ng/ l or greater of osteocalcin in gingival crevicular fluid ("GCF") is indicative of disease. The invention provides a quantitative measure of periodontal disease, and in addition can detect incipient and early stage disease, and not just advanced disease as current clinical techniques do. In addition, the method of the invention allows not just the detection and quantification of disease, but also permits precise localization of disease, so that treatment can be targeted to the tissues most severely affected.

Other features and advantages of the invention will be apparent from the following detailed description thereof, and from the claims.

Detailed Description GCF Collection

GCF is a secretion which is exuded from the sulcus between gum and tooth. When first formed, GCF does not mix with the saliva in the mouth, but only does so after it leaves the sulcus and enters the oral cavity. In collecting GCF for analysis according to the invention, it is desirable to minimize dilution of GCF with saliva. Thus the saliva in the vicinity of the sulcus from which GCF is to be collected is removed, preferably using cotton rolls in combination with compressed air, which is blown over the region.

Following air-drying, GCF is collected by placement of a strip of porous material such as methylcellulose filter paper into the sulcus and maintaining that material in the sulcus for about 30 seconds. The strip is inserted into the sulcus until slight resistance is detected. Following GCF collection, fluid volume is measured using a Peritron 6000 ™ measuring instrument or other similar device. The collected fluid is eluted from the filter strip using phosphate buffered saline containing proteinase inhibitors. Samples are stored frozen until needed for analysis. Prior to analysis, samples are allowed to thaw at room temperature and they are then assayed for osteocalcin by any conventional technique, such as radioimmunoassay (RIA) or some other standard detection method, e.g., ELISA. Canine Experiments Dogs (two one-year old male beagles) are used to assess the measurement of osteocalcin as a marker of periodontal disease activity. The right quadrants randomly serve as the experimental sites, while the left quadrants serve as control sites. Both animals receive an oral prophylaxis 14 days prior to initiation of the studies. The dogs are exposed to the following procedures.

Experimental periodontitis is induced in the right quadrants in both dogs in the following way: 3-0 silk ligatures are tied around the first through fourth maxillary and mandibular premolars in all four quadrants. The dogs are placed on a plaque-promoting diet of water- moistened Purina dog chow. The ligature induced periodontitis demonstrates heavy bacterial accumulation and leads to an immediate inflammatory response with subsequent resorption of bone. The control quadrants on the left do not have silk ligatures, but instead, oral hygiene procedures are routinely performed. The purpose of the monthly oral hygiene procedures is to maintain health of the gingiva in the control quadrants. There are 4 teeth/quadrant x 4 quadrants/dog x 2 radiographic sites/tooth = a total of 32 control sites and 32 experimental sites.

This condition of health and disease is verified by the use of subtraction radiography and nuclear medicine, which are utilized to quickly and accurately quantify bone resorption and to correlate these parameters with GCF levels of osteocalcin between experimental and control sites. Subtraction radiographic measurements are taken at the beginning of the ligature induced periodontitis period, denoted as baseline, and again at 1-month, 2- months, 4-months, and 6-months after onset of periodontitis, in both the experimental and control sites.

At baseline, 1-month, 2-months, 4-months and 6- months, both the experimental and control sites also have osteocalcin sample measurements taken. There are 4 teeth/quadrant x 4 quadrants/dog 3 sample sites/tooth = a total of 48 control sites and 48 experimental sites. In summary, the totals for sample analysis are as follows:

Time point Control Experimen¬ Quadrants tal Quadrants

Number of Number of Number of Number of

Osteocalcin Radio¬ Osteocalcin Radio¬

Samples graphic Samples graphic Sites Sites

BASELINE 48 32 48 32

1-MONTH 48 32 48 32 2-MONTHS 48 32 48 32

4-MONTHS 48 32 48 32

6-MONTHS 48 32 48 32

TOTALS 240 160 240 160

Ginσival Crevicular Fluid (GCF) Collection

GCF is collected from the mesial, buccal and distal locations of the first premolar through fourth premolar in each quadrant from both dogs. The GCF is collected as described above with methylcellulose strips, and the volume is determined with a Periotron 6000^™. Samples are placed in l mL Eppendorf vials in a solution containing 55 μl of proteinase inhibitors (15 nM aprotenin and 1 mM phenylmethylsulfonylfluoride (PMSF)) in phosphate buffered saline (PBS), pH 7.4. The fluid is subsequently stored on ice, followed by elution from the collection device for a period of 60 minutes at 25°C The samples are then stored at -20 to -80°C until needed for osteocalcin analysis. Osteocalcin Analysis

Frozen samples are allowed to thaw at room temperature for subsequent analysis of osteocalcin by RIA (Power et al., Clinical Chemistry (1989) 15:1408). Into each sample of 25 μ.1, 250 μl of ¹²⁵I human osteocalcin in borate buffer with 0.1% NaN₃ is added. 250 μl of primary anti-human osteocalcin rabbit antisera in borate buffer with 0.1% sodium azide is aliquoted into the mixture of sample/¹²⁵I-osteocalcin. The samples and standards are vortexed thoroughly and covered with Parafilm®. The reaction mixture incubates for 3 hours ± 10 minutes at room temperature. GCF samples are centrifuges at 1300- 1500 x g for 20 minutes at 2-8°C Sample mixtures are aspirated and counted for 1 minute in an auto gamma counter. Values are evaluated according to standard curves and expressed as ng/ l and total ng of osteocalcin/site. The sensitivity of this assay has a calculated value of 0.2 ng/ml and a typical ED90=0.5 ng/ml. Serum and saliva samples from both dogs are evaluated for osteocalcin content for comparison purposes.

Subtraction Radiographic Techniques Standard radiographs taken at Baseline, 2-months, 4-months, and 6-months are used to measure the loss of alveolar bone over time. Radiographs are exposed at 90 KVP, 15 Ma and 0.5 s and developed in an automatic film processor. The radiographs are then analyzed with a computer assisted method as follows: The radiographs are converted into subtraction images using a closed-circuit video system (GE model 4TE5) camera coupled to an analog digital converter capable of storing an entire video frame in solid-state memory in one time frame (1/30 s) . This device, a digital frame grabber (Imaging Technologies, PC Vision Frame Grabber) is capable of digitizing and storing image frames and has an information capacity of more than two million bits (512 x 512 pixels by 8 bits deep) utilizing an IBM PC AT™ as the central processing unit.

The images are placed in spatial register with the aid of a micromanipulator capable of orthogonal movement in two dimensions plus rotation in the same plane (Klinger) . The first radiograph is stored in the computer's fixed disk drive using the full 256 (8 bits) gray levels of resolution. Registration of the second film to be compared is facilitated by continuously "grabbing" the image of the second radiograph while simultaneously displaying the subtraction between the first and second radiograph on the same video screen. Since slight variations in film processing or voltage to the X-ray tube may result in differences in contrast in the resultant films, the non-parametric gamma correction algorithm of Ruttiman (Ruttiman, U.E. et al., J.Periodont.Res. (1986) 21-486) is used to correct differences in contrast between the two films. Registration is achieved when the video screen is a uniform gray and the anatomical structures such as teeth are not clearly discernible.

To measure the loss of bone between the two radiographs, the distance between the CEJ and the height of the alveolar crest is determined for the mesial and distal root surfaces. The alveolar bone height is taken either at the alveolar crest (for longitudinal bone loss) or at the point where the PDL space becomes indistinct (at the base of the apparent infrabony defects) . This measurement is converted for the magnification of the radiograph in the image processing system. Millimeters of bone loss at each site are determined from each radiograph. The radiographic images at 1-month are subtracted from the radiographs taken at baseline. Radiographs at 2-months are subtracted from the radiographs taken at baseline and so forth through 6 months. This provides multiple separate rates between each radiographic time interval (0, 1, 2, 3, 4, 5, and 6 months) . Previous studies have shown that the error of this method in estimating bone loss is less than 0.08 mm on the original radiograph.

Other embodiments are within the following claims.

What is claimed is:

Claims

1. A method of diagnosing severity of periodontal disease or peri-implant disease in a human patient, said method comprising a) obtaining a sample of tissue or gingival crevicular or other fluid from the mouth of said patient, and b) measuring osteocalcin or fragments thereof in said sample as a measure of severity of disease, wherein a higher osteocalcin or osteocalcin fragment measurement indicates greater severity of disease.

2. The method of claim 1, wherein an osteocalcin or osteocalcin fragment level above about 8 ng/ml in said tissue or fluid indicates the presence of a periodontal or peri-implant disease state.