CI N IFIC
TI LE
J Oral Maxillo'ac Surg 48:162-169, 1990
Purification of Rabbit Bone Morphogenetic Protein Derived From Bone, Dentin, and Wound Tissue After Tooth Extraction KAZUHISA BESSHO, DDS,* TOSHIROU TAGAWA, DDS, DMSc,t AND MUTSUO MURATA, DDS, MD, DMSc:f: Bone morphogenetic protein (BMP) was extracted from bone matrix, dentin matrix, and wound tissue after tooth extraction in rabbits, and purified. These purified fractions were shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (SDS-PAGE), and induced new bone in situ in 3 weeks when implanted into the calf muscles of Wistar rats. The dentin matrix-derived BMP was different from the other two types in molecular weight and the properties revealed in the process of purification. However, each tissue-derived BMP was shown to induce new bone growth in a bioassay of xenogenic implantation. For this reason, BMP is thought to have subunits with certain commonalities in different tissue.
After it was found that BMP can be solubilized with guanidine-HCl' -" and urea,' its isolation and purification have rapidly progressed. Each tissuederived BMP has been extracted from a variety of animals, and there are reports on the species specificity of these proteins,2.4·s However, no study has been carried out to compare BMP of different tissue origin. To examine similarities and differences in BMP of different tissue origin within a species, we extracted and purified BMP from the bone, dentin, and wound tissue after tooth extraction of rabbits.
Since Urist reported on bone morphogenetic protein (BMP) in 1965,1 many researchers have performed various studies for the purpose of clinical application of this protein. BMP exists in the bone matrix.I" osteosarcoma tissue,2.6.7 and dentin matrix,8.9 and induces differentiation of mesenchymal-type cells into cartilage and bone. If BMP could be easily obtained, its clinical applications would be very significant and wide-ranging, eg, jaw reconstruction, fracture reduction, orthognathic surgery, alveolar ridge augmentation, and dental implants. However, there are some unresolved problems such as amino acid sequences, synthesis in great quantities, and proper non biologic nonimmunologic delivery systems.
Materials and Methods DISSOCIATIVE EXTRACTION
Specimens were obtained from l-year-old Japanese White rabbits. The respective starting materials were the long bone immediately after the animals were killed, the dentin immediately after tooth extraction, and the wound tissue on day 14 after extraction of a mandibular molar. The long bone was prepared as follows: The epiphyseal end, periosteum, and bone marrow were mechanically removed, and the remaining cortical bone was washed in cold distilled water; the washed bone was then frozen in liquid nitrogen and crushed in a
Received from the Department of Oral Surgery, Faculty of Medicine, Mie University, Mie, Japan. * Graduate Student. t Lecturer. t Professor and Chief. Address correspondence and reprint requests to Dr Bessho: Department of Oral Surgery, Faculty of Medicine, Mie University, 2-174 Edobashi, Tsu , Mie, Japan . © 1990 American Association of Oral and Maxillofacial Surgeons
0278-2391/90/4802-0007$3.00/0
162
BESSHO, TAGAWA. AND MURATA Wiley mill (Ikemoto Scientific Technology, Japan) to a particle size of 1 mrrr'. The dentin was similarly crushed after mechanically removing the peripheral soft tissue and pulp. The wound tissue after tooth extraction was homogenized in cold distilled water with a Polytron homogenizer (Kinernatica, Switzerland). The three types of specimens were washed in distilled water, defatted in chloroform-methanol (1: 1) for 12 hours and demineralized in 0.5 N HCI for 72 hours at 4°C. The demineralized specimens were redefatted in chloroform-methanol (1: I) for 6 hours, rewashed in distilled water, chemically extracted with 10 vol of4 molJL guanidine-HClJ50 mmolJL Tris-HCI at pH 7.4 containing neutral protease inhibitors (100 mmol/L 6-aminohexanoic acid/5 mmol/L benzamidine HClIl mmol/L benzyl sulphonyl fluoride) for 48 hours at 4°C. These extracts were centrifuged (10,000 x g for 30 minutes at 4°C), and each extract was divided into G-S (guanidine-HCI soluble) and G-I (guanidine-HCl insoluble) fractions. The G-I fraction was washed in distilled water at 4°C and lyophilized. The G-S fraction was concentrated 1:5 by ultrafiltration (Diaflo membrane YM10, Amicon, Ireland) for separation into a concentrated fraction and a filtrated fraction of less than 10
FIGURE I. Implantation of the lyophilized pellet intothe calf muscle of a Wistar rat.
163 kd in molecular weight. The filtrated fraction was dialyzed against distilled water at 4°C with a molecular cutoff size of 3.5 kd, and lyophilized. Three volumes of chilled ethanol ( - 20°C) were added to the concentrated fraction, and the mixture was left standing for 4 hours at 4°C, separated by centrifugation (10,000 x g for 30 minutes at 4°C) into an ethanol-precipitate (Et-ppt) fraction and an ethanolsupernatant (Et-sup) fraction. Each of these fractions was dialyzed against distilled water at 4°C with a molecular cutoff size of 8 kd, and centrifuged (70,000 x g for 30 minutes at 4°C) for separation into W-S (water soluble) and W-I (water insoluble) fractions. These fractions were lyophilized. FRACTIONATION BY LIQUID CHROMATOGRAPHY The active fraction in this dissociative step was dissolved in 4 mmol/L guanidine-HCl at pH 5.2 (25 mg/5 mL) and was analyzed (flow rate, 20 mL/h) in a Sephacryl S-200 column (2.6 x 100 ern) which had
FIGURE 2. Softx-ray radiograph taken 3 weeks afterimplantation. A, This implant is the lyophilized specimen of bone rnatrix-derived G-S fraction dissolved in telopeptide-free type I collagen solution. B, This implant is the lyophilized specimen of bone matrix-derived fraction I obtained byTSKG2000 SWchromatography dissolved in telopeptide-free type I collagen solution.
164
PURIFICATION OF BONE MORPHOGENETIC PROTEIN
l.o
,
v,
,
25K
I
!
taining 10 mmol/L NaCl and was prepared and equilibrated with the same solution. The sample solution was applied to a MONO Q HR 515 column (5.0 X 50 mm; Pharmacia) and was analyzed (flow rate, 0.5 mUmin) at an NaCl concentration gradient of 10 mrnol/L to 1 mol/L. We prepared the bonederived material and the wound-derived material as follows: The active fraction obtained by SephacryI S-200 chromatography was dissolved in 6 rnol/L urea/50 rnmol/L sodium acetate at pH 4.8 (5 mg/ml.) containing 10 mmol/L NaCl and was applied to a TSK SP-5PW column (8.0 x 75 mm; Tosoh, Japan) which had been prepared and equilibrated with the same solution. Analysis was made (flow rate, 0.5 mUmin) at an NaCl concentration gradient of 10 to 500 mmol/L. The analyzed active fraction was' dissolved in 6 rnol/L urea/50 mmoUL phosphate buffer at pH 7.3 (5 mg/ml.) and was applied to a hydroxyapatite (HAP) column (5.0 x 100 mm), which had been prepared and equilibrated with the same solution. Analysis was made (flow rate, 0.1 mUmin) at a phosphate buffer concentration gradient of 50 to 300 mmollL. The analyzed active fraction was dis-
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TI LE
J Oral Maxillo'ac Surg 48:162-169, 1990
Purification of Rabbit Bone Morphogenetic Protein Derived From Bone, Dentin, and Wound Tissue After Tooth Extraction KAZUHISA BESSHO, DDS,* TOSHIROU TAGAWA, DDS, DMSc,t AND MUTSUO MURATA, DDS, MD, DMSc:f: Bone morphogenetic protein (BMP) was extracted from bone matrix, dentin matrix, and wound tissue after tooth extraction in rabbits, and purified. These purified fractions were shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (SDS-PAGE), and induced new bone in situ in 3 weeks when implanted into the calf muscles of Wistar rats. The dentin matrix-derived BMP was different from the other two types in molecular weight and the properties revealed in the process of purification. However, each tissue-derived BMP was shown to induce new bone growth in a bioassay of xenogenic implantation. For this reason, BMP is thought to have subunits with certain commonalities in different tissue.
After it was found that BMP can be solubilized with guanidine-HCl' -" and urea,' its isolation and purification have rapidly progressed. Each tissuederived BMP has been extracted from a variety of animals, and there are reports on the species specificity of these proteins,2.4·s However, no study has been carried out to compare BMP of different tissue origin. To examine similarities and differences in BMP of different tissue origin within a species, we extracted and purified BMP from the bone, dentin, and wound tissue after tooth extraction of rabbits.
Since Urist reported on bone morphogenetic protein (BMP) in 1965,1 many researchers have performed various studies for the purpose of clinical application of this protein. BMP exists in the bone matrix.I" osteosarcoma tissue,2.6.7 and dentin matrix,8.9 and induces differentiation of mesenchymal-type cells into cartilage and bone. If BMP could be easily obtained, its clinical applications would be very significant and wide-ranging, eg, jaw reconstruction, fracture reduction, orthognathic surgery, alveolar ridge augmentation, and dental implants. However, there are some unresolved problems such as amino acid sequences, synthesis in great quantities, and proper non biologic nonimmunologic delivery systems.
Materials and Methods DISSOCIATIVE EXTRACTION
Specimens were obtained from l-year-old Japanese White rabbits. The respective starting materials were the long bone immediately after the animals were killed, the dentin immediately after tooth extraction, and the wound tissue on day 14 after extraction of a mandibular molar. The long bone was prepared as follows: The epiphyseal end, periosteum, and bone marrow were mechanically removed, and the remaining cortical bone was washed in cold distilled water; the washed bone was then frozen in liquid nitrogen and crushed in a
Received from the Department of Oral Surgery, Faculty of Medicine, Mie University, Mie, Japan. * Graduate Student. t Lecturer. t Professor and Chief. Address correspondence and reprint requests to Dr Bessho: Department of Oral Surgery, Faculty of Medicine, Mie University, 2-174 Edobashi, Tsu , Mie, Japan . © 1990 American Association of Oral and Maxillofacial Surgeons
0278-2391/90/4802-0007$3.00/0
162
BESSHO, TAGAWA. AND MURATA Wiley mill (Ikemoto Scientific Technology, Japan) to a particle size of 1 mrrr'. The dentin was similarly crushed after mechanically removing the peripheral soft tissue and pulp. The wound tissue after tooth extraction was homogenized in cold distilled water with a Polytron homogenizer (Kinernatica, Switzerland). The three types of specimens were washed in distilled water, defatted in chloroform-methanol (1: 1) for 12 hours and demineralized in 0.5 N HCI for 72 hours at 4°C. The demineralized specimens were redefatted in chloroform-methanol (1: I) for 6 hours, rewashed in distilled water, chemically extracted with 10 vol of4 molJL guanidine-HClJ50 mmolJL Tris-HCI at pH 7.4 containing neutral protease inhibitors (100 mmol/L 6-aminohexanoic acid/5 mmol/L benzamidine HClIl mmol/L benzyl sulphonyl fluoride) for 48 hours at 4°C. These extracts were centrifuged (10,000 x g for 30 minutes at 4°C), and each extract was divided into G-S (guanidine-HCI soluble) and G-I (guanidine-HCl insoluble) fractions. The G-I fraction was washed in distilled water at 4°C and lyophilized. The G-S fraction was concentrated 1:5 by ultrafiltration (Diaflo membrane YM10, Amicon, Ireland) for separation into a concentrated fraction and a filtrated fraction of less than 10
FIGURE I. Implantation of the lyophilized pellet intothe calf muscle of a Wistar rat.
163 kd in molecular weight. The filtrated fraction was dialyzed against distilled water at 4°C with a molecular cutoff size of 3.5 kd, and lyophilized. Three volumes of chilled ethanol ( - 20°C) were added to the concentrated fraction, and the mixture was left standing for 4 hours at 4°C, separated by centrifugation (10,000 x g for 30 minutes at 4°C) into an ethanol-precipitate (Et-ppt) fraction and an ethanolsupernatant (Et-sup) fraction. Each of these fractions was dialyzed against distilled water at 4°C with a molecular cutoff size of 8 kd, and centrifuged (70,000 x g for 30 minutes at 4°C) for separation into W-S (water soluble) and W-I (water insoluble) fractions. These fractions were lyophilized. FRACTIONATION BY LIQUID CHROMATOGRAPHY The active fraction in this dissociative step was dissolved in 4 mmol/L guanidine-HCl at pH 5.2 (25 mg/5 mL) and was analyzed (flow rate, 20 mL/h) in a Sephacryl S-200 column (2.6 x 100 ern) which had
FIGURE 2. Softx-ray radiograph taken 3 weeks afterimplantation. A, This implant is the lyophilized specimen of bone rnatrix-derived G-S fraction dissolved in telopeptide-free type I collagen solution. B, This implant is the lyophilized specimen of bone matrix-derived fraction I obtained byTSKG2000 SWchromatography dissolved in telopeptide-free type I collagen solution.
164
PURIFICATION OF BONE MORPHOGENETIC PROTEIN
l.o
,
v,
,
25K
I
!
taining 10 mmol/L NaCl and was prepared and equilibrated with the same solution. The sample solution was applied to a MONO Q HR 515 column (5.0 X 50 mm; Pharmacia) and was analyzed (flow rate, 0.5 mUmin) at an NaCl concentration gradient of 10 mrnol/L to 1 mol/L. We prepared the bonederived material and the wound-derived material as follows: The active fraction obtained by SephacryI S-200 chromatography was dissolved in 6 rnol/L urea/50 rnmol/L sodium acetate at pH 4.8 (5 mg/ml.) containing 10 mmol/L NaCl and was applied to a TSK SP-5PW column (8.0 x 75 mm; Tosoh, Japan) which had been prepared and equilibrated with the same solution. Analysis was made (flow rate, 0.5 mUmin) at an NaCl concentration gradient of 10 to 500 mmol/L. The analyzed active fraction was' dissolved in 6 rnol/L urea/50 mmoUL phosphate buffer at pH 7.3 (5 mg/ml.) and was applied to a hydroxyapatite (HAP) column (5.0 x 100 mm), which had been prepared and equilibrated with the same solution. Analysis was made (flow rate, 0.1 mUmin) at a phosphate buffer concentration gradient of 50 to 300 mmollL. The analyzed active fraction was dis-
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