Alkaline phosphatase induces the mineralization of sheets of collagen implanted subcutaneously in the rat. W Beertsen, T van den Bos J Clin Invest. 1992;89(6):1974-1980. https://doi.org/10.1172/JCI115805. Research Article To determine whether alkaline phosphatase (ALP) can cause the mineralization of collagenous matrices in vivo, bovine intestinal ALP was covalently bound to slices of guanidine-extracted demineralized bovine dentin (DDS). The preparations were implanted subcutaneously over the right half of the rat skull. Control slices not treated with the enzyme were implanted over the left half of the skull of the same animals. Specimens were harvested after periods varying from 1 to 4 wk. It was shown that ALP-coupled DDS rapidly accumulated hydroxyapatite crystals. 4 wk after implantation, the content of calcium and phosphate per microgram of hydroxyproline amounted up to 80 and 60%, respectively, of that found in normal bovine dentin. Our observations present direct evidence that ALP may play a crucial role in the induction of hydroxyapatite deposition in collagenous matrices in vivo. Find the latest version: https://jci.me/115805/pdf Alkaline Phosphatase Induces the Mineralization of Sheets of Collagen Implanted Subcutaneously in the Rat Wouter Beortsen,* and Theo van den Bos** *Experimental Oral Biology Group, Department ofPeriodontology, Academic Centrefor Dentistry Amsterdam; and tDepartment of Cell Biology and Histology, Faculty ofMedicine, University ofAmsterdam, 1066 EA Amsterdam, The Netherlands Abstract that abolishes enzymatic activity causes profound skeletal hy- To determine whether alkaline phosphatase (ALP) can cause pomineralization. Also, the presence of the enzyme in matrix the mineralization of collagenous matrices in vivo, bovine intes- vesicles, the sites of early formation of mineral crystallites in tinal ALP was covalently bound to slices of guanidine-extracted cartilage and bone, suggests a role of ALP in mineraliza- demineralized bovine dentin (DDS). The preparations were im- tion (5-8). planted subcutaneously over the right half of the rat skull. Con- Several possible actions of the enzyme in mineralization trol slices not treated with the enzyme were implanted over the processes have been proposed (2, 3): increasing the local con- left half of the skull of the same animals. Specimens were har- centration of inorganic phosphate, destructing locally mineral vested after periods varying from 1 to 4 wk. It was shown that crystal growth inhibitors, acting as PI-transporter or acting as ALP-coupled DDS rapidly accumulated hydroxyapatite crys- Ca-binding protein. Robison (1) was first in proposing that tals. 4 wk after implantation, the content of calcium and phos- ALP hydrolyzes organic phosphate esters, thus producing an phate per microgram of hydroxyproline amounted up to 80 and excess of free inorganic phosphate that would cause local su- 60%, respectively, of that found in normal bovine dentin. Our persaturation and initiate the biomineralization process. Recently, Tenenbaum and Heersche (9) have shown in ex- observations present direct evidence that ALP may play a cru- plant cultures ofembryonic periosteum that in situ mineraliza- cial role in the induction ofhydroxyapatite deposition in collage- tion nous matrices in vivo. (J. Clin. Invest. 1992. 89:1974-1980.) of osteoid occurs upon the addition of external phos- * , * phates, such as ,B-glycerophosphate. In line with this, we have Key words: biomaterials dentin implants organic phos- adduced evidence that ALP can cause the remineralization of phates * remineralization decalcified dentin slices when incubated in media containing f3-glycerophosphate (10, 11). Introduction A criticism that applies to many in vitro mineralization Alkaline phosphatases (orthophosphoric-monoester phospho- studies is that exogenous phosphate esters are added to the hydrolase, alkaline optimum, EC 3.1.3.1.) are cell surface gly- culture systems in relatively high concentrations (up to 10 coproteins that hydrolyze a variety of monophosphate esters. mM). Although it has been argued that about 10 mM organic Usually three isoenzymes are distinguished: liver/bone/kidney phosphate occurs in circulation in vivo (9), other authors doubt (L/B/K),' placental, and intestinal. Although the bone isoen- whether these organic phosphates are hydrolyzable under physi- zyme has long been thought to play a role in the mineralization ological conditions (12), and whether there is sufficient sub- of bone and cartilage (1) and is widely used as a marker of strate in extracellular fluids to exert a significant effect on the biomineralization, its physiological significance is still a matter concentration of inorganic phosphate (13, 14). In addition, ac- of debate. The fact that the enzyme is widely distributed in the cording to some investigators, the rate of hydrolysis of phos- body, in calcifying as well as noncalcifying tissues, raises ques- phate esters by ALP at physiological pH would be too low to be tions as to the specificity of the relationship between alkaline relevant to the process of mineralization (15). phosphatase (ALP) and mineralization (2). It was the aim of the present study to determine whether Evidence for its role has come up from studies on hypo- ALP can cause the mineralization of collagenous substrates phosphatasia, an inherited disorder of osteogenesis character- under in vivo conditions. For this purpose ALP of intestinal ized by a deficient L/B/K ALP (3). Weiss and co-workers (4) origin was covalently bound to sheets of collagen, and thus have demonstrated that a mutation in the L/B/K ALP gene created biomaterial implanted subcutaneously in the rat. Methods Address correspondence and reprint requests to Dr. W. Beertsen, De- partment of Periodontology, Academic Centre for Dentistry Amster- Preparation of dentinal collagen sheets. Bovine permanent incisors dam, Louwesweg 1, 1066 EA Amsterdam, The Netherlands. were collected at the local slaughterhouse immediately after killing of Receivedfor publication 4 June 1991 and in revisedform 9 January the animals (age 1-3 yr) and frozen at -80°C until use. After thawing, 1992. the gingiva and periodontal ligament were removed and the roots cut with a diamond disk parallel to their longitudinal axis from the apex to 1. Abbreviations used in this paper: ALP, alkaline phosphatase; beta- the cervical area under constant irrigation with tap water and split with GP, beta-glycerophosphate; DDS, demineralized dentin slices; L/B/K, a chisel. The roots were then cleaned and freed from pulp. They were liver/bone/kidney; pNPP, p-nitrophenylphosphate. washed with ice-cold PBS in the presence of proteinase inhibitors (16), and the outer dentin (containing the mantle dentin layer and the ce- J. Clin. Invest. mentum) was removed with a diamond disk under cooling with tap © The American Society for Clinical Investigation, Inc. water. 0021-9738/92/06/1974/07 $2.00 Demineralized dentin slices (DDS) were prepared after demineral- Volume 89, June 1992, 1974-1980 ization of the roots with 0.6 M HO or 0.5 M acetic acid at 4°C (10). 1974 W. Beertsen and T. van den Bos After demineralization, sections were cut on a cryotome set at 30 Am. blue, or according to the Von Kossa method. Ultrathin sections were The DDS were further extracted with 4 M guanidine. HCl and 0.4 M cut with a diamond knife, stained with uranyl acetate and lead citrate, EDTA (pH 7.5) for 3 d at 4VC. Before use, the DDS were washed in and examined in an electron microscope (EM lOC; Carl Zeiss, Inc., double-distilled water for I h and placed in double-distilled water sup- Thornwood, NY). plemented with antibiotics (10) at 4VC overnight. They were then Enzyme histochemistry. To study the distribution ofALP bound to transferred to IMDM. The DDS were free ofcalcium but still contained the DDS, cryostat sections were treated according to the indoxyl-tetra- some collagen-bound phosphate residues (0.039±0.015 Mg/Mg hypro zolium salt method (18, 19). 5-Bromo4-chloro-3-indolylphosphate after demineralization with HCI). was used as a substrate. Control sections were incubated without sub- Preparation ofdura mater sheets. Pieces ofhuman dura mater were strate. used as supplied by the manufacturer (see last paragraph of Methods). Biochemical determinations. ALP activity was determined as fol- They contained 0.011±0.002 Ag collagen-bound phosphate/Mg hypro. lows: specimens were extracted in 0.2 ml glycine buffer (0.1 M glycine, Binding ofALP. Bovine intestinal ALP was covalently bound to the I mM MgCl2, 0.1 mM ZnCl2, pH 10.5) containing 0.1% Triton X-100 dentinal collagen sheets by using the coupling agents glutaraldehyde or for 1 h at 40C. Then, samples were supplemented with 2 ml glycine carbodiimide (17). buffer (370C). After 10 min, p-nitrophenylphosphate (pNPP) was Glutaraldehyde coupling. The DDS were incubated in PBS contain- added as a substrate (final concentration 6 mM) and the optical density ing 0.1% glutaraldehyde for 2 h at 20C in the presence of ALP (700 monitored at 405 nm by using a Beckman 25 spectrophotometer U/ml). The material was then extensively washed with PBS and stored (model 25; Beckman Instruments, Inc., Fullerton, CA). Enzyme activ- at 4VC in 0.1 M glycine buffer (pH 10.5) containing I mM Mg2" and ity (U) was expressed as micromole pNP released per minute (at 370C 0.1 mM Zn2". and pH 10.5). Carbodiimide coupling. The DDS were incubated for 2 d at 4°C in Hypro was determined colorimetrically (20, 21). Phosphate was 0.13 M 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. HCI (pH 4.5) determined according to the method of Kirkpatrick and Bishop (22), in the presence of ALP (700 U/ml). They were then exhaustively and and calcium by atomic absorption spectrometry. successively washed in distilled water, 1 M NaCG in 0.1 M Na-acetate X-ray diffraction. DDS were harvested and powdered with a pestle (pH 4.0), distilled water, 0.1 M NaHCO3 (pH 8.3), and finally distilled and mortar in liquid nitrogen.