Effect of aluminum on bone matrix inductive properties

Kidney International, Vol. 38 (/990), PP. 1141—1 145

Effect of aluminum on bone matrix inductive properties JIAN-MIN ZHU, WILLIAM HUFFER, and ALLEN C. ALFREY Departments of Medicine and Pathology, Denver V.A. Hospital, University of Colorado Medical Center, Denver, Colorado, USA, and Division of Endocrinology and Nephrology, Xian Medical University, Xian, People's Republic of China

Effect of aluminum on bone matrix inductive properties. The effect of aluminum on the bone inductive properties of implanted bone matrix

present at the osteoid junction between calcified and non-

was studied in rats. After decalcification femur sections were placed in either 0.1 or 0.01 M AICI3 or a solution of similar pH without Al for 24 hours. Following 28 days of implantation in subcutaneous pouches the 6 mg/kg in the matrix aluminum content was 3232 1020 and 51 pretreated with 0.1 and 0.01 M AId3. At the same time period following implantation the matrix calcium content was 794 539 and 3038 692 mmol/kg in the 0.1 and 0.01 M AId3 pretreated groups versus 4252 579 mmol/kg in the control group (P < 0.01). In the control group bone histology showed extensive osteoblastic and osteoclastic remodeling,

Historically aluminum has been used as a tanning agent because of its ability to cause intermolecular cross linkage of collagen fibrils [11]. Thus it would be expected that aluminum could also increase intermolecular cross linkage of bone collagen, bonding the aluminum to matrix, which could alter its ability to induce bone formation.

tetracycline labeling and bone formation. In contrast all of these histological features were virtually absent in aluminum treated matrix. Aluminum-induced resistance of bone matrix to collagenase degradation and restoration of bone inductive properties with chelation suggests that aluminum forms intermolecular cross links between collagen

calcified bone in aluminum-associated osteomalacia [10].

The present study was undertaken to determine how pretreatment of decalcified bone matrix with aluminum would affect its bone inductive properties and its ability to calcify.

Methods

fibrils. Aluminum-induced cross links of collagen fibrils and/or its

Preparation of bone matrix

effects on bone inductive proteins present in bone matrix could explain the mechanism by which aluminum induces osteomalacia.

Bone matrix was prepared using a modification of the method

described by Urist et al [121. Femurs were immediately removed from dead male Sprague-Dawley rats and sawed perpen-

The association between aluminum and osteomalacia in hu-

mans with renal disease has been well established [IL In addition, aluminum causes osteomalacia in a number of different animal species including dogs, rats and pigs [2—4]. Aluminum has been shown to produce a number of effects which could alter bone formation. It has been shown in organ cultures that aluminum inhibits the synthesis of bone alkaline phosphatase and acid phosphatase, implying that aluminum may affect osteoblast and osteoclast function [5]. Furthermore, aluminum has been shown to cause a reduction in the number of osteoblasts and osteoclasts suggesting it could be directly toxic to these cells (6]. Other studies have found that aluminum has a

dicular to the shaft to obtain sections approximately 2.4 mm thick. The bone sections were cleaned of periosteal and endosteal tissues, washed several times in sterile distilled water and decalcified in 0.6 M HCI (I g tissue in 100 ml HCI) at 2°C for 48 hours. Under these conditions bone has been shown to be almost completly decalcified and to have minimal denaturation of bone matrix [12]. The bone was then removed from the acid and washed with sterile distilled water. After washing, the bone matrix was placed in 0.1 M or 0.01 M AId3 or 0.001 M HCI for

24 hours at 2°C. The pH of the respective solutions was 3.6, 3.71 and 3.55 at the initiation of treatment and 3.67, 4.1 and 3.61 after the 24 hour incubation. In preparation for implantation the

bone matrix was washed with sterile distilled water. Additional bone sections were decalcified and placed in either 0.001 M HCI Two features make aluminum associated osteomalacia or 0.1 M AICI3 as described above. These sections were then unique. It is resistant to vitamin D treatment [8] and, unlike placed in a solution containing either 20 molIml deferroxamine most other osteomalacic states, it occurs in the setting of (DFO) or ethylenediamine tetracetic acid (EDTA) for an addihyperphosphatemia, mild hypercalcemia and an increased cal- tional 24 hours.

direct effect on preventing the physical and chemical events leading to calcium-phosphate crystal formation [7].

cium X phosphate product [9]. These findings imply that the Implantation of bone matrix

osteomalacia could result from an aluminum-induced alteration in bone matrix which renders it uncalcifiable. Consistent with

this is histochemical staining which reveals that aluminum is

Male weanling Spraque-Dawley rats approximately four weeks old (weighing 80 to 100 g) were used for the recipients of the bone matrix. Two aluminum-treated matrix specimens and

two control (HCI treated) specimens were implanted under sterile conditions into individual subcutaneous pouches (four)

Received for publication April 16, 1990 and in revised form July 23, 1990 Accepted for publication July 28, 1990

dissected into the ventral abdominal wall of each rat. The implants were removed at four and eight weeks following

© 1990 by the International Society of Nephrology

implantation. Matrix, untreated with aluminum, was implanted 1141

Zhu et a!: Aluminum effect on bone induction

1142

into four rats which received 1.5 mg/kg/day aluminum i.p. for 28 days following the implantation of matrix.

6000

Collagenase incubation Decalcified bone matrix was prepared as described above and

4500 4000

placed in 0.1, 0.01, 0.001 M Aid3 or 0.001 M HCI at 2°C for 24 hours. The matrix was then washed, dried at room temperature

3000

and weighted. Each matrix specimen was placed in 4 ml of water containing 25 mg of collagenase (Worthington Biochemical Corporation, Freehodi, New Jersey, USA) for 15 hours at 37°C. The residual matrix was removed from the collagenase, air dried at room temperature for 48 hours and reweighed.

In vitro nucleation of calcium-phosphate crystals A modification of the method described by Strates and

5500 5000

3500

2500

2000

< 1500 1000 500

0

0

28

56

Time, days concentration in matrix exposed to 0.1 M AId3 1. Aluminum Neuman [13] was used. The calcification solution consisted of Fig. 8) and 56 (N = 6) days before implantation (N = 4), and 28 (N 15 m barbital buffer, pH 7.4, 100 mM NaCI, 1.7 mM CaCl2 and following implantation.

1.7 m potassium phosphate. Three to four mg samples of bone matrix pretreated with either 0.1 or 0.01 M Aid3 or 0.001 M HCI were placed in the solution for 24 hours at 35°C. The specimens were then removed from the solution and analyzed for calcium as described below. Analytical procedures For aluminum and calcium determinations the bone matrix

was dried to a constant weight at 105°C. The matrix was extracted with a saturated solution of EDTA at 25°C for three days. Calcium and aluminum determinations were performed by previously described methods [14, 15]. For histological studies rats with matrix implants were given

tetracycline (16 mg/kg) five, three and one day(s) prior to removal of the implants. Following removal, the bone matrix was immediately placed into cold methanol. It was then embedded in glycol methacrylate for processing. The non-decalcified specimens were stained with either hematoxylin and eosin, acid fuschin or Von Kossa stain for routine histological studies or aurin tricarboxylic acid for aluminum. All results are given as mean I SD. Statistical comparisons

were carried out with Student's f-test for two groups and analysis of variance for multiple group comparisons. Results The tissue aluminum in unimplanted matrix pretreated with 1020 after 28 days of 0.1 si Aid3 was 4988 473, 3232 implantation and 2855 865 mg/kg after 56 days of implantation (Fig. 1). The tissue aluminum in unimplanted matrix pretreated with 0.01 M AICI3 was 395 4 prior to implantation and 51 6 mg/kg after 28 days of implantation. In the matrix treated only with HCI aluminum was less than 2 mg/kg both before and after implantation.

FIg. 2. Tetracycline labels (2 light lines on surface, arrows) in untreated matrix after 28-days of implantation.

Histological examination of the control tissue following 28 and 56 days of implantation revealed extensive tetracycline labeling, bone formation and remodeling with numerous osteo-

blasts and osteoclasts (Figs. 2, 3). In contrast in the matrix pretreated with 0.1 M AId3 following both 28 and 56 days of implantation, there was almost total absence of bone formation

as shown by no tetracycline labeling and little evidence of The tissue calcium content in unimplanted bone matrix cellular activity (Fig. 4). Of further interest was the fact that the treated with AId3 or HCI was less than I mmol/kg dry weight. implanted matrix pretreated with 0.1 M AICI3 was virtually Following 28 days of implantation the tissue calcium content was 4252 579 in the control tissue, 3038 692 in the tissue pretreated with 0.01 M AId3 and 794 539 mmol/kg in the tissue pretreated with 0.1 M AId3 (P < 0.01 between all groups). Following 56 days of implantation the calcium was 4642 242 in the control tissue and 593 380 in the tissue

unchanged from its pre-implanted appearance. It had incited little if any tissue reaction and had undergone virtually no degradation. In contrast the untreated matrix had assumed the appearance of the implanted femur section prior to decalcification (Fig. 4). In the matrix pretreated with 0.01 M AICI3 and removed 28 days following implantation, there were islands of

pretreated wth 0.1 M AICI3 as compared to 6354 in undecalcified normal rat bone.

bone formation within the matrix but much less bone than in the

242 mmol/kg

control tissue. The control group showed no staining with the

1143

½

•

Pt'

a

I

Fig. 4. Matrix treated with 0.1 si Aid3 (left) and control (right) after 28 days of implantation. There is no cellular activity or bone formation in the treated matrix. 100

S.

S

-g

'I

2

p

4'-

Zhu et a!: Aluminum effect on bone induction

90 80 70 60 50

- 40 30

Fig. 3. Osteoblastic and osteoclastic activity with bone formation and osteoid deposition in untreated matrix implanted for 28 days. Abbrevi-

ations are: Ost, osteoid; Osb, osteoblast; Cb, calcified bone; Oci, osteoclast; M, marrow.

20 10 0

I

iLii

A

Fig. 5. Percent of matrix remaining after 15 hours of incubation with

aluminum stain, whereas, the aluminum group showed diffuse staining of the entire matrix. Following chelation with DFO and EDTA the bone matrix aluminum was less than 3 mg/kg in both control and aluminum pre-treated matrix before and after implantation. In the DFO groups, calcium content of the implant was 2691 279 versus 2693 407 mmollkg in the aluminum pre-treated and control tissues (P = NS). Similarly in the EDTA groups there was no difference between calcium content of aluminum pre-treated and control matrix (2612 693 vs. 2789 104 mmollkg, P = NS).

collagenase in control matrix treated with HCI and matrix treated with 0.001 M, 0.01 M and 0.1 MAId3 (each group N = 4).

a metastable solution. The tissue calcium content was 109 18 and 34 5 mmol/kg in matrix pretreated with 0.1 and 0.01 M

AId3, respectively (P < 0.01) as compared to 14 2 mmol/kg in the control matrix (P <0.01). To determine the effect of extracellular aluminum on bone induction, bone matrix was implanted into four rats which received i.p. aluminum injections daily over a 28 day period following implantation. Plasma aluminum levels were 67.5 8.22 pg/liter six hours after aluminum injections as compared to 5 1 tgIliter in controls. At the end of the study matrix calcium was 3700 455 mmol/kg in the aluminum treated group as compared to 3790 226 mmollkg in the controls (P = NS).

Bone matrix aluminum prior to collagenase treatment was 4988 473, 395 4 and 115 13 mg/kg in the three groups pretreated with aluminum (0.1, 0.01 and 0.001 M AId3). The percent of bone matrix which had not undergone degradation after collagenase treatment was 93 4, 70 18, 33 22% in the three aluminum groups (Fig. 5) as compared to 15 8% in the controls (P < 0.05 between all groups). In contrast to the in vivo results, pretreatment of bone matrix

These studies show that aluminum treatment of decalcified bone matrix markedly inhibits its bone inductive properties. In

with aluminum enhanced the calcification resulting in vitro from

addition, aluminum treatment was found to increase bone

Discussion

1144

Zhu et a!: Aluminum effect on bone induction

matrix resistance to degradation by collagenase and enhance its remodeling was observed. However, in the aluminum treated in vitro calcification from a metastable solution. matrix there was virtually no cellular activity and very little An increased resistance of collagen to collagenase degrada- bone formation in the implant. This would suggest that alumition has been attributed to increased maturity and cross linking num treatment of matrix prevented calcification by suppressing [16]. In this case it seems highly likely that this resulted from matrix bone inductive ability and cellular involvement in this increased cross linkage caused by the aluminum. Additional process. This is also supported by the vitro data showing that support for aluminum cross linkage of collagen is the finding aluminum enhances the ability of collagen to calcify which that the aluminum pre-treated bone matrix was virtually total tends to exclude a basic alteration in collagen which prevents its intact after 56 days of implantation. It has previously been calcification. This loss of inductive ability of bone matrix could

shown that cross linked collagen is not only resistant to also explain why there is a decrease number and function of collagenase degradation in vitro but it is also resistant in vivo to osteoblasts and osteoclasts in organ cultures which have been treated with aluminum. degradation [17].

Mineral tannage has been extensively used in the tanning

industry. While chrome has been the most widely used element, aluminum and zirconium have also been employed [17]. In this

form of tannage cross links are through chrome or aluminum coordination complexes containing hydroxol, oxo, phosphate or sulfate bridges into which carboxyl groups on collagen enter forming the cross links. This provides the most hydrothermally stable and commercially important tannages [18]. In further support for aluminum-inhibiting bone matrix inductive proper-

The presence of aluminum in the matrix is required for this inhibitory effect since chelation of aluminum restores the bone inductive properties. This inhibitory effect could result from aluminum cross links of the collagen fibrils. However, it has

been suggested that collagen is not necessary for the bone inductive properties of matrix [21]. Somewhat against this is the

fact that the osteo-inductive components of bone have been shown to be ineffective in causing bone formation in tendons, also type I collagen [22]. Because of this, it has been suggested

ties by cross linkage of collagen fibrils is the fact that this that bone collagen might be a more suitable substratum for inhibitory property is completely reversed by the removal of anchorage-dependent proliferation and differentiation of cells aluminum from the matrix by chelation. This is a well known capable of bone formation [221. This would be consistent with the finding in this study showing that bone formation in the method of detanning metal tannages [11]. Additional evidence for the chemical bonding of aluminum in implanted matrix resembled the implant prior to being decalcibone matrix is the rather constant uptake of aluminum by fled. It is possible that aluminum-induced cross links of collagen matrix from solutions containing various concentrations of could either immobilize the bone inductive proteins or simply aluminum. Further support comes from the finding that at least the presence of aluminum in matrix could somehow have a one compartment of aluminum in matrix is very stable as direct effect on inhibiting bone induction. In further support supported by the minimal change in matrix aluminum concen- that aluminum bound in matrix is responsible for inhibiting bone tration which occurred between 28 and 56 days of implantation. induction is the fact that i.p. injected aluminum, which inThe fact that aluminum was found to enhance the calcifi- creased plasma aluminum and presumably extracellular fluid ability of matrix from an in vitro metastable solution is addi- aluminum, had no effect on the bone inductive property of tional evidence that aluminum alters the structure of bone matrix. Irrespective of the mechanism by which aluminum inhibits collagen probably through increasing cross linkage. Increased cross linking may predispose collagen to develop dystrophic or bone induction it requires only a small amount, less than 50 mg metastatic calcification. This is suggested by the fact that cross aluminumlkg matrix, to suppress bone formation. Whole bone linking collagen with glutaraldehyde predisposes pericardial aluminum obtained from uremic patients with osteomalacia bioprosthetic heart valves to calcify [19]. Bovine pericardium typically have much higher bone aluminum levels than this [23]. does not have bone inductive properties; therefore, its calcifi- In addition, the bone aluminum in osteomalacic bone tends to cation would appear to be of the dystrophic or metastic type. be concentrated in the osteoid, making it even more likely that However, aldehyde cross links are different from those formed it could alter the osteoid structure and its ability to promote by minerals. Aldehyde cross linking results from the formation bone formation. of stable covalent cross links associated with a relatively limited number of amine groups available on collagen [17]. Acknowledgments Irrespective of aluminum's effect on promoting dystrophic This study was supported by V.A. research funding. Dr. Zhu was calcification of collagen, which might account for most of the partially supported by a grant from Xian Medical University, Xian, calcium present in matrix treated with 0.1 M AId3, it would PRC. This work was presented in part at the American Society of appear that its major effect on bone matrix was the eradication Nephrology annual meeting in 1989, and published in Kidney mt of its bone inductive capabilities. Reddi, Wientroub and Muth- 37:1990. ukumaran have extensively studied the bone inductive properReprint requests to Allen C. Alfrey, M.D., Denver V.A. Hospital, ties of decalcified bone matrix [20]. Normally within the first 1055 Clerpnont Street, Denver, Colorado 80220, USA. week following implantation there is vascular invasion and the appearance of chrondroblasts and chrondrocytes with cartilage References formation in the implant and subsequent calcification of the I. WARD MK, FEEST TO, ELLIS HA, PARKINSON IS, Kniut DNS: cartilage. In a matter of days this is followed by the presences Osteomalacia dialysis osteodystrophy: Evidence for a water borne of osteoblasts and osteoclasts with extensive bone formation, aetiological agent; probably aluminum. Lancet i:841—845, 1978 remodeling and dissolution of the implanted matrix. In the 2. CHAN YL, ALFREY AC, POSEN S. LIs5NER D, HILLS E, DUNSTAN CR, EVANS RA: The effect of aluminum on normal and uremic rats: control implants the expected extensive bone formation and

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