Indian Journal of Biochemistry & Biophysics Vol. 43, June 2006, pp. 167-172

Augmented bone-matrix formation and osteogenesis under magnetic field stimulation in vivo XRD, TEM and SEM investigations

Praveen Singh1*Π, Rakesh C YashRoy1 and M Hoque2 1Biophysics and Electron Microscopy Section, 2Surgery Division, Indian Veterinary Research Institute,

Izatnagar-243122, UP, India

Received 02 June 2005; revised 25 January 2006

Bone is a composite biomaterial, which is formed, when proteins constituting collagen fibers attract calcium, phosphate and hydroxide ions in solution to nucleate atop the fibers. It grows into a hard structure of tiny crystallites of hydroxyapatite, aligned along the long axis of collagen fibers. The present work reports the stimulating effect of static magnetic field on microstructure and mineralization process of bone repair. A unilateral transverse fracture of mid-shaft of metacarpal was surgically created in healthy goats under thiopental sedation and xylocaine analgesia. Two bar magnets (~ 800 gauss/cm2 field strength) were placed across the fracture line at opposite pole alignment immobilized in Plaster of Paris (POP) splint bandage for static magnetic field stimulation. Radiographs were taken at weekly intervals up to 45 days. Results show that formation of extra-cellular matrix and its microstructure can be influenced by non-invasive physical stimulus (magnetic field) for achieving an enhanced osteogenesis, leading to quicker regeneration of bone tissue in goats. X-ray diffraction (XRD) patterns of treated (magnetic field-exposed) and control samples revealed the presence and orientation of crystalline structures. Intensity of diffraction peaks corresponding to 310 and 222 planes were enhanced with respect to 211 families of reflections, indicating preferential alignment of the crystals. Also, the percent crystallinity and crystal size were increased in treated samples. The study provides a biophysical basis for augmented fracture healing under the influence of semi-aligned static magnetic field applied across the fracture line.

Keywords: Fracture healing, Static magnetic field, Hydroxyapatite, Collagen fibers, XRD pattern, Electron microscopy

There has been an increasing interest to investigate the effect of magnetic and electric fields on the biological systems1-7. Clinical studies have demonstrated the usefulness of magnetic and electromagnetic fields, in stimulating healing of chronically disjointed fractures in humans/animals3-7. The devices generating such fields have been approved for clinical use. The osteo-stimulating effect of pulse magnetic field (MF) has been reported7,8. Magnetic field has also been successfully used in treating delayed union9, non-union10 and long-established cases of pseudo-arthroses11. Despite these studies, the biological processes underlying osteogenesis remain, only partially understood. In

fact, experimental approach to the magnetic field effect on living system is complicated by various non-linearities (intensity, frequency and time windows of the fields) and peculiarities (cell type, age and treatment). However, considerable progress has been made to understand the cellular and molecular changes associated with application of biophysical stimuli such as electromagnetic field11-15 and static magnetic field16. Each of these systems has shown substantial efficacy in controlled clinical trials17. In this paper, an attempt has been made to understand the effects of non-invasive stimulation by static magnetic field on morphology, ultra-microstructure of bone and on mineralization process of the fractured tissue. The results obtained are discussed in the light of existing models for coupling of magnetic field with bone tissue, bone cells and associated effects on extra-cellular matrix formation. The study throws light on modus operandi of augmented bone fracture healing process under the influence of static magnetic field.

_____________ *Corresponding author Tel: 91-09359108482; Fax: 91-581-2303284, E-mail: psingh67@yahoo.com ; ΠPresently on deputation to Department of Electronics, Kyushu University, Fukuoka, Japan Ph 81-92-6427057, Fax 81-92-6423967 E-mail singh@belab.ed.kyushu-u.ac.jp Abbreviations MF, magnetic field; HAP, hydroxyapatite; XRD, X-ray diffraction; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, JUNE 2006

168

Materials and Methods Non-descript healthy goats (8) of either sex, weighing between 30 and 40 kg were divided into two groups (A and B) of four animals each. They were fed standard diet. A unilateral transverse fracture of mid shaft of metacarpal was created in each animal under thiopental sedation and xylocaine local analgesia and it was then immobilized with Plaster of Paris (POP) splint bandage. In animals of group A, two bar magnets (~ 800 gauss/cm2 field strength), placed one each in proximal and distal fracture fragments, across the fracture line at opposite pole alignment were included in POP casts. The animals were observed for lameness and weight-bearing. Radiographic assessment at different intervals (0, 7, 15, 30 and 45 days) was made in this arrangement of magnets (Fig. 1). Most of field lines would pass through the fracture-line obliquely. Animals were sacrificed after 45 days and grown tissue was collected for conducting SEM (scanning electron microscopy), TEM and X-ray diffraction (XRD) examinations. For SEM examination, callus specimens were rinsed for 30 min in three changes of 0.1 M phosphate buffers at pH 7.2. After three rinses for 30 min in distilled water, tissues were passed through (30 min each) of progressive concentrations of acetone for dehydration. Finally, the tissues were immersed in 100% acetone with two changes, 1 h each. The tissue pieces were air-dried first, and sputter-coated by Jeol Ion Sputter model JFC 1100 with an approximately 20 nm thick layer of pure gold in vacuum at 7 mA. For TEM, the tissues fixed in trump’s fixative (1% gluteraldehyde and 4% formaldehyde in 0.1 M phosphate buffer pH 7.2) were partially decalcified in a 10% acetic acid, 0.85% NaCl, and 10% formalin

solution. The tissues were further processed in 0.1 M phosphate buffered 1% OsO4 for post-fixation and staining. After three rinses for 30 min in distilled water, specimens were stained enbloc with 2% uranyl acetate for 30 min. Thin (60 nm) sections were cut with ultramicrotome, placed on 200-mesh copper grids and stained with lead citrate. Electron-micrographs were taken on CM10 Philips electron microscope operating at 60 kV. XRD examinations were made on air-dried, dehydrated and non-decalcified tissue pieces with Philips diffractometer (model PW 1730/10) using Cu-Kα (λ = 1.54056 Å) and Ni filter in wide range of Bragg’s angles 20° < 2θ < 70° at room temperature. The details of X-ray spectrometer parameters and conditions used in the study are given in Table 1.

Results and Discussion Fig. 1a and b show the X-ray radiographs of fractured tibia of goat exposed to magnetic field and control respectively on day 45. In Fig. 1a, the grown callus is quite visible, as marked by arrow on both the fracture fragments. Fig. 1b shows that there is very little callus growth at the periosteum of bone fragments. The SEM study was undertaken to observe the microstructure of bone matrix formed in grown callus at the fracture site. The study provided a topographic view of the healing site and thus proved a valuable parameter for evaluating the osteogenic activities under magnetic field. Fig. 2 shows SEM micrograph of the fracture gap in MF-exposed case. The advancing bundles of collagen fibres appear to bridge the fracture gap and hydroxyapatite (HAP)-like crystalline structures appear on fiber bundles. The fiber bundles oriented almost parallel to one another

Fig. 1—X-ray radiographs showing the state of fracture on day 45 (a) in MF-exposed; and (b) control. [Arrows in Fig. 1a indicate the grown callus at periosteum in fracture line. The lack of callus at fracture line is clearly seen in Fig. 1b]

Table 1—X-ray spectrometer operational parameters

Parameters Value in units Radiation type, source X-rays

Cu, 40 kV, 30 mA, Nickel filtered

Wavelength used 1.540598 Å Kα1, Monochromatic λ Discriminator Diffracted beam, graphite-mono λ Detector Scintillation Temperature 22° ± 1° Range (2θ) 10°-70° Specimen motion None Step size 0.02° 2θ Scan rate 2°/min External 2θ standard Si Lattice parameter of 2θ standard

5.4310 Å

SINGH et al.: AUGMENTED BONE-MATRIX FORMATION UNDER MAGNETIC FIELD

169

Fig. 2—SEM micrograph showing columnar collagen fiber bundles oriented almost parallel to each other with hydroxyapatite crystals laid over it forming osteon [The fracture line is clearly visible (marked by arrow) which is semi-repaired; the alignment of matrix below and above fracture line is distinguished (MF-exposed)

in the fracture gap, apparently for mediating repair, under the influence of static magnetic field (Fig. 2). Collagen, the structural protein of connective tissue appears to be constituted of very fine fibrils of thickness varying from 100 to 150 Å. The magnetic torque alone may only orient the fibrils into planes normal to the field direction, which in turn is conducive to fill up the fracture gap. Thus, magnetic orientation is considered useful in fibrillogenesis and in the production of highly oriented collagen bundles18. It is seen that collagen columns thus formed have a high degree of uniaxial alignment (Fig. 2). Collagen molecules self-assemble into collagen fibrils which have large number of peptide planes, contributing to diamagnetic anisotropy with energy, sufficient enough to have ordering effects under magnetic alignment even at physiological temperature levels18. The micro-organisation of bone consists of haversian canals (small channel formations that house the blood vessels, nerves and control networks of bone cells). The osteoblasts deposit the bone forming materials, the secreted bone-matrix eventually surrounds them. The cells then live in tiny spaces in the bone matrix called ‘lacunae’. Osteocytes, the other bone cells use canalculi to facilitate maintenance of bone, as well as the contact between other osteocytes. Fig. 3 shows SEM micrograph revealing bone matrix with crystalline structures in a tissue that is not exposed (control) to constant magnetic field. The grown matrix is under-aligned and containing fewer collagen bundles. The grown callus normally has 60 per cent extra-cellular mass called matrix, the main structural component is collagen fibers. The collagen fiber has a diameter of about 40-60 nm and length of

several micrometers. Individual collagen fibril has a diameter of 10-15 nm. Fig. 4 (SEM micrograph) shows the secreted bone matrix in rings (lamellae) around the haversian canals, as marked by arrow. This shows formation of osteons, which are the cylinders of bone consisting concentric layers of lamellae. These lamellae are made of bone matrix and radiate outward from the central haversian canals. In TEM micrograph (Fig. 5) of MF-exposed sample, collagen fibers are lying partially aligned longitudinally in most of the section. HAP microcrystals are deposited over the fibers. There is extra-deposition of HAP crystals of various sizes. In TEM micrograph of control sample (Fig. 6), the fibres are seen in cut section and of 30-40 nm in diameter. Also, fewer HAP crystals deposited atop fibres are seen, compared to those in Fig. 5. The width of HAP crystal varies from 10-15 nm. In longitudinal fibre (marked), the length of crystal is approximately 30-40 nm, which is consistent with the calculated size of crystals from XRD spectrum. Fig. 7 shows the conspicuous vascularization of the callus formed under the influence of magnetic field, compared to their insignificant presence in the control callus tissue

Fig. 3—SEM micrograph showing bone matrix with crystalline structures [The grown matrix shows fewer collagen bundles and no alignment (control)]

Fig. 4—SEM micrograph showing secretion of bone matrix in rings (lamellae) around the haversian canal, as marked by arrow (MF-exposed)

INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, JUNE 2006

170

Fig. 5—TEM micrograph of collagen fibers with calcium micro-crystal (black after staining) over fibers. [Bigger crystals (marked by arrow) are seen over bunched fibers (MF-exposed)]

Fig. 6—TEM micrograph of collagen fibers cut across in the section with micro black spot on some of fibers indicating the hydroxyapatite present over fibers. The size of hydroxyapatite crystals is almost uniform. Nowhere, extra deposition of calcium salts is seen (control)

Fig. 7—TEM micrograph showing grown callus and vascular network (MF-exposed)[VS refers to vascular space]

(Fig. 6). Vascularization would apparently quicken the fracture healing process due to augmented blood supply. The XRD pattern of MF-exposed bone tissue is shown in Fig. 8a. The pattern exhibits diffraction lines superposed on a broad hump extending from 20° to 35°, indicating the amorphous nature of examined callus sample. The X-ray diffractogram of bone callus is indexed with X-ray powder diffraction pattern of standard HAP19 and as reported by other studies20-24. Other diffraction lines are produced due to crystalline phases. The intensity versus angle (2θ) plots reveal that 310 and 222 peaks of HAP are strongly enhanced in static magnetic field exposed sample (Fig. 8a), as compared to control (Fig. 8b). It appears that intensity of 211 and 310 planes are interchanged due to crystal orientation under the magnetic field. It is found that initially micro-crystals form on collagen fibers at intervals of 670 Å. This is exactly the spacing of the gaps, between the heads and tails of each of successive tropocollagen molecules in the quarter-staggered array, which plays a role in bone formation25. Earlier, it was found that hexagonal HAP unit cells contain Ca10(PO4)6(OH)2 belonging to the space group C26h (P63/m)

25-27. Thus, unit cell has two equal edges ‘a’, inclined at 120° to one another and a third, ‘c’,

Fig. 8—XRD spectrum of bone samples (a) MF–exposed showing well-resolved, high intensity peaks of various planes of HAP crystals; and (b) Control sample, showing low intensity, diffused peaks of various planes of HAP crystals

SINGH et al.: AUGMENTED BONE-MATRIX FORMATION UNDER MAGNETIC FIELD

171

perpendicular to these. Control samples give a rather diffuse X-ray diffraction pattern, depending on the small crystals sizes28, as evidenced from the data given in Table 2. Another striking feature of the diffraction pattern of bone is the background scatter. This back-ground scatter has been measured in order to assess the relation between crystalline and “amorphous” portions of apatite. In normal bones, as much as one-third of the apatite mass could appear in ‘amorphous’ form. There is a marked meridional orientation of the 002 reflection at 26° from apatite in MF-exposed bone tissues only. Furthermore, the hk0 reflections are sharper then 00l reflections. Thus, one can conclude that the apatite crystals are also elongated in the direction other than c direction. Most of HAP crystals are parallel to the fiber axis of the collagen fibrils, as also confirmed by electron microscopy studies. XRD results indicate that the elongated HAP particles in the form of either continuous or closed-packed aggregate are aligned along the collagen fibers. Many of these particles seem to have a width of about 100 Å and a length of about 350 Å, placed along the fiber axis as also endorsed by other researchers29-31. One common feature is that the elongated apatite particles seem to be aligned along the collagen fibrils. Sometimes, a sub-structural division could be seen among the particles. In thicker collagen bundles (probably composed of several fibrils), apatite crystals are located within these bundles. There also seems to be a close relationship between the periodic banding of collagen and location of apatite particles as confirmed by various studies19-22. According to Scherrer’s equation, crystal size is given as:

D = K λ/B Cos θ … (1)

where D is the crystal thickness (in the same units as λ) in the direction perpendicular to the lattice planes corresponding to the diffraction line in question, B is

the half-width (in radians) of diffraction line, θ is the Bragg angle, λ is the wavelength of X-rays and constant K has a value close to 0.9. The crystallite size corresponding to each diffraction maxima can be determined from the measurement of the half width of diffraction peak. The data of crystal size corresponding to different peaks are shown in Table 2. The size of crystals in MF-exposed samples is larger as compared to control. Largest size of order of 373 Å is obtained corresponding to 323 (hkl) peak of exposed samples. Thus, bigger crystals are formed on application of static magnetic field, which is likely to provide early toughness to fractured bone and, therefore, expedite the bone fracture healing process. Estimation of crystallinity

The well-resolved sharp diffraction peaks of Fig. 8a indicate high crystallinity of MF exposed callus samples. For estimation of crystallinity, judicious extrapolation was made to separate the amorphous and crystalline peak areas. The total area of diffraction pattern is divided into two parts — the crystalline (Ac) and amorphous (Aa) components. In Figs 8a and b, area between dotted baseline and solid curve is the amorphous component Aa, while area between the solid line and diffraction curve is crystalline component Ac. The percent crystallinity Xc (%) of the samples is thus determined from the ratio of crystalline area to the total area through the following expression

Xc(%) = Ac /(Ac+ Aa) × 100(%) … (2)

The crystalline component in control bone on day 45 is only 16%, whereas in MF-exposed bone sample it is 31%. This suggests that mineralization process gets accelerated as a result of application of static magnetic field, under our experimental design, which in turn, explains the expeditious bone fracture healing process observed by us. Our crystalline per cent in control case is also vindicated by a recent study in rat32. In conclusion, the evidence obtained from this study indicates that static magnetic field intervention appears to stimulate the natural bone healing process via aligned collagen growth, faster mineralization and vascularization of new grown callus tissues, apparently due to enhanced cellular activities. As a whole, general picture of collagen-apatite organization as revealed by electron microscopy

Table 2—XRD peak and crystal thickness calculated from Scherrer’s equation

Control/ exposed

Peak (hkl)

B (radians)

2θ (degree)

θ (degree)

Crystal size

(D) in Å Control Control Exposed Exposed Exposed

222 323 310 400 323

0.0105 0.0087 0.0087 0.0078 0.0044

46.50 64.80 38.40 44.60 64.80

23.25 32.40 19.20 22.30 32.40

143.74 188.76 168.77 191.35 373.24

INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, JUNE 2006

172

appears to be in accordance with the results obtained by X-ray diffraction studies. Electron microscopy observations indicate expeditious collagen alignment across the fracture gap with extensive blood vascularization and enhanced HAP crystal formation in callus specimen under the influence of static magnetic field. The present study, therefore, provides the biophysical and ultra-structural mechanisms underlying the process of augmented bone fracture healing by application of non-invasive static magnetic field, first observed16 in our laboratory.

Acknowledgement Authors are thankful to Dr. M P Yadav, Director, IVRI, for encouragement in the project. Support extended by Dr G R Singh, Head, Surgery Division, IVRI for X-ray film facility at Animal Hospital and Dr Navin Kumar for his fruitful discussion are duly acknowledged. Officer-in charge, E M facility, All India Institute of Medical Sciences and O/C XRD facility at Dept. of Textile Engineering, IIT Delhi are thanked for instrumental support.

References 1 Tenforde T S (1995) in Handbook of Biological Effects of

Electromagnetic Fields (Polk C & Postow E, eds), 2nd edn, pp. 185-197, CRC Boca, Raton, USA

2 Eraslan G, Akdogan M, Yarsan E, Essiz D, Sahindokuyucu F & Altintas L (2004) Indian J Biochem Biophys 41, 57-59

3 Brighton C T & Conolly J F (1981) J Bone Jt Surg 63-A, 2-7 4 Basset C A L, Mitchell S N & Gaston S R (1981) J Bone Jt

Surg 63-A, 511-516 5 Black J & Brighton C T (1979) in Electrical Properties of

Bone and Cartilage (Brighton C T, Black J, Pollack, eds), pp. 215-234, Grune and Stratton, New York

6 Burchandt H (1983) Clin Orthop 174, 28-30 7 Burwell R G (1964) J Bone Jt Surg 46 B, 110-115 8 Sedel L, Christel P & Duriez J (1981) Rev Chirl Orthop

Reparatrice Apar Mot 67, 11-23 9 Basset C A L, Pilla A A & Pauluk R J (1982) Clin Orthop

Relat Res 247, 623-625 10 McDowell E M & Trump B F (1976) Arch Pathol Lab Med

100, 405-407

11 Watson J & Downes E M (1978) Jp Jn Appl Phys 17, 215-217

12 Kotani H, Iwasaka M & Ueno S (2000) J Appl Phys 87, 6191-6193

13 Murray J C & Ferndale R W (1985) Biochim Biophys Acta 838, 98-105

14 Sabrina R, Vanessa M, Antonella L and Settimio G, Renato G, Marco L, Paolo P, Antonio C, Deleana P & Giuliani L (2002) Appl Phys Lett 81, 2890-2892

15 Richard A L, Christopher D C, Monica C Y C, David M R, Rosen W & Ross W A (1982) Proc Natl Acad Sci (USA) 79, 4180

16 Singh P, YashRoy R C & Hoque M, Proceedings on Discussion Meeting on Structural Biology and Symposium on

Biophysics, Chennai, Jan. 2002, India, P-81 17 Hoque M, Naveen Kumar, Aithal H P, Singh G R, Singh P &

Kalicharan (2003) Indian J Anim Sci 73, 275-277 18 Torbet J & Ronziere M C (1984) Biochem J 9, 1057-1059 19 Tucker B E, Cottel C M, Auyering R C V, Spector M &

Nicollas G H (1996) Biomaterials 17, 631-637 20 Sinha A, Ingle A, Munim K R, Vaida S N, Sharma B P &

Bhisey A N (2001) Bull Mat Sci 24, 653-657 21 Shi D & Jiang G (1998) Mat Sci Eng C6, 175-182 22 Rama Krishna D S, Chaitanya C K, Seshadri S K & Sampath

Kumar T S (2002) Trends Biomat Artif Organs 16, 15-17 23 Cuneyt T A (2001) Powder Diffraction 16, 102-106 24 Saraswathy G, Pal S, Rose C & Sastry T P (2001) Bull Mat

Sci 24, 415-420 25 Freifelder D M (1987) in Molecular Biology, 2nd edn, pp.

174-178, Jones and Bartlett, Boston, USA 26 Slavica L, Slavica Z, Nada M & Slobodan M (2001)

Thermochim Acta 374, 13-22 27 Zhang S & Gonsalves K E (1997) J Mat Sci Mat Med 8,

25-28 28 Engstrom A (1972) in The Biochemistry and Physiology of

Bone (Geoffery H B, ed), Vol. I, pp.237-258, Academic Press, New York

29 Verde C G, Guarino A & Gonzalez G (2004) Eur Cells Mat 7, 58-59

30 Hartgerink J F, Elia B & Samuel I S (2001) Science 294, 1684-1688

31 Cameron D A (1972) in The Biochemistry and Physiology of Bone (Geoffery H B, ed) Vol. I, pp.191-236, Academic Press, New York

32 Rai D V, Darbari R & Aggarwal L M (2005) Indian J Biochem Biophys 42, 127-130