J Neurosurg Spine 6:141–149, 2007
The effects of magnesium particles in posterolateral spinal fusion: an experimental in vivo study in a sheep model RAMAZAN ALPER KAYA, M.D.,1 HALIT ÇAVUS¸ OG˘LU, M.D.,1 CANAN TANIK, M.D.,2 ALI ARSLAN KAYA, ASSOC. PROF.,3 ÖZGÜR DUYGULU, M.SC.,3 ZIHNI MUTLU, D.V.M., PH.D.,4 EBRUHAN ZENGIN, D.V.M.,4 AND YUNUS AYDIN, M.D.1 Neurosurgery Clinic and 2Pathology Department, S¸is¸ li Etfal Education and Research Hospital, Istanbul, Turkey; 3Materials Institute, TÜBI˙ TAK Marmara Research Center, Gebze-Kocaeli, Turkey; and 4Veterinarian Faculty, Surgery Department, Istanbul University, Istanbul, Turkey 1
Object. Magnesium has recently become a material of interest as a biocompatible and biodegradable implant metal. Authors of several reports have noted the potential bone-cell activating or bone-healing effect of high Mg ion concentrations. The classic method for achieving intertransverse process fusion involves using an autologous iliac crest bone graft. Several studies have been performed to investigate enhancement of this type of autograft fusion. To the authors’ knowledge, no research has been conducted in which the efficacy of pure Mg particles in posterolateral spinal fusion has been investigated. The objective of this study was to determine whether Mg particles enhance the effectiveness of intertransverse process lumbar fusion in a sheep model. Methods. Sixteen skeletally mature female sheep were subjected to intertransverse process spinal fusions with pedicle screw fixation at L2–3 and L5–6. Each animal was given a 5-cm3 bone autograft at one fusion level, and a combined 5-cm3 bone autograft with the addition of 1 cm3 Mg at the other level. Six months after surgery, bone formation was evaluated by gross inspection and palpation, and by radiological, histological, scanning electron microscopic, and x-ray diffraction analyses. Radiological results were graded from 0 to 4 according to the status of the bridging bone, which was determined by evaluating both x-ray films and computed tomography scans. The quality of the spinal fusion was assigned a histological score of 0 to 7, in which a score of 0 represented an empty cleft and a score of 7 represented complete bridging of bone between the transverse processes. The trabecular bone formation at each fusion level and the Ca hydroxyapatite crystalline structure in core biopsy specimens were evaluated using scanning electron microscopy and x-ray diffraction analyses, respectively. The rate of rigid bone fusion, according to both palpation and radiological assessment, in the combined Mg and autologous bone treatment group was higher (81.25%) than in the autograft bone treatment group (62.5%), but this difference was not statistically significant. The quality of bone fusion, according to the histological grading system and scanning electron microscopy inspection, was higher in the bone fusion segments of the Mg and autologous graft combined group than in the group with autograft-only arthrodesis, and this difference was statistically significant. The xray diffraction analyses further confirmed the effect of Mg in promoting the formation of the crystalline portion of the bone (hydroxyapatite). Conclusions. Based on the results of this study, adding Mg particles to autologous corticocancellous bone in a posterolateral intertransverse process fusion enhances the quality of bone formation. However, radiological findings did not reveal a statistically significant effect of Mg on the rate of solid bone fusion formation between the two transverse processes.
KEY WORDS • magnesium • autologous bone graft • spinal fusion • animal model • sheep
spinal fusion is commonly performed for spinal stabilization. Increasing the efficacy and speed of stable spinal fusion are primary goals in spinal surgery. Autologous bone is considered to be the most effective bone graft material for posterolateral lumbar arthrodesis, yet nonunions occur in up to 30% of patients6,33 and donor site complications may occur in 25 to
P
OSTERIOR
Abbreviations used in this paper: ACB = autologous corticocancellous bone; CT = computed tomography.
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30% of patients.9,16,41 These bone graft difficulties have prompted investigations of bone graft extenders, enhancers, and substitutes. A considerable number of clinical and experimental studies have explored the use of materials such as bone marrow, demineralized bone matrix, collagen, ceramics, bone morphogenetic proteins, and others for use in bone graft procedures.1–3,8,10,12,13,17–19,22–25,28 A growing interest in the role of Mg in bone metabolism has arisen in recent years.26,30,37,39,40,43 Magnesium is essential to human metabolism and is naturally found in bone tissue. It is a light metal of low toxicity and is the 141
R. A. Kaya et al. fourth most abundant cation in the human body.27,36,38 The estimated amount of Mg present in an average human body weighing 70 kg is about 1 M, with approximately half of it stored in bone tissue.27,36,38 Another important feature of Mg is its in vivo degradation through corrosion from the body’s electrolytic environment.11,35,37,40,42 Authors of some published reports have also noted the potential bone-cell activation or bone-healing effect of high Mg ion concentrations.14,26,30,40,43 Given these characteristics of Mg as a biocompatible and biodegradable metal, we sought to investigate its possible effects in an animal model. In this study, we used a sheep model to evaluate the effectiveness of Mg particles when used in conjunction with autologous bone to facilitate posterior intertransverse process lumbar spinal fusion. In 16 sheep, the formation and quality of the fusion using an Mg and autologous bone mixture at one level were compared with another level fused only with autologous bone. The fusion formation was evaluated radiologically using both x-ray films and CT scans and by gross inspection and palpation. In addition, a histological analysis was conducted using light microscopy and scanning electron microscopy to determine the quality of the new bone within the fusion masses. Moreover, the effects of Mg on the crystalline structure of the newly formed bone were also investigated using an x-ray diffraction technique. To our knowledge, this is the first report in which the effect of Mg particles on spinal bone posterolateral fusion is investigated in an in vivo animal model. Materials and Methods Study Design and Surgical Procedures Sixteen skeletally mature female sheep (mean age 1.5 years, and mean weight 50–75 kg) underwent unilateral intertransverse process spinal fusions with pedicle screw fixation at L2–3 and L5–6 via a standard posterior midline approach. The preparation stage of each operation was performed similarly to the method previously described by Kim et al.13 Each animal was preanesthetized with 0.4 mg/kg atropine intramuscularly, anesthetized with 10 mg/kg ketamine and 0.5 mg/kg diazepam intravenously, and placed prone on the surgical table. Isoflurane and intravenous fluids were administered throughout the surgery. All anesthesia procedures were performed by veterinary surgeons. Each sheep was shaved, prepared, and draped in a sterile manner. Lateral and anteroposterior fluoroscopic views were used to determine the optimal incision location. A midline skin incision was made followed by a unilateral fascial incision, and the L2–6 paraspinal muscle was dissected. The transverse processes were exposed and a high-speed drill was used to decorticate the transverse process and lateral aspect of the lamina at L2–3 and L5–6. The graft materials were prepared as described later and placed into the transverse process interval. In all animals, autografts were harvested from the iliac bone through a separate incision made on the iliac crest. A total of 10 cm3 of corticocancellous bone was harvested from the posterosuperior iliac crest of each animal; half of the bone was separated for one fusion site and the remainder was blended with 1 cm3 Mg particles for treatment at the other fusion site. The crest sites were then irrigated, packed with Gelfoam, and closed. Thus, in each animal, one fusion level was treated with 5 cm3 ACB (ACB group), and the other level received 1 cm3 Mg particles in the form of chippings (250–300 mm wide and long and several mm thick; Fig. 1) and 5 cm3 of ACB mixture (MgACB group). Treatments were alternated between the cephalad and caudad levels. Single-sided pedicle screws (35 mm long and 5 mm in diameter) were inserted at each fusion level by using the standard technique
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FIG. 1. Scanning electron micrograph showing the size and morphology of the Mg particles used. Original magnification 3 50. Bar = 100 mm. WD = Working Distance.
under fluoroscopic guidance, and a rod was placed over the screws and secured with nuts (Tasarim Medikal). The surgical site was examined for bleeding, and hemostasis was attained in all animals. The fascial incision was closed with 2-0 absorbable sutures, and the skin incision was closed using a surgical stapler. A 20-mg/kg dose of ceftriaxone was administered for 5 days as a prophylactic antibiotic. The animals were allowed to recover and move freely, and were fed regularly. Blood serum samples were collected just before and at 2 weekly intervals after surgery; serum levels of Mg, Na, K, Ca, urea, and creatinine were measured; and liver function was tested as a follow-up check for probable systemic effects. All animals were killed after 6 months with an intravenous injection of sodium pentobarbital. The fused bone segments were then removed for evaluation. Assessment of Spinal Fusions Gross Inspection and Manual Palpation. After the specimens were retrieved en bloc, the instrumentation was removed and each spine was inspected and manually palpated by two blinded observers to determine whether fusion formation was solid. Only levels assessed as solid were recorded as fused. Radiographic Assessment. Posteroanterior plain radiographs and CT scans were obtained for all of the dissected spine specimens immediately after removal. The corresponding fused segments as seen on radiographs and CT scans were evaluated together, and were graded in a blinded fashion by two independent radiologists who were not aware of either the type of treatment or the gross findings. The fusion observed radiologically was graded using a scale devised to assess the amount of intertransverse bone graft, as previously described in an experimental study34 in the following manner: Grade 0, no bone present between the transverse processes; Grade 1, small islands of bone between the transverse processes; Grade 2, bridging bone with two or more radiolucent lines or one large gap within the mass of the fusion; Grade 3, bridging bone with one radiolucent line within the mass of the fusion; and Grade 4, bridging bone with no gaps or radiolucent lines. Histological Analysis. Histological analysis was performed to compare the new bone formation in the MgACB group with the formation in the ACB group. After gross inspection, manual palpation, and radiographic assessment, each fusion mass between the two transverse processes was excised and divided into two equal parts for both light microscopy and scanning electron microscopy studies. For light microscopy analyses, samples were fixed in a 10% formalin solution for 48 hours, decalcified in 30% formic acid, and washed in phosphate-buffered saline. Tissues were processed routinely and were embedded in paraffin. Sections were cut longitudi-
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Effects of magnesium on spinal fusion nally to a length of 10 mm, deparaffinized, rehydrated, and stained with H & E and trichrome. Five sequential coronal sections of each specimen were examined to avoid a sampling error. The quality of the fusion was graded by assigning a histological score from 0 to 7, as described previously by Emery et al.7 (Table 1); the higher the score, the greater the amount of bone present between the transverse processes. Scanning Electron Microscopy and X-Ray Diffraction Analyses. After the samples had been removed from the animals they were fixed in a 10% formalin solution for 48 hours. Samples were each kept in 30, 70, 95, and 100% ethanol for 10 to 30 minutes and then in 33, 50, and 67% amyl acetate (Aldrich) for 10 minutes, after which they were stored in amyl acetate for 1 week. A Bio-Rad E3000 critical-point dryer device was used at a temperature of 36.1˚C and 1100 psi to dry the specimens. The scanning electron microscopy studies used a JEOL JSM6335 field-emission gun scanning electron microscope operated at 3 kV and equipped with an Oxford energy dispersive microanalysis system and Inca software (version 4.05). For x-ray diffraction studies, powder specimens were ground to an average particle size of 50 mm, and a Shimadzu XRD-6000 diffractometer operating with Cu-Ka radiation at 40 kV and 30 mA was used. Radiographs used to determine the peak positions were obtained using a 0.02˚-step size and a 7-second dwell time over the 25- to 45˚-2 t range where major peaks were observed.
Results Surgical Outcome and Gross Observations
The surgical procedures were well tolerated, and all animals resumed normal activity within 24 hours of surgery. No complications during the early or late postoperative period were observed from the surgeries. Blood samples taken at 2 weekly intervals also showed no significant abnormality. Because of data from other in vivo studies involving Mg,15,37 we were anticipating a wound complication of subcutaneous H gas accumulation due to the reaction of Mg in the electrophysiological environment. The dissociation of H2O released H gas, thus forming subcutaneous bubbles as Mg was oxidized using O2 from the H2O molecules. This phenomenon was observed to a varying degree in each animal. Subcutaneous gas accumulation appeared within 1 week after surgery and disappeared after 2 to 3 weeks. Gas bubbles were punctured in sterile conditions when extreme subcutaneous tension was suspected. No adverse effects from the gas bubbles were observed in any of the sheep. After the fused segments had been removed, the instrumentation was removed for plain x-ray films and CT scans. Using manual palpation, fusion was detected in 10
TABLE 1 Histological scoring of the fusion mass* Score
Amount of Bone Between the Transverse Process
7 6 5 4 3 2 1 0
bone only more bone than fibrocartilage more fibrocartilage than bone fibrocartilage only more fibrocartilage than fibrous tissue more fibrous tissue than fibrocartilage fibrous tissue only empty cleft
* From Emery et al.
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(62.5%) of the ACB fusion segments and in 13 (81.25%) of the MgACB fusion segments. Fusion rates between the ACB and MgACB groups, as determined by manual palpation, did not show statistically significant differences (p = 0.433, chi-square test). Radiographic Analysis
All x-ray films of the 16 sheep showed fusion in different stages according to the grading system used in this study (Grade 0 was not detected in any of the animals), and radiographic findings correlated well with the CT findings (Figs. 2 and 3). Three of the ACB fusion segments and two of the MgACB fusion segments showed Grade 1 fusion (only small islands of bone). Results of fusion formation examined radiologically in both treatment segments are shown in Fig. 4. The palpation assessment prior to radiological examination was consistent with the radiological grading, which recorded all grades below 1 and 2 as nonfused by manual palpation. The number of fusions given a radiological Grade 4 (the best fusion) was also higher in the MgACB group. However, the difference in overall solid bone fusion between the MgACB and the ACB groups, according to the radiological grading system, was not statistically significant (p = 0.119, Mann– Whitney U-test). However, the number of segments given a radiological Grade 3 or 4 in the MgACB group was higher (81.25%) than those in the ACB group (62.5%). Histological Analysis
According to the histological grading system used in this study, the quality of fusion in both treatment groups was consistent with the radiological findings. Results of the histological grading of fusion formation are given in Fig. 5. The segments in the ACB and MgACB groups each had only one histological score of 3 and 4, and the remaining scores were all higher. The best bone formation grade (score of 7) was observed in four segments of the ACB group and in 11 segments of the MgACB group. Overall, the quality of bone fusion according to the histological grading system was better in the MgACB fusion segments than in the ACB fusion segments, and the difference between the two treatment groups was also statistically significant (p = 0.027, Mann–Whitney U-test). The fusion masses in the MgACB group were characterized mainly by cortical and trabecular woven bone, osteoid seams, and fatty marrow (Fig. 6). Although small amounts of cartilaginous material were present in a few samples, the fusion masses in the MgACB group were predominantly mature bone (Fig. 7). No significant inflammatory response was observed in either of the treatment groups, and no Mg particles were observed in any of the specimens, indicating that all Mg particles had degraded and been resorbed. In addition, no ectopic bone formation, neurological deficits, pathological abnormalities, or evidence of osteosarcoma was observed in association with the segments in the MgACB group. Scanning Electron Microscopy and X-Ray Diffraction Analyses
Numerous detailed scanning electron microscopy examinations showed that the trabecular bone formation in 143
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FIG. 2. Sheep 9. An x-ray film (upper) and CT scan (lower) obtained from an animal in the MgACB group showing a solid bone fusion at the left side of the L2–3 segment.
the fusion segments of the MgACB group was greater than in those from the ACB group in all specimens. A scanning electron microscopy image representative of the areas examined for this comparison is given in Fig. 8. These scanning electron microscopy examinations revealed no Mg particles in the fusion segments of the MgACB group, confirming the complete degradation of Mg. Results of the x-ray diffraction study indicated the presence of hydroxyapatite to a greater extent in the segments from the MgACB group than in segments from the ACB group (Fig. 9). As seen in Fig. 9 hydroxyapatite counts were greater in the samples from the MgACB group, despite identical quantities of bone with equal powder size in each case, as well as identical sampling sizes during xray diffraction. A more detailed assessment of x-ray spectra also revealed unconventional relative peak intensities, compared with the established relative peak intensities from different crystal planes of the hydroxyapatite structure, which may indicate preferential involvement of new atomic species in its structure. The major peak from hydroxyapatite is expected at 31.8˚ (hkl = 211 planes) according to the International Center for Diffraction Data. 144
FIG. 3. Sheep 11. Upper: An x-ray film of the L2–3 spinal segment from a sheep in the MgACB group, interpreted as radiological Grade 3. Note the one radiolucent area within the fusion mass. Lower: A CT scan showing sufficient bridging for consideration of solid fusion.
The same x-ray diffraction data also show that the peak height at 43.8˚ (hkl = 113 planes) should be much smaller (ratio 8:100) than the major peak for hydroxyapatite. Analysis of our study has shown that what is expected to
FIG. 4. Graph showing the radiological grades of fusion formation in both treatment groups. Although the number of Grade 4 segments in the MgACB-treated masses is higher than in the ACBtreated masses, the difference is not statistically significant (p . 0.05).
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Effects of magnesium on spinal fusion
FIG. 5. Graph showing the histological scores in both treatment groups. The quality of bone within the MgACB-treated fusion masses was higher than in the ACB-treated masses, and the difference was statistically significant.
be a relatively minor peak appears to be the major peak in sheep bone (Fig. 9 upper). Savarino and colleagues29 also reported the same major peak position in x-ray diffraction from human bone. Because of the nature of hydroxyapatite, the position of the major peak can be expected to change depending on the age of the bone, as well as on the atomic species present and their arrangement in the hydroxyapatite crystals.4,14,21 The relative height of the peaks at both 31.8 and 43.8˚ in the sample from the MgACB group is greater than those from the sample of the ACB group, which may be indicate that the Mg promoted bone growth and resulted in greater amounts of hydroxyapatite formation than when Mg was not present. Discussion Bone is a composite structure made up of collagen and noncollagenous proteins, such as organic matter and inorganic or crystalline mineral components. The mineral part of a bone comprises approximately 60 to 70% of its overall structure. The crystalline mineral component is usual-
FIG. 6. Photomicrograph of a fusion segment in the MgACB group that was classified as a histological score of 7. A cortical rim of bone surrounding the trabecular bone is shown. H & E, original magnification 3 40.
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FIG. 7. Photomicrographs of fusion segments from the MgACB (upper) and ACB (lower) groups with a histological score of 6. Small amounts of cartilaginous tissue (upper, arrow) are visible within the predominantly matured bone in both images. H & E, original magnification 3 100.
ly described as a Ca phosphate with an apatitic structure (Ca,X)10(PO4,HPO4,CO3)6(OH,Y)2), commonly referred to as hydroxyapatite (Ca10(PO4)6(OH)2).17 In this expression of the mineral constituent, X designates the cations (Mg, Na, and Sr ions) that can substitute for the Ca ions, whereas Y represents the anions (Cl2 or F2) that can substitute for the hydroxyl group.14,17 Because of the ability of Mg to substitute for Ca and the natural presence of Mg in bone, it may be expected to facilitate the formation of bone and/ or the growth of new bone tissue. Several published studies have noted the potential bone-cell activating or bone-healing effect of a high Mg ion concentration.14,26,30,40,43 A careful review of the use of Mg as a biomaterial32 indicates that these studies have been conducted since the first half of the twentieth century. Nevertheless, the first attempt to use Mg to promote bone healing and growth failed because of the rapid cor145
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FIG. 8. Scanning electron micrographs of fusion segments from the MgACB (left) and ACB (right) groups. The trabecular bone formation in MgACB fusion segments is greater than in the ACB fusion segments. Original magnification 3 500. Bar = 10 mm.
rosion of Mg and a large amount of gas accumulation beneath the skin.15 Since then, alloying elements have been added to inhibit the rapid corrosion of Mg, and thus Mg alloys have been used in experimental studies. When Mg was used, a hard callous formation was reported at the fracture sites, which was interpreted as the osteoconductive effect of Mg at such sites.20,35,37,42 Hydrogen gas accumulation due to the corrosion of Mg was observed without exception, but was easily treated by puncturing the bubbles with a subcutaneous needle, as we have also performed.37 These early results indicate that Mg-based materials are nontoxic and may actually stimulate bone tissue healing. In recent years, authors of a number of studies have investigated both the mechanism of bone–Mg implant interaction, and the effect of enriching the surface of a biomaterial such as hydroxyapatite with Mg ions.26,40,43 In an in vivo animal study, Witte and coworkers37 demonstrated that the corrosion layer of all implants made of different Mg alloys was in direct contact with the surrounding bone during degradation, and that it displayed an accumulation of biological Ca phosphates, implying an osteoblastic response to the degrading Mg alloys. Another in vitro study by Zreiqat et al.43 concluded that Mg2+ supplementation of bioceramic substrata may be a promising way to improve the integration of implants in orthopedic and dental surgery. Revell and colleagues26 observed increased interfacial strength of implants with hydroxyapatite surfaces enriched with Mg. In two studies by Yamasaki and colleagues,39,40 in which they used Mg-enriched apatite or collagen materials, reported similar beneficial effects of Mgenriched materials for bone cell attachment and tissue growth. All of these investigators in the aforementioned studies examined either in vivo or in vitro effects of Mg on bone metabolism as an orthopedic implant. However, in our study we investigated the direct effects of pure Mg on new bone formation, fusion, and bone mineral apposition in vivo. The results of this study are consistent with those of the previous studies. One of the important results of this 146
study is the histologically higher quality of fusion segments in the MgACB group than in the ACB group, implicating an osteoinductive effect of pure Mg in our sheep spinal fusion model. Moreover, fusion masses from the MgACB group were characterized histologically by a predominant remodeling of bone that was more mature than that associated with the ACB group, indicating that the fusion process occurred more rapidly with MgACB than with ACB alone. The radiological results, which are the direct evidence of the presence of a solid fusion between the two transverse processes, did not show a statistically significant difference between the two treatments, even though the rate of fusion in the MgACB group was higher than that in the ACB group. Thus, it can be concluded that adding Mg particles to ACB in the posterolateral intertransverse process fusion area enhances the quality of bone formation, but does not create a statistically significant effect on solid bone fusion between the two transverse processes. Another important finding of this study is the complete degradation of Mg particles within 6 months, which was confirmed by both light microscopic histological analysis and scanning electron microscopy imaging analysis. The Mg particle size used was expected to have a very significant effect on the dissolution time of Mg, as the corrosion reaction is a surface phenomenon that accelerates with increasing surface area and with decreasing particle size. Complete degradation must also be time and dose dependent. The lack of an increase in the serum levels of Mg, systemic toxic effects, or inflammatory response to the MgACB fusion segments also supports the biocompatibility of Mg. The nontoxic effect of Mg can be expected to vary with the amount used. In this study we tested a single dose of Mg (1 cm3) for a single time period (6 months), although in previous studies the authors did not provide a comparable example in this regard. Therefore, interpretation of all the results in this study should be considered only for this dose and time period. Magnesium is a fast-corroding metal, which hinders its greater immediate use in many cases. In biomedical appliJ. Neurosurg: Spine / Volume 6 / February, 2007
Effects of magnesium on spinal fusion
FIG. 9. Graphs showing the results of x-ray diffraction analysis of fusion segments in the MgACB (upper) and ACB (lower) groups. Note that the peak heights in corresponding locations are higher in the MgACB segments, indicating a greater amount of hydroxyapatite formation.
cations, however, this feature of Mg need not inhibit its use altogether, but the Mg needs to be formed carefully to realize the benefit of its bone formation promoting effect. Corrosion of Mg in aqueous and electrophysiological environments occurs by the formation of a hydroxide layer on the metal. This reaction, in essence, results from the dissociation of H2O molecules, which in turn releases H gas. The presence of chlorine in the medium has been reported to enhance this process, as is the case in physiological environments. Possible reaction sequences given in → the literature are as follows:31,32,37 Mg(s)12H2O→ →MgCl2; Mg(OH)2 Mg(OH)2(s)+H2(g); Mg(s)12Cl2(aq)→ (s)12Cl2→MgCl2. The evolution of H gas may be suppressed by inhibiting the corrosion of Mg, or at least reducing the reaction rate with suitable alloying elements added to the metal.31,32,37 The presence of free gas that causes swelling in soft tissues may be confused with the presence of wound infection. However, an awareness of such consequences when using Mg facilitates differentiating free-gas swelling from a possible infection. The swelling in the wound area develops approximately 1 week after the application, which J. Neurosurg: Spine / Volume 6 / February, 2007
is a period similar to purulent infection collection formation. However, the local wound infection features such as erythema, heat, tenderness, fluctuation, and detachment of the suture line due to discharge were not observed in the swollen wounds of the animals in this study. Purulent wound drainage during the puncture was not observed either, nor were systemic infection findings (such as fever, fatigue, lack of appetite, and others) concomitant with this condition. Hydroxyapatite occurs both geologically as a mineral, and as the predominant inorganic component of bones.17 Although geological hydroxyapatite may occur as large single crystals, bone mineral consists of small crystals a few angstroms long, containing impurities such as carbonate, Mg, and acid phosphate.5 Using x-ray diffraction is a very good method to determine the crystalline compound type(s) existing in a sample, to make a qualitative assessment of the quantities of phases (different crystal structures) present in the sampling volume, and to assess if there is preferential orientation (texture).5 Other than these commonly practiced uses of x-ray diffraction, more elaborate assessments are also possible. For example, changes in the crystal’s structure in terms of atomic positions and/ or involvement of new atomic species in an otherwise known crystal, and the amount of deformation in a known crystalline phase, can also be established quantitatively if comparative studies are conducted using formal mathematical treatments and base standard samples, and if not, qualitative assessments are still possible. The main features in a radiographic spectrum that have to be considered in all of these approaches are the peaks’ positions, relative heights, and widths (full width half-maximum). Peak positions establish the type(s) of crystalline phases present, whereas relative peak heights are informative in terms of a qualitative quantification of crystalline components in a sample even without resorting to formal treatments. Likewise, if the possibility of a texture is eliminated, then comparing the relative peak heights to the intensity levels established in the literature can indicate involvement or exclusion of atomic species in the sample without changing the crystal’s structure. Peak widths, on the other hand, can yield information regarding the grain size and/or the amount of deformation in a polycrystalline structure under examination. The x-ray diffraction spectra presented here clearly demonstrate that Mg promoted the formation of hydroxyapatite as revealed by greater peak heights at both major and minor peak positions. Unconventional relative peak heights, such as an expected minor peak appearing greater than an expected major peak, have been attributed to the nature of the hydroxyapatite crystal. Unexpected peak heights have also been reported by others depending on the age of the bone, the hydroxyapatite type (organic or nonorganic origin), and the solute content present in the hydroxyapatite studied.4,21,29 Conclusions Magnesium and its alloys are under consideration as possible biocompatible and biodegradable materials for use in bone-related surgery. The sheep intertransverse process fusion model in this study showed that the rate of 147
R. A. Kaya et al. rigid Mg particle–induced fusion was 81.25%, compared with 62.5% in the autograft-induced fusion group. The quality of bone formation was higher in Mg particle– treated segments than in autografts alone, which implies an osteoblastic response to the degrading Mg. Radiographic results confirmed that Mg promoted hydroxyapatite formation, possibly by supplying substitutes for Ca present in this crystal. This Mg promotion accelerated bone formation and fusion, possibly supported by the histological findings of this study. However, further research is needed to establish critical Mg doses and timerelated results to fully understand the phenomenon. References 1. Boden SD, Martin GJ Jr, Morone MA, Ugbo JL, Moskovitz PA: Posterolateral lumbar intertransverse process spine arthrodesis with recombinant human bone morphogenetic protein 2/hydroxyapatite-tricalcium phosphate after laminectomy in the nonhuman primate. Spine 24:1179–1185, 1999 2. Boden SD, Schimandle JH, Hutton WC: Lumbar intertransverse-process spine arthrodesis using a bovine-derived osteoinductive bone protein. A preliminary report. J Bone Joint Surg Am 77:1404–1417, 1995 3. Cook SD, Dalton JE, Prewett AB, Whitecloud TS III: In vivo evaluation of demineralized bone matrix as a bone graft substitute for posterior spinal fusion. Spine 20:877–886, 1995 4. Dalconi MC, Meneghini C, Nuzzo S, Wenk R, Mobilio S: Structure of bioapatite in human foetal bones: an X-ray diffraction study. Nucl Instrum Methods Phy Res B 200: 406–410, 2003 5. Danilchenko SN, Kukharenko OG, Moseke C, Protsenko IY, Sukhodub LF, Sulkio-Cleff B: Determination of the bone mineral crystallite size and lattice strain from diffraction line broadening. Cryst Res Technol 37:1234–1240, 2002 6. DePalma AF, Rothman RH: The nature of pseudarthrosis. Clin Orthop Relat Res 59:113–118, 1968 7. Emery SE, Brazinski MS, Koka A, Bensusan JS, Stevenson S: The biological and biomechanical effects of irradiation on anterior spinal bone grafts in a canine model. J Bone Joint Surg Am 76:540–548, 1994 8. Emery SE, Fuller DA, David A, Stevenson S: Ceramic anterior spinal fusion: biologic and biomechanical comparison in a canine model. Spine 21:2713–2719, 1996 9. Fernyhough JC, Schimandle JH, Weigel MC, Edwards CC, Levine AM: Chronic donor site pain complicating bone graft harvesting from the posterior iliac crest for spinal fusion. Spine 17:1474–1480, 1992 10. Grauer JN, Patel TC, Erulkar JS, Troiano NW, Panjabi MM, Friedlaender GE: 2000 Young Investigator Research Award winner. Evaluation of OP-1 as a graft substitute for intertransverse process lumbar fusion. Spine 26:127–133, 2001 11. Heublein B, Rohde R, Kaese V, Niemeyer M, Hartung W, Haverich A: Biocorrosion of magnesium alloys: a new principle in cardiovascular implant technology? Heart 89:651–656, 2003 12. Kai T, Shao-qing G, Geng-ting D: In vivo evaluation of bone marrow stromal-derived osteoblasts-porous calcium phosphate ceramic composites as bone graft substitute for lumbar intervertebral spinal fusion. Spine 28:1653–1658, 2003 13. Kim DH, Jahng TA, Fu TS, Zhang HY, Novak SA: Evaluation of HealosMP52 osteoinductive bone graft for instrumented lumbar intertransverse process fusion in sheep. Spine 29: 2800–2808, 2004 14. Kim SR, Lee JH, Kim YT, Riu DH, Jung SJ, Lee YJ, et al: Synthesis of Si, Mg substituted hydroxyapatites and their sintering behaviors. Biomaterials 24:1389–1398, 2003
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42. Znamenskii MS: Metallic osteosynthesis by means of an apparatus made of resorbing metal. Khirurgiia 12:60–63, 1945 43. Zreiqat H, Howlett CR, Zannettino A, Evans P, Schulze-Tanzil G, Knabe C, et al: Mechanisms of magnesium-stimulated adhesion of osteoblastic cells to commonly used orthopaedic implants. J Biomed Mater Res 62:175–184, 2002 Manuscript submitted August 3, 2006. Accepted October 23, 2006. Financial support for this study was provided by the State Planning Department of Turkey through TÜBI˙ TAK, the Turkish Scientific and Technological Research Council. Address reprint requests to: R. Alper Kaya, M.D., Göktürk Cad. No: 46/14 S ¸ amat Apt., 34077 Göktürk, Istanbul, Turkey. email:
[email protected].
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