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European Journal of Trauma and Emergency Surgery

Original Article

Bone Fracture Healing with Umbilico-Placental Mononuclear Cells: A Controlled Animal Study Onur Polat, Gurur Polat, Sercin Karahuseyinoglu, Nüket Yörür Kutlay, Arzu Gül Tasci, Esra Erdemli, Ajlan Tukun, Mustafa Cihat Avunduk, Sükrü Küplülü, Mehmet Demirtas1

Abstract Background: Fracture healing is a significant process in orthopedics. In this controlled animal study, our aim is to expose the healing effects of cord blood umbilico-placental mononuclear cells (UPMNCs) on bone fractures. Materials and Methods: Caesarean sections were performed on five pregnant New Zealand rabbits at term. Placentas and cords were collected. Standard closed transverse shaft fractures were created on both tibial bones of 15 baby rabbits. The right tibias were given UPMNCs; the left tibias were the control group. Histological examinations, osteoblast and osteoclast cell counts, and mechanical stabilities were compared. Anchorage of the donor cells was shown by the fluorescence in situ hybridization (FISH) technique. Results: In the group injected with UPMNCs, histopathological fracture healing was faster, osteoblast and osteoclast counts were significantly increased, and the maximum load capacity was higher. The presence of XX and XY chromatins on the same slide revealed the anchorage of female donor cells on male tissues. Conclusion: The effects of umbilico-placental mononuclear cells on bone healing are histopathological healing priority, increased osteoblastic and osteoclastic activities (bone turnover), and better mechanical stability. Key Words Bone fracture Æ Mononuclear Æ Stem cell Æ Cord blood Æ Placenta Eur J Trauma Emerg Surg 2010;36:60–6

results in optimal skeletal repair and restoration of skeletal function. Delayed fracture healing is a significant problem in orthopedics. Einhorn reported that 5.6 million fractures occur annually in the United States [1]. Praemer et al. reported that 5–10% of these patients had delayed or impaired healing [2]. Researchers have arduously sought alternatives to the current treatments, with tissue engineering receiving much recent attention. Bone tissue engineering refers to the science of creating viable tissue that will replace, repair or augment diseased or defective bone tissue. To achieve successful bone tissue engineering, four critical parameters must be met for osteogenesis: osteoproduction, osteoinduction, osteoconduction, and mechanical stimulation [3]. Osteoproduction refers to the ability of a cell to produce the actual bone material (osteoid matrix). Traditionally, osteoblasts are responsible for osteogenesis; mesenchymal stem cells can differentiate to become an osteoblast, which can subsequently form bone [4]. Mesenchymal stem cells have been shown to have osteogenic potency, and as the technology to purify and deliver these cells is perfected, it will become possible to treat diseases such as osteogenesis imperfecta and osteoporosis [5]. It has been shown that mononuclear cells derived from umbilical cord blood give rise to fibroblast-like cells that express mesenchymal stem cell related antigens [6]. The objective of this study was to evaluate the healing effects of umbilico-placental mononuclear cells (UPMNCs) on cortical bone regeneration.

DOI 10.1007/s00068-009-9038-8

Introduction Fracture healing is a complex, well-organized regenerative process initiated in response to injury, which 1

Materials and Methods Surgical Procedure Following University Ethics Committee approval, caesarean sections were performed on five pregnant

Ibni Sina Hospital Emergency Department, Ankara University Faculty of Medicine, Ankara, Sihhiye, Turkey.

Received: February 15, 2009; revision accepted: July 20, 2009; Published Online: September 11, 2009

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Eur J Trauma Emerg Surg 2010 Æ No. 1 URBAN & VOGEL

Polat O, et al. Bone Fracture Healing with Umbilico-Placental Mononuclear Cells

New Zealand rabbits at term. All baby rabbits were marked to identify their mothers and the order in which they were born. Placentas and cord blood were collected in a 50 ml conic Falcon tabbed tube that contained 10 ml of cord blood stem cell feeding medium (TX-ES, Thromb-X, Leuven, Belgium). Separating and Freezing the Mononuclear Cells After 3 min of vortexing, the extract was transferred to a Falcon tube. It was then filtered to get rid of large tissue pieces. The filtered extract was transferred to another Falcon tube containing 2 ml of Ficoll. After centrifugation (at 10C, 1,300 rpm for 30 min), the upper layer of serum was transferred to another tube for cryopreservation. The intermediate part, which was rich in mononuclear cells, was mixed with 2 ml of hydroxyethyl starch (HES) and centrifuged at 1,200 rpm for 10 min. The supernatant was separated and the bottom part was resuspended with 1 ml HES. The suspension was placed in a 4.5 ml cryo-vial and 1 ml of serum and 0.2 ml of a dimethyl sulfoxide (DMSO)-containing cryo solution were poured slowly onto the cell suspension and left in a liquid nitrogen tank for vitrification. Thawing Procedure Frozen vials were taken out of the nitrogen tank, rotated manually for 10 s, and placed into a 37C water bath until little pieces of crystal could be seen. The thawed suspension was transferred to another tube. After 5 ml of HES solution had been slowly added, the solution was mixed by swinging the tube. The tube was then centrifuged at 1,200 rpm for 5 min. The cell pellets formed at the bottom were resuspended with stem cell culture media (TX-ES) and placed in a 5 ml syringe to be injected into the fracture area. Tibial Fracture Procedure and Cell Injections The baby rabbits that survived were nurtured for four weeks. At the end of this period, fifteen male rabbits were anesthetized with intraperitoneal ketamine 20 mg/kg and xylazine 5 mg/kg, and standard closed transverse shaft fractures were created on both tibial bones. To produce the standardized fractures, a metal rod that weighed 500 g and was pointed at one end in order to exert pressure on a single point was left to fall freely for 10 cm between two parallel rods that were 3 cm apart, and there was a hole at the bottom of the parallel rods to insert the rabbit’s leg. A tibial fracture was chosen for this study because the subcutaneous localization of the tibia makes it easy to identify the callus and provides easy access for injections near the fracture line in rabbits.

Eur J Trauma Emerg Surg 2010 Æ No. 1

After thawing, 1 ml of the umbilico-placental fluid of the female rabbit born to the same mother were injected into the right tibias of their male siblings at the fracture line immediately after the fractures had been generated. The fluid contained 3.2 ± 0.8 · 106 mononuclear cells, with a viability of 96.2 ± 1.9%. The same amount of TX-ES was injected near the fracture lines of the left tibias of the same males, forming the control group. A sterile wound dressing was applied to the site of injection and the fracture was immobilized in a circular cast extending well over the proximal and distal joints of the tibial bone. To obtain a cumulative effect, the UPMNC injections into the leg were repeated three times on the study legs, with an injection performed every other day. The casts were removed and prophylactic antibiotics were injected intramuscularly 30 min before cell injection. The fracture hematoma was palpated and injections were performed through the posteromedial soft tissue near the fracture hematoma to decrease the risk of infection. The same procedure was applied and the same amount of TX-ES was injected into the control tibia. One week after the last injections were completed, five rabbits were sacrificed for histological examination. This step was repeated every week for two weeks. At the end of the third week, three of the last five rabbits sacrificed were chosen randomly and a threepoint bending test was performed on their tibias on both sides (study and controls). Cross-sectional tissue samples were also obtained from these animals for histological examination. The remaining two rabbits were sacrificed for direct histological examination. Tissue sections obtained from these five animals were prepared for fluorescence in situ hybridization (FISH) to analyze the donor cell anchoring. The weekly plans and histological examinations were conducted according to a study by Kilicoglu et al. [7]. Histological Examination and Quantification of Osteoblasts and Osteoclasts After decalcification, tissue specimens were prepared in an autotechnicon, embedded in paraffin, and sectioned with a microtome. The sections (5 lm) were stained with hematoxylin–eosin and Mallory’s azan dyes. Stained specimens were investigated by a Nikon Eclipse E400 light microscope. After examining the histopathological healing site, four different areas were photographed for each specimen after staining using a Nikon Coolpix 5000 camera attachment. All of the images were transferred to a PC and analyzed using Clemex Vision Lite 3.5 image analysis software. The

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osteoblasts and osteoclasts in a 1,000 lm2 area were marked using the same image analysis software. Cross Sex Fluorescence In Situ Hybridization (FISH) Method In order to expose the UPMNCs in the fracture area, paraffin-embedded tissue sections were prepared for fluorescence in situ hybridization (FISH) according to the previously described protocol, with 5 min heat (in 2 · SSC at 75C) and 10 min of enzyme proteinase K (in TEN buffer at 37C) treatment. FISH was performed on pretreated slides using X centromere (DXZ1, Oncor P5060-D or P5060-B) and Y centromere (DYZ3, Oncor P5065-B or P5065-D) probes [8]. Biomechanical Analysis Tibia specimens (n = 6) were placed on a Lloyd Instruments (Segensworth, UK) standard miniature three-point bending set-up. Mechanical experiments were carried out with Lloyd Instruments LS500 materials testing machine. Data were collected via Dapmat software from Lloyd Instruments. A load cell with a capacity of 500 N and a resolution of 0.5% was used during the experiments. The distance between the lower supports was set to 11 mm. The crosshead speed was 1 mm/min. The force/displacement data were collected every 0.1 s. The bones were placed on the anterior– posterior plane and loaded through the midshaft, as performed previously. Experiments were continued until fractures appeared. At the end of each experiment, load versus deflection curves were obtained. By combining the geometric calculations and the biomechanical test results, the following geometric and mechanical properties of each specimen were calculated: femur length, a/p width, m/l width, maximum load, deflection at maximum load, and energy absorption. Statistical Analysis Using the mechanical test, the geometric and mechanical properties of each specimen were calculated. Comparisons between two groups in terms of maximum load and energy absorption were performed using the Mann-Whitney U-test. The Bonferroni correction was applied for all possible multiple comparisons. Because of the number of tests undertaken, the level of significance was set to 0.0167. Differences between the histopathological data obtained during the three consecutive weeks were evaluated by Kruskal-Wallis variance analysis. When the p value from the Kruskal-Wallis test statistic was statistically significant, a multiple comparison test was performed to discern which of the weeks differed from the others [9]. SPSS for Windows 15.0 was used for statistical evaluation.

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Histologic examinations, the FISH method, mechanical tests, and statistical analyses were performed by investigators who were blind to group allocation.

Results Histopathological Analysis and Quantification of Osteoblasts and Osteoclasts The histopathological analysis revealed that there were no significant differences between the control and experimental groups at the end of the first week of the healing period. At the end of the second week, both groups showed similar histopathological changes, such as prominent fibrocartilage and hyaline cartilage tissue formation. Bilateral subperiosteal hyaline cartilage callus formation and increased vascularization were detected in particular, but osteoid tissue formation was not observed. On the other hand, the group injected with UPMNCs healed slightly better, showing hyaline cartilage callus permanence and trabecular bone regularity, in contrast to the interruption of the hyaline cartilage callus and trabecular bone connections by connective tissue that was observed in the control group (Figure 1). At the end of the third week, remodeling of the bone continued with hyaline cartilage callus formation. The callus was more regular in the group injected with UPMNCs. Osteoid formation had started, and the restoration and remodeling had already produced firm callus (Figures 2, 3). The osteoblastic and osteoclastic activities of the fractured area injected with mononuclear cells were compared with those of the controls. Osteoblast counts were different for each of the the weeks for the group injected with UPMNCs (p < 0.001 for all comparisons). There was a statistically significant increase in osteoblast counts at the end of the third week in this group; there was also a statistically significant difference between the osteoblast count in the first and the second weeks, but not between the second and the third weeks for the group injected with UPMNCs (p = 0.017 and p = 0.087, respectively). There was no statistically significant difference between the osteoclast counts in the first and second weeks, but it was higher at the end of the third week than during the second week for the group injected with UPMNCs (p = 0.543 and p < 0.001, respectively). There was no statistically significant difference between the osteoclast counts during the three weeks for the control fractures (p = 0.661). There were also statistically significant differences between the study and control groups in terms of the osteoblast and osteoclast counts during the third week, but this was not the case for the first and second weeks (for the osteoblast count:



Figure 1. At the end of the second week, the group injected with mononuclear cells is one step ahead of the control group in terms of the permanence of the hyaline cartilaginous callus (HC) and the regularity of trabecular bone (B). In the control group, hyaline cartilaginous callus and trabecular bone connection is interrupted by connective tissue (up arrow). A, control group; A¢, group injected with mononuclear cells (Mallory azan, 40·). Figure 2. At the end of the third week, remodeling of the bone continues with the hyaline cartilaginous callus and the remodeling of the callus is more regular in the group injected with mononuclear cells. HC, hyaline cartilaginous callus; C, control group; C’, group injected with mononuclear cells (HE, 40·).

Figure 3. Third week: remodeling of the bone continues with the hyaline cartilaginous callus (HC) in manner, but in the group injected with mononuclear cells osteoid (up arrow) formation has started, so restoration and remodeling had already produced firm callus. FB, fractured bone; C, control group (HE, 100·); C¢, group injected with mononuclear cells (HE, 200·).

p < 0.001, p = 0.686, p = 0.570, respectively; for the osteoclast count: p = 0.015, p = 0.686, p = 0.683, respectively (Figures 4, 5). Cross-Sex Fluorescence In Situ Hybridization (FISH) Method XX and XY chromatins at the same fracture can be seen on the screen (Figure 6). It showed that donor (female) UPMNCs joined male rabbit’s for bone repair.


Biomechanical Testing With respect to geometrical properties such as tibia length, anterior/posterior length, and medial/lateral length, there were no meaningful differences between the right and the left tibia groups (p > 0.05). There was also no significant difference between the right and left tibia groups for energy absorption (p = 0.83, p = 0.71) (Table 1). Regarding the maximum load capacity, there was a statistically meaningful difference between

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70 60 50 40 30 20 10 0

Experimental Control

1

2 Weeks

3

Mean Osteoclast

Figure 4. Quantification of the osteoblasts and osteoclasts at the end of each week.

Mean Osteoblast


4 3.5 3 2.5 2 1.5 1 0.5 0

Experimental Control

1

2 Weeks

3

Figure 5. Osteoblast and osteoclast counts.

Figure 6. XX chromatins from different parts of the same fracture (FISH) in a male rabbit. XX nucleated cells from the female rabbits can be seen at the male rabbit’s bone fracture area (left). XX and XY chromatins together on the same slide showed the female cells were seized by the male tissues (right).

the UPMNC-injected right tibias and the control tibias (p = 0.04, Table 1).

Discussion Bone, which undergoes continuous remodeling throughout life, is one of the few organs that retain

the potential for regeneration in adult life [10, 11]. During a fracture repair process, the pathway associated with normal embryonic development is recapitulated with the coordinated participation of several cell types [12]. The healing process involves the coordinated participation of hematopoietic and immune cells within the bone marrow, and vascular and

Table 1. Descriptive statistics for maximum load, energy absorption, and a comparison between groups. Experimental (n = 3)

Maximum load Energy absorption

Control (n = 3)

p

X ± SD

Median (min–max)

X ± SD

Median (min–max)

37.85 ± 12.38 40.53 ± 2.29

32.34 (29.17–52.03) 41.6 (37.9–42.1)

14.20 ± 5.73 37.97 ± 19.65

14.64 (8.26–19.7) 37.6 (18.5–57.8)

0.049 0.513

(X, mean; SD, standard deviation; min, minimum; max, maximum)

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skeletal cell precursors including mesenchymal stem cells [13]. In this study, we injected UPMNCs obtained from the umbilico-placental units of siblings with similar immunogenic activities to males born from the same mother, thus avoiding a graft-versus-host reaction. However the collection and preparation of cord blood mononuclear cells from the rabbits was not an easy procedure, because each placenta was small and each umbilicus was short and narrow. Collecting the umbilical and placental blood separately does not appear to be possible. This is why the cells that were used have been termed ‘‘umbilico-placental.’’ There is some evidence to suggest that stem cells may home into sites of injury. The fusion of stem cells to resident cells, followed by reductive cell division, may be one mechanism by which stem cells become differentiated [14, 15]. Cord blood stem cells are superior in that they have been shown to reduce immune reactions. The reason for this diminished immune reaction is the decreased number of CD8(+) lymphocytes in the cord blood stem cell, which has made those cells popular for research in animal studies [16]. Additionally, fewer cytokines and cytotoxic effector cells can be found in cord blood than in peripheral blood [17]. Whether these properties account for the reduced capacity of transplanted cord blood cells to modulate graft-versus-host disease remains to be determined [18]. Erices et al. [19] showed that umbilical cord bloodderived mononuclear cells, when set in culture, gave rise to adherent cells that exhibited either an osteoclast or a mesenchymal-like phenotype. Their results suggest that preterm (as compared with term) cord blood is richer in mesenchymal progenitors, similar to hematopoietic progenitors. The reason for using UPMNCs in this study was to benefit from these advantages. The study showed, with the help of the FISH method, that these cells anchor to the fractured area [8]. Fracture healing involves two processes: one is direct or primary healing, and the other is indirect or secondary healing [20]. The majority of the fractures heal by the indirect route. This involves a combination of intramembranous and endochondral ossification with the subsequent formation of callus [21]. In this study, intramembranous and endochondral ossification was observed on both the study and control tibias; moreover, the inductive effects of the mononuclear cells were noted in the experimental group. Intramembranous ossification involves the formation of bone directly, without cartilage formation. Callus formation was compared between the experimental (those injected with UPMNCs) and control


groups. Callus formation can be seen earlier on histological slides in the experimental group than in the control group. Osteoid formation begins during the third week of the healing period in the experimental group, but cannot be seen in the control group. The permanence of the hyaline cartilagenous callus and the regularity of trabecular bone both point to an advanced healing process in the experimental group. The biomechanical evaluation of fracture healing was performed with two different parameters: load to failure (maximum load) and energy absorption capacity. The load to failure capacity of the treatment group was significantly higher than that of the control group (p < 0.05). The load to failure capacity is highly recognized in the literature as a major parameter that needs to be checked for the biomechanical assessment of fracture healing [22, 23]. The load to failure capacity of the treatment group was twice as much as that of the control group. This difference demonstrates that the fractures treated with the UPMNC suspension actually gained a considerable amount of structural integrity during the healing process. This result also supports the existence of proper callus and osteoid formation, which was observed during histopathologic analysis. The second parameter used in the analysis is the energy absorption capacity. This parameter shows the resilience of a material during material testing. According to the results, the energy absorption capacity of the group treated with UPMNCs was higher than that of the control group. Even though the difference was not significant, the higher absorption capacity of the treatment group illustrated the trend that this particular treatment affords more resilience than observed for the controls. This trend may become more significant in the later stages of the healing process, after three weeks. The overall biomechanical evaluation shows that the fractures treated with UPMNCs gained twice as much strength and better resilience than the control ones. It can therefore be said that the healing of the group treated with UPMNCs had better structural integrity than the healing of the control group. Bone tissue engineering, which combines the application of the principles of orthopedic surgery with basic science and engineering, has been heralded as an alternative when bone regeneration is required to replace or restore the function of traumatized, damaged, or lost bone. Stem cells have a potential of constructive effects on bone regeneration [24]. Orthopedic surgery is moving towards minimally invasive techniques; in the future, minimally invasive techniques and stem cells may be used together for seriously damaged patients to obtain complete and rapid bone healing.

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In this study, the increase in the osteoblastic and osteoclastic activity (bone turnover) and the earlier healing of fractures in rabbits given umbilico-placental cells demonstrated the constructive effects of induction. Similar effects on bone healing can be expected for delayed unions and nonunions. Easy application is another advantage of this approach, since injection near to the area is sufficient. Therefore, the incidence of infection is expected to be low. To date, it has not been proven that UPMNCs that have been injected into bones, bone marrow, fracture lines, or intramuscularly can travel to any other affected parts of the body. Therefore, in our study, it was assumed that the injected cells did not contribute significantly to the healing of the control tibia, which was found to have healed to a much smaller extent in comparison with the treated one. For the same reason, female chromatins were not sought in the control tibia. This issue is currently being investigated under the scope of another of our research projects. In summary, fracture healing is a complex, wellorchestrated biological event in which multiple factors and pathways are involved and new developments are anticipated in the future. The results of this animal study show that the injection of UPMNCs near the fracture site speeds up bone healing as the histological priority, increases osteoblastic and osteoclastic activity (bone turnover), and increases mechanical stability. Considering the potential applications of UPMNCs to bone fractures (trauma) and possibly metabolic diseases, the results of this animal study are especially encouraging. Of course, more studies of animals and humans are needed to determine their ultimate effect on bone union and on the restoration of mechanical strength and histological architecture after bone fracture.

4.

5. 6.

7. 8.

9. 10. 11. 12. 13. 14. 15.

16.

17. 18. 19. 20. 21.

Acknowledgments Part of this study was presented at the First Joint Congress of EATES and ETS, 23–26 May 2007, Graz (Austria), as an oral presentation. The authors have not received any financial support or benefit in association with the present article.

Conflict of interest statement The authors declare that there is no actual or potential conflict of interest in relation to this article.

References 1. 2.

3.

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Einhorn TA. Enhancement of fracture-healing. J Bone Joint Surg Am 1995;77:940–56. Praemer A, Furner S, Rice D. Musculoskeletal injuries: musculoskeletal conditions in the United States. Park Ridge: American Academy of Orthopaedic Surgeons, 1992:85–124. Young BH, Peng H, Huard J. Muscle-based gene therapy and tissue engineering to improve bone healing. Clin Orthop Rel Res 2002;403S:243–51.

22. 23.

24.

Bruder SP, Fink DJ, Caplan Al. Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J Cell Biochem 1994;56:283–94. Caplan AI, Goldber VM. Principles of tissue engineered regeneration of skeletal tissues. Clin Orthop 1999;367:12–6. Gang EJ, Jeong JA, Hong SH, Hwang SH, Kim SW, Yang IH, Ahn C, Han H, Kim H. Skeletal myogenic differentiation of mesenchymal stem cells isolated from human umbilical cord blood. Stem Cells 2004;22:617–24. Erdemli B, Kilicoglu-Serin S, Erdemli EA. New approach to the treatment of osteoporosis. Orthopedics 2005;28:59–62. Nilsson SK, Hulspas R, Weıer HUG, Quesenberry PJ. In situ detection of individual transplanted bone marrow cells using FISH on sections of paraffin-embedded whole murine femures. J Histochem Cytochem 1996;44:1069–74. Conover WJ. Multiple comparison test (in Section 5.2). In: Practical nonparametric statistics, 2nd edn. New York: Wiley, 1980:229–239. Olsen BR, Reginato AM, Wang W. Bone development. Annu Rev Cell Dev Biol. 2000;16:191–220. Reddi AH. Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol 1998;16:247–52. Ferguson C, Alpern E, Miclau T, Helms JA. Does adult fracture repair recapitulate embryonic skeletal formation? Mech Dev 1999;87:57–66. Dimitriou R, Tsiridis E, Giannoudis PV. Current concepts of molecular aspects of bone healing. Injury 2005;36:1392–404. Korbling M, Estrow Z. Adult stem cells for tissue repair – a new therapeutic concept. N Engl J Med 2003;349:570–82. Spees JL, Olson SD, Ylostalo J, Lynch PJ, Smith J, Perry A, Peister A, Wang MY, Prockop DJ. Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma. Proc Natl Acad Sci USA 2003;100:2397–402. Rainaut M, Pagniez M, Hercend T, Daffos F, Forestier F. Characterisation of mononuclear cell subpopulations in normal fetal peripheral blood. Hum Immunol 1987;18:331–7. Roncarolo MG, Bigler M, Ciuti E, Martino S, Tovo PA. Immune responses by cord blood cells. Blood Cells 1994;20:573–85. Moise KJ Jr. Umblical cord stem cells. Obstet Gynecol 2005;106:1393–1407. Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 2000;109:235–42. McKibbin B. The biology of fracture healing in long bones. J Bone Joint Surg Br 1978;60B:150–62. Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop 1998;355S:7–21. Turner C, Burr D. Basic biomechanical measurements of bone: a tutorial. Bone 1993;14:595–608. Schriefer JL, Robling AG, Warden SJ, Fournier AJ, Mason JJ, Turner CH. A comparison of mechanical properties derived from multiple skeletal sites of mice. J Biomech 2005;38:467–75. Rose FR, Oreffo RO. Bone tissue engineering: hope vs. hype. Biochem Biophys Res Commun 2002;292:1–7.

Address for Correspondence Onur Polat, MD Ibni Sina Hospital Emergency Department Ankara University Faculty of Medicine Ankara 06106 Sihhiye Turkey Phone (+90/312) 5083030, Fax -5083032 e-mail: [email protected]
