Growth Factors in Human Serum During Operative Tibial Lengthening With the Ilizarov Method Maksim V. Stogov,1 Svetlana N. Luneva,1 Konstantin I. Novikov2 1 Clinical and Experimental Laboratory Department, Russian Ilizarov Scientific Center for Restorative Traumatology and Orthopedics, 6, M. Ulianova Street, 640014, Kurgan, Russian Federation, 2Laboratory of Limb Deformity Correction and Lengthening, Russian Ilizarov Scientific Center for Restorative Traumatology and Orthopedics, 6, M. Ulianova Street, 640014, Kurgan, Russian Federation
Received 6 December 2012; accepted 5 July 2013 Published online 24 July 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22454
ABSTRACT: Despite the widespread clinical use of distraction osteogenesis for limb lengthening, the cellular and molecular mechanisms by which this surgical treatment promotes new bone formation in humans are not well understood. The aim of the research was to study the levels of growth factors (GFs) in the serum of patients that were undergoing tibial lengthening with the Ilizarov method of distraction osteogenesis. Those were patients with unilateral congenital discrepancy of the tibia (n ¼ 12), unilateral posttraumatic tibial shortening (n ¼ 7), and healthy patients that underwent cosmetic bilateral tibial lengthening (n ¼ 10). The study established that unlike the congenital group, the posttraumatic group and healthy subjects showed a significantly evident increase in the levels of angiogenic GFs in their serum on day 10 of distraction. In the congenital group, the changes were not significant at this time point. The levels of TGF-a, TGF-b1, and TGF-b2 tended to decrease on day 10 of distraction and on day 30 of the post-distraction period in the cosmetic and posttraumatic groups while they grew in the congenital group. Most dynamic changes in the GFs levels during tibial lengthening were noted in the subjects undergoing cosmetic lengthening, and the least ones were in the congenital group. ß 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1966–1970, 2013 Keywords: Ilizarov method; tibial lengthening; serum; growth factors
The Ilizarov method of distraction osteogenesis has been widely used for managing orthopedic diseases and injuries as well as in somatically healthy individuals that desire to increase their height.1,2 However, few studies have been devoted to comparing the features of the lengthening process in different pathological conditions. Several authors assessed clinical and radiographic features of distraction osteogenesis used for limb length discrepancy (LLD) due to congenital and acquired pathology and their conclusions stated that there were differences in the groups in regard of the healing process.3–5 Moreover, the cellular and the molecular mechanisms that regulate distraction osteogenesis during the Ilizarov operative lengthening in humans have been studied insufficiently. Our previous study revealed that the metabolic processes in operative lengthening depended on the disease etiology and differed significantly during lengthening in LLD and cosmetic cases.6 Those metabolic differences may have an effect on the rate of distraction regenerate formation.7 The metabolic processes in the tissues of the limb segment under lengthening are mainly regulated with humoral mechanisms. Their agents are numerous growth factors (GFs). Some authors investigated several GFs expressed during dosed bone distraction in humans.8 However, the majority of the available studies were focused on investigating the biochemical processes during experimental distraction osteogenesis. In particular, it was shown that bone regeneration during distraction osteogenesis was accompanied by the rise in the expression of angiogenic GFs in serum, Correspondence to: Maksim V. Stogov (T: þ7-3522450538; F: þ7-3522454060; E-mail:
[email protected]) # 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
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and specifically of the vascular endothelial growth factor (VEGF) and angiopoietins (ANG-1 and ANG2).9–12 The platelet-derived growth factor (PDGF) was also noted to participate in reparative bone regeneration.13 It was revealed that several factors impeded bone formation, and the studies on mice showed that the impediment was due to the tumor necrosis factoralpha (TNFa).14 It was reported that the insulin growth factor 1 (IGF-1) levels rose during tibial lengthening in dogs.15 Nevertheless, we have not found any thorough description of the human GFs spectrum expressed by applying the method of distraction osteogenesis in the reported studies. Also, none of the papers available has compared the change in the GFs concentrations in the human serum during Ilizarov limb lengthening procedures indicated for varied orthopedic pathologies. Our study objective was to investigate the changes in the GFs levels in the blood serum of the patients that were undergoing Ilizarov tibial lengthening procedures and to compare the dynamics of their levels during tibial elongation in congenital, posttraumatic and somatically healthy cases in order to detect the differences that can occur in humoral regulation of distraction osteogenesis in those cases of varied etiology.
METHODS Serum GFs were studied in three groups of consecutive patients that were treated at our institution within the period of 2011–2012. Group 1 (n ¼ 12) had unilateral congenital idiopathic tibial discrepancy. According to their medical histories, the pathology in this group of patients had been diagnosed at birth but none of the known genetic disorders had been identified in them. Their mean LLD was 5.68 0.9 cm. Functions of their limb joints were not limited. Group 2 (n ¼ 7) had a mean posttraumatic residual
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discrepancy of 3.64 1.0 cm unilaterally as a consequence of united tibial factures (4–15 years following injury). Group 3 (n ¼ 10) included healthy patients were undergoing tibial lengthening for cosmetic reasons. The groups matched in the age range of 19–39 years (group 1), 18–37 years (group 2), and 20–35 years (group 3). All the patients had not had any previous lengthening procedures. The lengthening was performed using the Ilizarov method and apparatus. Bone distraction in all the groups was manual and similar at the beginning of the distraction process. It started on days 4 or 5 postoperatively with the rate of 1 mm per day made with four steps 0.25 mm each. The distraction rate was corrected according to radiographic findings that evaluated the regeneration process, and according to clinical examination that assessed joint contractures in the limbs under elongation. The regular tendency was the decrease of the distraction rate, and the corrected mean values by monofocal distraction were 0.76 0.14 mm/day in group 1, 0.81 0.08 mm/day in group 2, and 0.68 0.27 mm/day in group 3. The average lengthened amount in group 3 was 6.1 2.7 cm by monofocal variant of lengthening. The posttraumatic group 2 had the mean lengthened amount of 4.52 1.9 cm, and in group 1 of congenital cases the average lengthening achieved was 6.48 1.8 cm. The levels of the following GFs in patients’ serum were assessed: insulin-like growth factors 1 and 2 (IGF-1, IGF-2), vascular endothelial growth factor (VEGF), angiopoietin 1 (ANG-1), platelet-derived growth factor AA (PDGF-AA), epidermal growth factor (EGF), and transforming growth factors (TGF-a, TGF-b1, TGF-b2). Blood was collected for tests at three time points in each patient: one day prior to the surgery, on day 10 after the start of bone distraction for lengthening, and on day 30 in the post-distraction period when the leg length had been already equalized but the regenerated bone continued maturation with the fixator on place. The immune enzyme analyzer ELX808 (Bio-Tek Instruments, Inc., Winooski, VT) and the reagents kits of the Research&Diagnostics Systems, Inc. (Minneapolis, MN) were used for studying GF concentrations.
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The levels of serum GFs in the studied groups were compared with the GF levels of the reference group (n ¼ 20) of recruited healthy volunteers (age range from 18 to 38 years) at a single time point. Non-parametric Wilcoxon W-test for independent samples was used for comparing the reliability of difference of the values at the mentioned stages in the patients’ groups with the levels of the reference group. The reliability of differences between the groups was assessed using the non-parametric Kruskal–Wallis H-test followed by multiple comparisons using the Dunn criterion. All patients and healthy subjects gave their informed consent for inclusion into the study. The study was approved by the ethic board of the institution.
RESULTS Before Surgery Our tests revealed that the concentration of IGF-2 was 25% lower while the levels of EGF and ANG-1 were elevated by 60% and 70%, respectively, in group 1 relative to the reference group (Figs. 1–3). In group 2 at the same time point, the serum IGF-1 level was 20% lower but the concentrations of EGF and TGF-a were increased by 70% and 150%, respectively, as compared with the reference group. Group 3 patients had the concentrations of the studied GFs within the reference group values. The levels of EGF, ANG-1, and TGF-a showed a statistically significant difference between three groups of patients before surgeries. Day 10 of Distraction The levels of IGF-1 and TGF-b1 in group 1 were significantly higher relative to the reference group. In group 2, the concentrations of angiogenic factors VEGF, ANG-1, and PDGF- were statistically higher than the reference levels. A significant increase in agiogenic GFs was observed in group 3 (VEGF by
Figure 1. Dynamic changes in IGF-1, IGF-2, and EGF at studied time points. Note: BS, before surgery; D10, on day 10 of distraction; PD30, on day 30 post-distraction; HN, highest level in the reference group; LN, lowest level in the reference group; , reliability of difference with the reference group, p < 0.05; #, reliability of difference between three groups of treated patients, p < 0.05. JOURNAL OF ORTHOPAEDIC RESEARCH DECEMBER 2013
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Figure 2. Dynamic changes in VEGF, ANG-1, and PDGF-AA at studied time points. Note: BS, before surgery; D10, on day 10 of distraction; PD30, on day 30 post-distraction; HN, highest level in the reference group; LN, lowest level in the reference group; , reliability of difference with the reference group, p < 0.05; #, reliability of difference between three groups of treated patients, p < 0.05.
Figure 3. Dynamic changes in TGF-a, TGF-b1, and TGF-b2 at studied time points. Note: BS, before surgery; D10, on day 10 of distraction; PD30, on day 30 post-distraction; HN, highest level in the reference group; LN, lowest level in the reference group; , reliability of difference with the reference group, p < 0.05; #, reliability of difference between three groups of treated patients, p < 0.05.
120%, ANG-1 by 70%, PDGF- by 20%, and EGF by 60%). Interestingly enough, but the TGF-b1 level decreased while the TGF-b2 level rose in group 3. The majority of the studied factors (EGF, VEGF, ANG-1, PDGF-A, TGF-b1, and TGF-b2) showed significant differences in their concentrations when three groups of patients were compared. Day 30 of Post-Distraction Period Group 1 had a considerable growth in TGF-a and TGF-b2 concentrations and group 2 showed the rise of IGF-1 concentration while its IGF-2 and TGF-b1 JOURNAL OF ORTHOPAEDIC RESEARCH DECEMBER 2013
levels decreased when compared with the reference values. In group 3, the levels of VEGF and TGF-b2 remained elevated while the mean concentrations of IGF-1 and TGF-b1 were statistically lower by 40% relative the reference values. Statistically significant differences between our three groups of patients were observed for IGF-1, IGF-2, VEGF, TGF-a, and TGF-b1.
DISCUSSION GFs are synthesized in a variety of cells that are located in all the tissues and fulfill a wide range of
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biological actions in the human organs and tissues. Therefore, it is difficult to make a sufficient analysis of the revealed findings and make definite conclusions. However, we suppose that the program of humoral regulation of distraction osteogenesis that is realized through the GFs that were studied by us depends on LLD etiology. Specifically, according to our findings the mechanisms of humoral regulation of distraction osteogenesis were mainly directed to the activation of angiogenesis in the cosmetic and posttraumatic groups. Groups 2 and 3 revealed a considerable rise in the angiogenic GFs while this phenomenon was not observed in the congenital group. The regulation of distraction osteogenesis in group 1 was influenced by IGF-1 as only this factor level grew in that group in the distraction period. One can suppose that the initially low level of IGF-1 in group 2 would mean that the bone ability for reparation is reduced. However, the concentration of IGF-1 in group 2 during the treatment was elevated. It proves that the synthesis of this GF was activated and was caused by distraction strain.16 It was noted that EGF activated the osteolytic mechanism through its interaction with osteoclasts and promoted the increase in the release of calcium from the bone thus stimulating resorption.17 It is important to note that group 3 showed the changes that could have caused osteolysis as far as the concentration of IGF-1 tended to decrease during treatment and the level of EGF grew considerably by day 10 of distraction. The differences in the humoral mechanisms of distraction osteogenesis regulation in our three groups of varied etiologies were also evident via the effects that were realized by transforming GFs. Our groups of patients showed different changes in the concentrations of TGFs at the time points studied. It has been difficult to define the roles of separate TGFs in the regulation of distraction osteogenesis in our study. We can suppose that the differences in TGFs concentrations in our groups were directly associated with the differences in the mechanisms of reparative osteogenesis resulting from the etiology of the conditions. It is known that the TGF group (TGF-a, TGF-b1, TGF-b2) plays an important role in stimulation of osteogenesis by promoting the formation and remodeling of the extracellular bone matrix.18,19 We can summarize that the response to lengthening in group 1 was mainly influenced by the action of the systemic anabolic IGF-1. Distraction strain in groups 2 and 3 resulted in the response reaction that predominantly caused the expression of the GFs that activated angiogenesis and finally promoted the ingrowth of new vessels into the regenerate in support of active osteogenesis. However, the increase in EGF and the decrease in IGF-1 proved the osteolytic tendency in group 3. Therefore, according to our study the potential use of GFs will depend on the etiology of
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pathologies, and the stimulation of distraction osteogenesis could be implemented by such potential agents as IGF-1, VEGF, and ANG-1. The detection of the differences that occurred in humoral regulation of distraction osteogenesis in our cases of varied etiology broadens the fundamental knowledge and may result in new approaches to stimulation of bone regeneration. The basic research that studies GFs, preclinical data and some clinical reports on platelet-rich plasma that contains a variety of GFs support their use for orthopedic problems and congenital disorders.20,21 We conclude that the humoral regulation during tibial lengthening with the Ilizarov method is dependent on the etiology of the condition and is influenced by specific GFs in each of the conditions studied. Further research should reveal the mechanisms of GFs relationship with the cells that participate in distraction osteogenesis.
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