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Keywords:

  • distraction osteogenesis;
  • bone growth factors;
  • matrix metalloproteinases;
  • human growth hormone;
  • systemic biochemical regulation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This study investigates the systemic biochemical regulation of fracture healing in distraction osteogenesis compared with rigid osteotomy in a prospective in vivo study in humans. To further clarify the influence of mechanical strain on the regulation of bone formation, bone growth factors (insulin-like growth factor [IGF] I, IGF binding protein [IGFBP] 3, transforming growth factor [TGF] β1, and basic FGF [bFGF]), bone matrix degrading enzymes (matrix-metalloproteinases [MMPs] 1, 2, and 3), human growth hormone (hGH), and bone formation markers (ALP, bone-specific ALP [BAP], and osteocalcin [OC]) have been analyzed in serum samples from 10 patients in each group pre- and postoperatively. In the distraction group, a significant postoperative increase in MMP-1, bFGF, ALP, and BAP could be observed during the lengthening and the consolidation period when compared with the baseline levels. Osteotomy fracture healing without the traction stimulus failed to induce a corresponding increase in these factors. In addition, comparison of both groups revealed a significantly higher increase in TGF-β1, IGF-I, IGFBP-3, and hGH in the lengthening group during the distraction period, indicating key regulatory functions in mechanotransduction. The time courses of changes in MMP-1, bone growth factors (TGF-β1 and bFGF), and hGH, respectively, correlated significantly during the lengthening phase, indicating common regulatory pathways for these factors in distraction osteogenesis. Significant correlation between the osteoblastic marker BAP, TGF-β1, and bFGF suggests strain-activated osteoblastic cells as a major source of systemically increased bone growth factors during callus distraction. The systemic increase in bFGF and MMP-1 might reflect an increased local stimulation of angiogenesis during distraction osteogenesis.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

DISTRACTION OSTEOGENESIS is a method of producing large quantities of bone using local host tissues stimulated by mechanical distraction forces. After an osteotomy, the continuously enlarging gap is filled with living bone mainly because the intramembranous ossification of the newly built bone regenerates.(1–3) Because of the law of tension-stress as outlined by Ilizarov,(2) living tissues become metabolically activated by slow, steady traction, a phenomenon characterized by the stimulation of both proliferative and biosynthetic cellular functions called mechanotransduction.

However, the molecular mechanisms that translate the mechanical forces into the cellular events during distraction osteogenesis still remain to be understood fully. Limb lengthening by distraction osteogenesis is an ideal model to investigate the in vivo correlation between mechanical stimulation and biological response of the distracted cells, because the callus is stretched by a standardized rate and rhythm of mechanical strain.(1)

Development of new bone from an avascular collagenous network into a high vascular lamellar intact bone via intramembranous ossification requires coordination of programs for cellular growth-like proliferation, differentiation, formation, and degradation of the extracellular matrix (ECM), angiogenesis, and remodeling. During distraction osteogenesis, strain-stimulated cells have to produce enormous amounts of collagen fibers and ECM, which, in turn, undergo degradation, angiogenesis, and ossification.(2–5)

It is well known that these processes are affected by various local and systemic biochemical factors including hormones, vitamins, growth factors, or enzymes. The rapid progress in skeletal cellular and molecular biology led to the identification of various crucial regulators of bone metabolism able to enhance and accelerate bone formation. Among these factors are the group of bone growth factors such as bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF) β superfamily, the FGFs, platelet-derived growth factors (PDGFs), and the insulin-like growth factor (IGF) system.(6–12)

The physiological pathways inducing metabolic degradation of the ECM during developmental tissue disassembly and remodeling consist of a family of matrix metalloproteinases (MMPs). MMPs are zinc-containing proteinases with distinct specificities for the individual components of the ECM. Recent studies have stressed the pivotal role of theses proteinases in the development of new bone.(13–17) In addition, the importance of growth factors and MMPs in the local regulation of bone metabolism acting in an autocrine or paracrine manner has been documented.(7,10,12,14,15) In vivo studies have suggested that the regeneration of bone tissue is affected by humoral mechanisms.(8,9,18–20) However, there is still little information about the function of growth factors and proteinases acting as systemic regulatory molecules.

To document further the systemic regulatory biochemical cascade of distraction osteogenesis, we investigated the serum levels of osteoinductive bone growth factors (IGF-I, IGF binding protein [IGFBP] 3, TGF-β1, and basic FGF[bFGF]) and members of the three main groups of MMPs, the collagenases (MMP-1), gelatinases (MMP-2), and stromelysins (MMP-3) in patients undergoing callus distraction. Patients with nondistraction rigid osteotomy fracture healing for correction of axis deviations served as controls. Furthermore, because of its importance in the regulation of IGF-I and IGFBP-3 and of its well documented importance in skeletal formation,(21–24) serum levels of growth hormone (GH) have been analyzed.

To our knowledge, until today, no other studies have documented changes in serum levels of MMPs, bFGF, or human GH (hGH) during distraction osteogenesis in humans.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Ten otherwise healthy patients undergoing elective osteotomy of the lower limb for callus distraction using an internal lengthening device were recruited for our experiments and assigned to a distraction group. Ten patients with rigidly fixed also internal osteosynthesis (plates, locked intramedullary nails) after osteotomy for elective correction of axis deviations of the lower limbs served as controls in an osteotomy group. Organic diseases like hypo- or hyperthyroidism, diabetes mellitus, liver disease, chronic inflammatory disease, malignancy, or extreme obesity were exclusion criteria in this study. None of the patients were taking steroids during the investigation period. The clinical data of all patients are summarized in Table 1.

Table Table 1.. Clinical Data for Patients of Distraction Group (D) and Osteotomy Group (O)
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The daily distraction rate of 1 mm in average was performed continuously using a fully implantable motorized intramedullary distraction nail.(25)

Serum samples were collected three times before surgery and in a fixed schedule (Fig. 1) six times after surgery in both groups. After this period, five additional samples were collected in the distraction group. Each sample was drawn after an overnight fasting and aliquoted and stored at −70°C until analysis. Weekly clinical examination and routine laboratory tests including complete blood count (CBC), C-reactive protein (CRP), blood urea nitrogen (BUN), creatine, protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transferase (GGT), calcium, and phosphorus have been performed to control the clinical status and to estimate the amount of stress evoked by different techniques of surgery.

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Figure FIG. 1. Time schedule for collection of blood samples. Time point 0 presents sampling of three serum probes within 1 week.

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Quantitative analysis of serum samples for each factor was performed at the end of the individual investigation period, which was 24 weeks in the experimental group and 10 weeks in the control group. Samples of each individual have been assayed in one run to minimize the influence of intra- and interassay variability.

Measurement of serum hGH and hGH binding protein

Serum levels of circulating GH were measured by an in-house immunofluorometric sandwich assay (IFMA) involving two monoclonal antibodies. The assay was calibrated against the International Reference Preparation (IRP) 80/505. Intra-assay CV was 5.2% and 6.8% at 0.5 ng/ml and 6.2 ng/ml, respectively. Interassay CV was 7.2% and 6.0% at concentrations of 0.5 ng/ml and 10.0 ng/ml, respectively. The lower limit of detection was 0.2 ng/ml.

GH binding protein (GHBP) was measured by a ligand immunofunctional assay (LIFA) as described previously.(26)

Measurement of serum IGF-I and IGFBP-3

Serum levels of IGF-I have been measured by a commercially available IGFBP-blocked IGF-I radioimmunoassay (Mediagnost, Tübingen, Germany). Serum IGFBP-3, which represents the most abundant IGFBP in the circulation, was analyzed by a standardized IFMA (DSL, Houston, TX, USA).

Measurement of plasma TGF-β1 and bFGF

Systemic concentrations of TGF-β1 and bFGF have been measured using a commercially available ELISA (R & D Systems, Minneapolis, MN, USA) detecting specifically the biological active forms of the factors. Platelets contain both factors in high concentrations. To minimize the risk of biasing influences through platelet release of these factors, EDTA plasma samples have been used.

Measurement of serum MMPs

MMPs are secreted in an inactive form and require in vivo activation by other proteases. Therefore, in this study an ELISA against the proform of the MMPs (MMP-1 to MMP-3), which are the predominant forms in human serum, have been used (pro-MMP1-3; The Binding Site Heidelberg, Germany).

Measurement of markers of osteoblastic activity (ALP, bone-specific ALP, and osteocalcin)

Serum levels of ALP have been measured using the optimized standard method according to the recommendations of the Deutsche Gesellschaft für Klinische Chemie (HiCo alkaline phosphatase optimized; Boehringer Mannheim Systems, Mannheim, Germany). For the detection of bone specific ALP (BAP), the total activity of ALP was determined first. Thereafter, BAP was precipitated using a lectin from wheat germ, and the remaining ALP activity in the supernatant was measured (Bone-ALP; Boehringer Mannheim Systems).

Serum osteocalcin (OC) was analyzed using the commercially available ELISA detecting only the intact de novo OC and not fragments of the protein from resorbing bone tissues (R & D Systems).

Statistical analysis

Postoperative serum or plasma values are expressed as a percentage (±SEM) of the corresponding preoperative baseline levels. Baseline levels represent the mean of at least two measurements collected within 5 days before surgery. The nonparametric Friedman test was used in each group to detect significant changes over time during the entire investigation period. If significant, the Wilcoxon signed rank test for paired samples was used to determine significant differences between the pre- and postoperative levels in both groups. The confidence limit was predetermined at an α value of 0.05.

For statistical comparison of each factor at a certain postoperative time point between the distraction group and the control group during the first 10 weeks after surgery, the nonparametric Mann-Whitney U test for unpaired samples was used. In addition, area under curve (AUC) values, using linear interpolation, have been calculated and compared between both groups (Wilcoxon, two-sided).

For further statistical investigation, repeated measurement analysis was performed during the first 10 weeks after the procedure to analyze treatment- or time-related effects in both groups. Correlation of time courses between growth factors (TGF-β1 and bFGF), GH, bone formation markers (BAP), and MMPs (MMP-1) was evaluated statistically during the lengthening phase in the distraction group by use of linear regression analysis (Spearman's r, p < 0.05). To detect a potentially temporal delayed correlation between serum parameters caused by regulatory retardation effects, time-shifted correlation of MMP-1 or BAP and the other factors was performed. For this purpose, MMP-1 or BAP levels were compared with the levels of the other factors 1 week later.

The clinical investigations of the study presented here have been conducted in accordance with the Declaration of Helsinki. Ethical clearance certification was confirmed by the ethic human research committee of the Ludwig-Maximilians-University of Munich, Germany.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Changes in serum markers over time

In the distraction group, a significant increase in MMP-1, bFGF, ALP, and BAP could be observed over time compared with the preoperative baseline levels.

MMP-1 levels showed a significant increase over the entire investigation period in patients undergoing callus distraction with an initial marked peak in the lengthening period (p < 0.05). In contrast, no significant changes in MMP-1 have been detected in the control group (Fig. 2A).

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Figure FIG. 2. Systemic analysis (mean ± SEM) of concentrations of MMPs (A) MMP-1, (B) MMP-2, and (C) MMP-3 and bone formation markers (D) ALP, (E) BAP, and (F) OC from patients undergoing distraction osteogenesis or osteotomy fracture healing before (week 0 = 100%) and after surgery. Significant differences compared with baseline levels are *p < 0.05 and **p < 0.01. Significances at a certain time interval between the distraction and the osteotomy group are #p < 0.05 and ##p < 0.01. Δ, Significant differences of AUC values; ⊕, significant treatment effects; ⊗, significant time effects in repeated measurement analysis (weeks 0-10, p < 0.05).

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Plasma levels of bFGF peaked between weeks 4 and 10 of the lengthening period in the distraction group followed by a marked decrease at the end of distraction. A second peak could be observed in the late consolidation phase (week 20, Fig. 3B). In contrast, bFGF values in the osteotomy group did not differ significantly from the preoperative values during the whole observation period.

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Figure FIG. 3. Systemic analysis (mean ± SEM) of concentrations of bone growth factors (A) TGF-β1, (B) bFGF, (C) IGF-I, and (D) IGFBP-3 and (E) hGH from patients undergoing distraction osteogenesis or osteotomy fracture healing before (week 0 = 100%) and after surgery. Significant differences compared with baseline levels are *p < 0.05 and **p < 0.01. #Significances at a certain time interval between the distraction and the osteotomy group are #p < 0.05 and ##p < 0.01. Δ, Significant differences of AUC values; ⊕, significant treatment effects; ⊗, significant time effects in repeated measurement analysis (weeks 0-10, p < 0.05).

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Serum ALP (Fig. 2D) and BAP (Fig. 2E), interestingly, showed time courses similar to those observed for bFGF. In the distraction group, BAP was significantly increased throughout the entire investigation period reaching two postoperative maxima, one during distraction (weeks 6-8) and one in the late consolidation phase. In contrast, in the control group, serum ALP and BAP postoperatively showed a small but significant decrease below baseline levels and subsequently increased during the healing process.

Postoperative changes in hGH, IGF-I, IGFBP-3, and TGF-β1 (Fig. 3) showed a tendency to similar time courses as described for bFGF and BAP in the distraction group; however, these changes failed to reach significance using the Friedman test. Repeated measurement analysis revealed significant time effects for IGF-I (p < 0.05) and IGFBP-3 (p < 0.0001), indicating significant change over time for these factors in both groups (interaction term group/time was not significant). The remarkable increase in serum levels of hGH in the distraction group during the lengthening period up to 1400% of the preoperative baseline values during the 6th postoperative week (Fig. 3E) was not statistically significant. In the osteotomy group, clearly, there was no significant increase in hGH serum levels after surgery.

Serum concentrations of the high-affinity hGHBP are thought to reflect the status of the GH receptor in peripheral tissues.(27) To exclude a potential resistance of hGH receptor, which could lead to higher serum levels of the hormone itself, serum levels of the hGH high-affinity BPs were measured before and in 6 weeks after surgery when hGH concentrations in the distraction group apparently reached their maximum. There were no significant alterations of the serum hGHBP in both groups after surgery (data not shown).

Serum levels of IGF-I (Fig. 3C) and IGFBP-3 (Fig. 3D) in the distraction group also showed an increase during the lengthening period. Cessation of traction caused a drop of serum concentrations followed by a smaller peak at the end of the investigation period.

Fracture healing in the osteotomy group was accompanied by insignificantly increased serum deviations of IGF-I and IGFBP-3 in the 10th week also. In contrast to the distraction group, in control, serum values fell below the baseline levels immediately after surgery.

Analogous to bFGF, systemic TGF-β1 showed a characteristic double-peak-type time course in the distraction group, reaching twofold and threefold, respectively; the baseline values in the 4th week and 10th week time interval followed by a marked decrease at the end of distraction and a subsequent peak in the consolidation period (Fig. 3A). Controls again fell under the preoperative level in the first 2 weeks after surgery, reached a maximum in week 4 (not significant), and successively reached the starting level after 3 months.

The mineralization marker OC did not react significantly in both groups but showed a tendency to an increase over time with a maximum of ∼200% (p = 0.051) compared with baseline in the distraction group at the end of the consolidation phase (Fig. 2F).

Serum concentrations of MMP-2 (gelatinase A) and MMP-3 (SL-1) showed no remarkable alterations in both groups after surgery. A slight increase of both factors in osteotomy fracture healing (Figs. 2B and 2C) was not significant. In the distraction group, MMP-2 and MMP-3 remained unchanged during the entire investigation period.

Comparison of serum markers between distraction group and osteotomy group

For calculation of differences between both groups, only the first 10 weeks after surgery have been considered. Postoperatively, significant differences could be detected for MMP-1, IGF-I, IGFBP-3, bFGF, TGF-β1, ALP, hGH (p < 0.05), and BAP (p < 0.01) between both groups, independently from the fact that these factors did not significantly change from baseline levels (Figs. 2 and 3). Interestingly, except for hGH, significant differences in serum markers between the distraction group and the controls have been observed exclusively during the initial investigation period, which was concomitant with the beginning of mechanical strain in the distraction group.

In addition, AUC values have been significantly higher for MMP-1 (p = 0.039), hGH (p = 0.01), and BAP (p = 0.05) in the distraction group. Repeated measurement analysis revealed significant treatment effects between both groups for MMP-1 (p = 0.03), hGH (p = 0.03), bFGF (p = 0.04), and TGF-β1 (p = 0.03).

Correlation of time courses between serum markers

For correlation of time courses between serum parameters, only the lengthening phase in the distraction group has been taken into account. For that reason values of hGH and bone growth factors with the highest postoperative deviations (bFGF and TGF-β1) have been compared with those of MMP-1 and the osteoblastic parameter BAP (Table 2). Besides significant correlation between the bone growth factors and TGF-β1 to the GH, interestingly, there was correlation of the type I collagenase (MMP-1) to TGF-β1, bFGF, and hGH (p < 0.05) but not to BAP.

Table Table 2.. Correlation (Spearman's R) of Time Courses between Bone Growth Factors TGF-β1 and bFGF, hGH, Bone Formation Markers (BAP) and MMP-1 in the Distraction Group during the Lengthening Phase
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Time-shifted correlation of MMP-1 and BAP in advance of one time point to hGH, TGF-β1, and bFGF revealed significant correlation of BAP to both TGF-β1 and bFGF (p < 0.05) and no correlation between MMP-1 and the bone growth factors (Table 2).

Clinical status of the patients has been controlled by weekly clinical examinations and by a routine laboratory program that was performed at each blood sample. CRP was significantly increased during the first week after surgery in both groups, but no differences between the groups could be detected and no significant alterations over time or between the investigation groups were seen in any other parameter of the laboratory program except for phosphorus, which was significantly increased in the first week after surgery in the distraction group (data not shown). There was no serious local or systemic infection in any patient and no other serious complication due to organic failure of a patient or failure of an implant, which would have interrupted the treatment schedule.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The presence of humoral osteoinductive factors during fracture healing has been described by many investigators,(8,9,18–20) and several in vitro studies revealed that mechanical traction forces are able to enhance bone cell proliferation and metabolic activities.(28–31) Furthermore, immunohistological studies on human specimens could show that bone growth factors, that is, IGF-I and TGF-β, are located at the site of the distraction regeneration.(32) In a recent study, Holbein et al.(33) showed that cyclic stretching stimulates the production of biologically active TGF-β in primary human osteoblast cultures. In addition, they found increased serum levels of active TGF-β during the distraction phase when compared with the preoperative baseline values in humans. Lammens et al.(34) supported these findings in a canine tibia-lengthening model. Compared with a nonlengthened tibia osteotomy canine model, local concentrations in the distracted bone and systemic concentrations of TGF-β1 were found to be increased. IGF-I was increased not only in the osteotomized bones, but also in the contralateral nonosteotomized tibias in the distraction and the osteotomy group. However, on a systemic level, serum IGF-I concentrations were increased in dogs undergoing distraction osteogenesis only.

In our study, systemic concentrations of bone growth factors (TGF-β1, bFGF, IGF-I, and IGFBP-3), hGH, MMPs (MMP-1, MMP-2, and MMP-3), and bone formation markers (ALP, BAP, and OC) have been investigated in patients undergoing either lengthening by distraction or correction of axis deviation of the lower limb by rigid osteotomy. To our knowledge, this is the first study investigating systemic concentrations of hGH, bFGF, and MMP-1 to MMP-3 during callus distraction, and few information exists about the systemic occurrence of these factors in fracture healing.(35)

Compared with the preoperative baseline level, significant systemic increases in MMP-1 and bFGF could be detected in the distraction group but not in the osteotomy group. This indicates that mechanical strain induces secretion of theses factors and suggests a key regulatory function of these factors in the translation of traction forces into a biological response of bone cells.

In accordance with the results of Lammens et al.,(34) ALP was found to be significantly increased in both the distraction and the control groups. In addition, our study shows that after an initial decrease (week 1 and week 2) in the osteotomy group, BAP increases in both groups at week 6 after surgery. These factors seem to be involved in fracture healing in distraction osteogenesis and osteotomy.

In contrast to the findings by Holbein et al. (in humans) and Lammens et al. (in dogs), the apparent increase in serum concentrations of TGF-β1, IGF-I, and IGFBP-3 failed to reach significance when compared with the baseline levels (Friedman test, p > 0.05). As we (in contrast to the other studies) investigated more time points after surgery, the between- and within-individual variability in these factors together with the limited number of subjects might influence this statistical result. However, repeated measurement analysis revealed significant time effects for IGF-I and IGFBP-3, which may indicate borderline significance for the changes in these factors in both groups over time. As seen by Lammens et al., in the osteotomy group, a significant decrease in IGF-I concentrations occurred immediately after surgery (week 1).

Comparison of postoperative serum concentrations between both groups revealed significantly higher levels of MMP-1, IGF-I, IGFBP-3, bFGF, TGF-β1, bFGF, ALP, and BAP in the distraction group during the initial lengthening period (first week and second week) and of hGH during the late (10th week) lengthening period. Therefore, even though no significant systemic alterations in IGF-I, IGFBP-3, TGF-β1, and hGH could be detected in the postoperative time course when compared with baseline levels within the distraction group, significant differences in serum concentrations between both groups during the distraction phase provide further evidence for the importance of these factors in the regulation of mechanotransduction during distraction osteogenesis. We postulate that strain-activated osteoblastic cells induce the secretion of bone formation markers, GH, bone growth factors, and type I collagenase in the distraction phase, which leads to a significant increase in serum values of these factors in distraction compared with rigid osteotomy.

However, other stretched tissues like nerves, vessels, and muscles might contribute to the systemically measured concentrations of biochemical parameters. To get more information about osteoblastic cells as a source of serum factors, we correlated the time course of the specific osteoblastic marker BAP with that of other growth factors (TGF-β1 and bFGF), hGH, and MMP-1. Interestingly, a significant correlation between BAP and growth factors (but not hGH and MMP-1) could be detected during the distraction phase when the BAP concentrations were compared with the other factors 1 week later (time-shifted correlation). No correlation between BAP and other factors was seen for other time shift periods or when analyzing the factors simultaneously (Table 2). From these observations, we hypothesize that the delayed correlation between the osteoblastic marker BAP and growth factors TGF-β1 and bFGF points to common regulatory pathways for these factors. Furthermore, it supports the concept of osteoblasts as a major source of bone growth factors during callus distraction. No direct correlation between time courses of BAP and hGH or MMP-1 could be observed in our study. There are either different ways of regulation or a temporal delay between the biological effects of BAP on hGH or MMP-1 that was not detectable by our statistical approach. In addition, weekly sampling might not be sufficient to detect all physiological effects of one hormone to another. Therefore, the thesis that strain-activated osteoblasts are responsible for the release of MMP-1 and hGH remains to be proven.

According to Stein et al.(36) and Lammens et al.,(34) the cellular biology in bone regeneration can be divided into the stages of proliferation, maturation, and mineralization. Each of these periods is reflected by the appearance of certain biochemical markers. Lammens et al. related TGF-β1 and type I collagen to the proliferation stage, related ALP to the maturation phase, and related OC to the stage of mineralization.

In our study, in the distraction group we also found a sequence in the time courses of the factors measured. An increase during the lengthening period was followed by a drop of serum concentrations at the end of the mechanical traction stimulus and a subsequent increase in the late consolidation phase (Figs. 2 and 3). The MMP-1 values reached their highest concentrations after surgery earlier (week 2/4) than those of TGF-β1 (week 4), ALP/BAP (weeks 2-8), hGH (week 6), and bFGF, IGF-I/IGFBP-3 (week 8). This sequence in the release of MMP-1, bone growth factors, ALP, and GH might reflect the local biochemical cascade of fracture healing during distraction osteogenesis on a systemic level. According to the three stages of fracture healing, in our series, MMP-1 and TGF-β1 could be related to the proliferation stage; ALP, BAP, hGH, bFGF, IGF-I, and IGFBP-3 could be related to the maturation period. The mineralization marker OC did not increase until the late consolidation period. This suggests that mechanical strain keeps the regeneration in the proliferation and maturation stage, delaying the process of mineralization. These findings support the thesis of Lammens et al.,(34) who postulated that a suppression of OC by TGF-β is responsible for the delayed ossification of the regeneration.

The early increase of MMP-1 in distraction osteogenesis was a major finding in our series. Histological studies from Ilizarov(2) and Vauhkonen(5) could show that type I and type II collagen, together with noncollagenous components of the ECM, represent the major part of the regeneration in initial distraction osteogenesis. According to Hauschka et al.,(37) bone growth factors are deposited in the ECM from where they can be released and activated. From our findings, we hypothesize that MMP-1-mediated degradation of bone matrix could induce the release of growth factors and thereby be a major regulator in bone formation during callus distraction. This thesis is supported by a significant correlation of the time courses of MMP-1 and the bone growth factors TGF-β1 and bFGF during the lengthening phase. Interestingly, time-shifted correlation of MMP-1 concentrations with the growth factors 1 week later revealed no significant relationship (Table 2). Thus, we postulate coordinated effects of MMP-1 on the growth factors, that is, digestion of ECM and release of bone growth factors, without serious temporal delay. MMP-1 might be one of the initial inductive factors in the biochemical cascade in distraction osteogenesis.

Following the somatomedin-hypothesis,(21,38) GH results secreted by the pituitary gland induce secretion of IGF-I and IGFBP-3 from liver cells. In our study serum hGH reached its maximal postoperative concentrations during distraction about 1 week before IGF-I and IGFBP-3. This could support the concept of an impact of endocrine regulatory mechanisms on bone formation during distraction osteogenesis. However, stress after fracture of long bones or injury to bone marrow has been shown to induce alterations of serum parameters (i.e., nitrogen balance and albumin), which could influence serum levels of osteogenic factors, especially hGH, IGF-I, and IGFBP-3 itself.(8,19,39,40) Therefore, differences in measured concentrations between patients undergoing callus distraction and osteotomy for axis correction might be explained by a different degree of stress rather than by strain-related effects of distraction. However, comparison of serum values for CBC, CRP, BUN, protein, and albumin revealed no significant differences between the distraction group and the osteotomy group. Furthermore, no differences in any measured serum parameter were seen in the control group, regardless of whether intramedullary nailing or internal fixation by a plate has been used. From that we conclude that the significant differences in hormonal responses between the treatment groups represent strain-related effects rather than different amounts of stress.

Sufficient local blood supply is a basic requirement for the initial steps of bone formation. Under experimental circumstances in dogs, Aronson(41) could show that distraction osteogenesis induces a sevenfold increase in regional blood flow. However, the mechanism by which mechanical strain induces neovascularization remains unknown. Recent studies suggested effects of MMPs and bFGF as potent initiators of angiogenesis. For example, digestion of ECM by MMPs followed by the release of angiogenic factors has been shown to be a crucial step in the initiation of angiogenesis.(15,42) On the other hand, bFGF has been found to stimulate MMP-1 production in human vascular smooth muscle cells(43) and serves as a potent mitogen on capillary endothelial cells, thereby stimulating neovascularization.(44) In accordance with these findings, we hypothesize from our study that a systemic increase in the angiogenic factors MMP-1 and bFGF during callus distraction could reflect increased local neovascularization during distraction osteogenesis.

The potential regulatory actions of the hormonal cascade of mechanotransduction during distraction osteogenesis are summarized in Fig. 4.

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Figure FIG. 4. Model illustrating secretion of bone growth factors, hGH, and MMP-1 from strain-activated osteoblastic cells toward ECM in distraction osteogenesis (step 1). MMP-1 may induce release of bone growth factors from their storage form by partial degradation of ECM (step 2). Growth factors and hGH may then have autocrine and paracrine regulatory functions on osteoblasts in the regeneration (step 3), initiating the biochemical cascade of fracture healing or diffuse in the circulation (step 4), and act systemically on target cells at distant skeletal sites. Thus, mechanical strain may stimulate osteoblastic proliferation (TGF-β1) and maturation (ALP/BAP, hGH, IGF-I/IGFBP-3, and bFGF) but inhibit differentiation into mineralization stage (i.e., suppression of OC expression by TGF-β1; step 5). GH, either produced locally or secreted systemically from the pituitary gland, may induce release of IGF-I and IGFBP-3 from liver cells and contribute to systemic concentrations of these factors (step 6). MMP-1-guided digestion of ECM might be a crucial step in neovascularization of the regeneration supported by bFGF-mediated stimulation of mitogenic and metabolic activity (increased secretion of MMP-1) of capillary endothelial cells (step 7).

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In conclusion, our results show a pronounced systemic increase in concentrations of several bone growth factors and MMPs in callus distraction, which is not seen after rigid osteotomy. A significant postoperative increase in MMP-1, bFGF, ALP, and BAP in the circulation during distraction osteogenesis in a certain time sequence indicates a biochemical cascade involved in distraction osteogenesis fracture healing. We hypothesize that strain-induced secretion by local osteoblastic cells and MMP-mediated degradation of bone ECM are major sources of the increased serum concentrations. These changes might reflect the local biochemical regulation of distraction osteogenesis. Systemic increase of MMP-1 and bFGF during leg lengthening might indicate crucial regulatory functions in angiogenesis during callus distraction for these factors.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This work was supported by the Friedrich-Baur-Institute of the medical faculty of the Ludwig-Maximilians-University of Munich.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  • 1
    Aronson J, Harrison BH, Stewart CL, Harp JH Jr 1989 The histology of distraction osteogenesis using different external fixators. Clin Orthop 241:106116.
  • 2
    Ilizarov GA 1989 The tension-stress effect on the genesis and growth of tissues. Part I. The influence of stability of fixation and soft-tissue preservation. Clin Orthop 238:249281.
  • 3
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