Systemic regulation of angiogenesis and matrix degradation in bone regeneration—Distraction osteogenesis compared to rigid fracture healing

Aim of this study was the investigation of systemic biochemical regulation mechanisms of bone regeneration by angiogenic and matrix-degrading enzymes during distraction osteogenesis compared to rigid osteotomy bone healing.Serum samples of 10 otherwise healthy patients with callus distraction for lower limb-lengthening and 10 osteotomy patients undergoing elective axis correction have been collected prospectively in a standardized time schedule before and up to 6 months after the procedure. At the end of the individual investigation period, concentrations of metalloproteinases (MMP-9, -13), tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) and the angiogenic factors angiogenin and VEGF have been detected by use of commercially available enzyme immunoassays. Results have been compared to our preliminary study on proMMP-1–3.In distraction osteogenesis, significantly elevated serum concentrations compared to baseline could be detected postoperatively for proMMP-1, MMP-9, TIMP-1, angiogenin and VEGF but not for proMMP-2, proMMP-3 or TIMP-2. In patients with rigid osteotomy healing, MMP-9, TIMP-1, TIMP-2, angiogenin and VEGF were significantly increased respectively. Comparison of both patient collectives revealed significantly higher increases of serum proMMP-1, VEGF and TIMP-1 in distraction patients during the lengthening period and significantly higher serum concentrations of TIMP-2 in late fracture healing period in osteotomy patients. Serum levels of MMP-13 were below the lowest standards, and therefore quantitative analysis was not possible. Bone regeneration in distraction osteogenesis and rigid osteotomy healing is accompanied by systemic increase of matrix-degrading and angiogenic factors in a certain time course and quantity. This might reflect biochemical regulation of local bone healing in the circulation. ProMMP-1, VEGF and TIMP-1 seem to be key regulatory factors during distraction osteogenesis.     

Systemic regulation of angiogenesis and matrix degradation in bone regeneration--distraction osteogenesis compared to rigid fracture healing

Aim of this study was the investigation of systemic biochemical regulation mechanisms of bone regeneration by angiogenic and matrix-degrading enzymes during distraction osteogenesis compared to rigid osteotomy bone healing.

Serum samples of 10 otherwise healthy patients with callus distraction for lower limb-lengthening and 10 osteotomy patients undergoing elective axis correction have been collected prospectively in a standardized time schedule before and up to 6 months after the procedure. At the end of the individual investigation period, concentrations of metalloproteinases (MMP-9, -13), tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) and the angiogenic factors angiogenin and VEGF have been detected by use of commercially available enzyme immunoassays. Results have been compared to our preliminary study on proMMP-1–3.

In distraction osteogenesis, significantly elevated serum concentrations compared to baseline could be detected postoperatively for proMMP-1, MMP-9, TIMP-1, angiogenin and VEGF but not for proMMP-2, proMMP-3 or TIMP-2. In patients with rigid osteotomy healing, MMP-9, TIMP-1, TIMP-2, angiogenin and VEGF were significantly increased respectively. Comparison of both patient collectives revealed significantly higher increases of serum proMMP-1, VEGF and TIMP-1 in distraction patients during the lengthening period and significantly higher serum concentrations of TIMP-2 in late fracture healing period in osteotomy patients. Serum levels of MMP-13 were below the lowest standards, and therefore quantitative analysis was not possible. Bone regeneration in distraction osteogenesis and rigid osteotomy healing is accompanied by systemic increase of matrix-degrading and angiogenic factors in a certain time course and quantity. This might reflect biochemical regulation of local bone healing in the circulation. ProMMP-1, VEGF and TIMP-1 seem to be key regulatory factors during distraction osteogenesis.     

Expression of growth factors in the mandibular distraction zone: a sheep study.

Interest in craniofacial osteodistraction has increased in recent years parallel with the growing attention given to the role of growth factors in tissue healing and regeneration. This study was embarked upon to investigate the expression of bFGF, TGF-beta and IGF-1 in the distraction zone of the mandible. Fourteen growing sheep were allocated to three experimental groups. Six animals were allocated to Groups A and B (n = 12) and underwent bilateral mandibular corticotomies with fixation of an external lengthening device. The distraction protocol consisted of a rate of 1.0 mm/day (twice daily) for 20 days followed by a consolidation phase of 20 days after which the sheep were sacrificed. Group C comprised of age matched sham operated animals (n = 2). Bone histochemistry for growth factors were performed in the harvested mandibles. A strong staining of bFGF was seen in the osteoblasts, osteocytes and osteoid matrix following 20 days of distraction and 20 days of consolidation compared to the control group. TGF-beta and IGF-1 demonstrated mild but clear staining in osteocyte and osteoblast cells and TGF-beta stained positively in the osteoid seam in the experimental groups. These finding suggest that bFGF, IGF-1 and TGF-beta may play different roles in the remodelling phase of distraction osteogenesis.     

Expression of angiogenic factors during distraction osteogenesis

Distraction osteogenesis is a unique and effective way to treat limb length inequality resulting from congenital and posttraumatic skeletal defects. However, despite its widespread clinical use, the cellular and molecular mechanisms by which this surgical treatment promotes new bone formation are not well understood. Previous studies in distraction osteogenesis have noted increased blood flow and vessel formation within the zone of distraction. These observations suggest that distraction osteogenesis may be driven in part by an angiogenic process. Using immunohistological analysis, the expression of two different angiogenic factors (VEGF and bFGF) was shown to localize at the leading edge of the distraction gap, where nascent osteogenesis was occurring. These cells were spatially adjacent to new vessels that were identified by staining for factor VIII. Microarray analysis detected maximal mRNA expression for a wide variety of angiogenic factors including angiopoietin 1 and 2, both Tie receptors, VEGF-A and -D, VEGFR2, and neuropilin 1. Expression of these factors was found to be maximal during the phase of active distraction. Expression of mRNA for extracellular matrix proteins and BMPs was also maximal during this period. A comparison between the patterns of gene expression in fracture healing and distraction osteogenesis revealed similarities; however, the expression of a number of genes showed selective expression in these two types of bone healing. These data suggest that bone formation during distraction osteogenesis is accompanied by the robust induction of factors associated with angiogenesis and support further investigations to elucidate the mechanisms by which angiogenic events promote bone repair and regeneration.     

Growth factors in distraction osteogenesis

Although growth factors have been demonstrated during bone healing, their presence has not yet been confirmed in callus distraction. Therefore, in 3 patients we searched for cytokines during callus distraction. Bone biopsies were immuno-histochemically stained for TGF-beta1, IGF-I, TGF-beta type II receptor, IGF receptor, and proliferating cell nuclear antigen (PCNA). Histologically we found immature woven bone in the middle of the callus zone and increasing calcification and lamellar bone in the re-modelling zone. Osteoblasts and fibroblast-like cells in the middle zone, and osteoblasts in all zones stained for TGF-beta and its receptor. The number of positive staining cells related to proliferous activity as assessed both by PCNA, and by bone density in radiographs. IGF-I could be detected everywhere. In conclusion, growth factors are present in bone formation and in areas of re-modelling during callotasis. Their relation to proliferous activity and radiographic density supports their involvement in osteogenesis.     

激活素A、TGF-β1在兔下颌骨牵引中的表达

目的 检测下颌骨牵引过程中激活素A(ACT A)及转化生长因子β1(TGF-β1)的表达情况，了解ACTA、TGFβ1在下颌牵引成骨中作用的异同点。方法 采用兔下颌骨牵引模型在牵张不同时期取牵引组织用免疫组化和RT-PCR检测ACTA、TGFβ1蛋白及mRNA在不同时期的表达。结果 牵引过程开始ACT A mRNA的表达量逐渐上升，固定10d及20d达峰值，ACT A蛋白主要分布在成骨细胞内。延迟期末TGFβ1表达量达峰值，其蛋白主要分布于间质及间质细胞。结论 在兔下颌骨牵引过程中ACT A与TGFβ1的作用有很大不同，二者联合可能促进新骨形成。     

Enhancing bone healing during distraction osteogenesis with platelet-rich plasma

Gradual limb lengthening with external fixators using distraction osteogenesis principles is the gold standard for treatment of limb-length discrepancy. However, long treatment time is a major disadvantage of the current lengthening procedures. Efforts to decrease the treatment include biological and biomechanical factors. Injection of platelet-rich plasma (PRP) is a biological method to enhance bone healing during distraction osteogenesis. We hypothesised that PRP can enhance bone healing during limb lengthening. We report our experience with the use of PRP during distraction osteogenesis. This retrospective study included 19 patients divided into the standard group of 10 patients who did not receive PRP and the PRP group of nine patients who received PRP at the end of the distraction phase. The study variables included external fixator time, external fixation index, and complications during treatment. The PRP group had statistically significantly shorter treatment time (p = 0.0412). Injection of PRP into regenerate bone might be an effective method to shorten treatment time during limb lengthening and lead to better functional outcomes and improved patient satisfaction.Level of evidence: Level IV, therapeutic study.     

Increased lengthening rate decreases expression of fibroblast growth factor 2, platelet-derived growth factor, vascular endothelial growth factor, and CD31 in a rat model of distraction osteogenesis

BACKGROUND: The rate of lengthening has a profound impact on bone regeneration during distraction osteogenesis. Rapid distraction can delay or completely inhibit union, whereas distracting too slowly may lead to premature consolidation. However, the mechanisms responsible for retardation of healing due to rapid distraction have not been elucidated. This study explored whether rapid distraction alters the expression of certain angiogenic growth factors, in particular, fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-AA), and subsequent new vessel formation as evidenced by platelet endothelial cellular adhesion marker expression (CD31), an indicator of vascular budding. METHODS: Unilateral femoral lengthenings were performed in 60 male Sprague-Dawley rats using a protocol that involved a 7-day latency period and distraction rates of either 0.5 (slow distraction) or 1.5 mm/d (fast distraction) for a total of 7.0 mm of lengthening. Animals were euthanized on postoperative days 8, 10, 12, 14, and 21 (n = 6 per time point and distraction rate). Expression of FGF-2, VEGF, PDGF-AA, and CD31 was characterized immunohistochemically. RESULTS: Cellular staining of FGF-2, PDGF-AA, VEGF, and CD31 was reduced on days 8 to 12 in the regenerate of the fast-distraction animals compared with the slow-distraction animals. Staining of all growth factors was weak on days 14 and 21 at the slow rate and absent at the fast rate. Regardless of time point, a similar spatial localization of growth factor expression was observed at the 2 rates of distraction. CONCLUSIONS: The reduced expression of angiogenic growth factors and CD31, a marker of new vessel formation, indicates that the angiogenic cascade and new vessel formation required for effective bone healing is disrupted at a distraction rate of 1.5 mm/d in a rat model of limb lengthening. CLINICAL RELEVANCE: Delayed bone healing with rapid distraction may be due in part to decreased cellular signaling required for angiogenesis. It may be possible to improve bone healing at increased distraction rates with the appropriately timed administration of growth factors.     

Creation and characterization of a mouse model of mandibular distraction osteogenesis

While the histological and ultrastructural changes associated with distraction osteogenesis have been extensively characterized using various animal models, the molecular mechanisms governing this technique remain poorly understood. In the current study, for the first time, we describe a mouse mandibular distraction osteogenesis model. Development of this model will allow assessment of factors involved in normal vs. abnormal healing (especially in non-unions) of craniofacial skeletal elements. Complete osteotomies were created on the right hemimandibles of 51 adult male CD-1 mice and customized distraction devices attached. Thirty-three animals underwent gradual distraction (5 days latency, distraction at 0.2 mm BID × 8 days, 28 days consolidation), while the remaining 18 mice underwent acute lengthening (immediate distraction to 3.2 mm) at the time of surgery. Mandibles were harvested at time points corresponding to the latent (POD 5), distraction (POD 9, 13), and consolidation (POD 28, 41) periods and processed for histological or quantitative real-time RT-PCR analysis. Specimens from each group were processed for μCT analysis. Histological and radiological data demonstrated that all mandibles undergoing gradual distraction achieved complete bony union by the end of consolidation, while those undergoing acute lengthening formed a fibrous non-union. Quantitative real-time RT-PCR demonstrated upregulation of mRNA for VEGF, FGF-2, collagen I, and osteopontin during gradual distraction but not during acute lengthening. These data validate our novel mouse mandibular distraction model and demonstrate its utility in elucidating the molecular mechanisms regulating bone formation during distraction osteogenesis as compared to those that are expressed during the formation of fibrous non-unions.     

Expression of growth factors in canine flexor tendon after laceration in vivo

Growth factors, transforming growth factor beta (TGF-beta), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), are critical components of the cutaneous wound healing process. Little is known, however, about the expression of these growth factors in normal flexor tendon healing. In this study, we wished to examine which of these growth factors are present at 10 days following tendon injury in a canine flexor tendon repair model. Using immunohistochemical analysis, we found positive staining for all growth factors in both timing groups. TGF-beta was detected around the repair site and proximal to it. PDGF-AA, PDGF-BB and VEGF appeared in the whole tendon section following repair. EGF, IGF and bFGF were not seen in tenocytes but were present in inflammatory cells surrounding the repair site. These findings provide evidence that TGF-beta, EGF, PDGF-AA, PDGF-BB, IGF, bFGF and VEGF are all expressed at 10 days after tendon injury but by different cell types and in different locations. The time course of growth factor expression is an important element in wound healing, and a better understanding of where and when such factors are expressed may help in the development of methods to manipulate this expression, accelerate healing, and reduce adhesions.     

Deformation across the zone of callotasis during loading. radiostereometric analysis in a patient with achondroplasia.

The present study demonstrates that high-resolution radiostereometric analysis (RSA) can be used to assess global longitudinal compressive deformation across the callotasis zone during loading. In an achondroplastic patient operated with bifocal lengthening of the tibia by use of the Ilizarov external fixator, the axial compressive intersegmental strain in the proximal lengthening zone under a load of 71% of body weight was 7.7 mm. The proximal lengthening zone was 51.0 mm, and accordingly the overall linear strain across the callotasis was 15.1%. This large strain value found in distraction osteogenesis 6 weeks after end of distraction is not consistent with classical theory of the magnitude of micromotion needed for adequate stimulation of bone formation in fracture healing. The increased axial displacement did not stimulate bone healing and delayed union was observed. This one single observation does not allow for any conclusions to be drawn about the relationship of strain to fracture healing, but further and refined use of the RSA method will certainly improve our understanding of the role of axial strains in distraction osteogenesis.     

Effects of locally applied transforming growth factor-beta1 on distraction osteogenesis in a rabbit limb-lengthening model

In this study we tested the effect of locally applied transforming growth factor-beta1 (TGF-beta1) on distraction osteogenesis in rabbits. A total of 61 rabbits were studied. Seven days after tibial osteotomy, distraction was started at a rate of 0.25 mm/12 h for 3 weeks. Starting with distraction, TGF-beta1 was applied in four different dosages (0, 10, 20, and 40 ng/day) at the site of osteotomy through a catheter connected to a subcutaneously implanted miniosmotic pump. Rabbits were killed at the end of the distraction period or 3 weeks later, and histological, densitometric, and biomechanical parameters were assessed. TGF-beta1 treatment had no detectable effect on bone mineral density or histologically determined bone volume in the distraction gap but it increased the amount of fibrous tissue in the callus region. Load to failure in uniaxial tension tended to be lower in TGF-beta1-treated animals. In conclusion, TGF-beta1 treatment during distraction osteogenesis did not have a beneficial effect in this model.     

Noninvasive Quantitative Assessment of Bone Healing After Distraction Osteogenesis

One of the greatest challenges of limb lengthening and deformity correction is deciding when the bone has healed enough to remove the external fixator. Standard radiography is the most common imaging method used to assess bone healing after distraction osteogenesis because it is widely available, cheap, and relatively safe. However, other imaging technologies and methods are being investigated that will help quantify bone healing after distraction osteogenesis, providing an objective method for deciding when it is appropriate to remove an external fixator. This review will examine the latest techniques used to assess bone healing after distraction osteogenesis including dual-energy X-ray absorptiometry scans, ultrasound, quantitative computed tomography, and digital radiography (X-ray). Recommendations for clinical practice will be outlined.     

Gradual reduction of distal radial malunion using distraction osteogenesis

PURPOSE: To evaluate gradual distraction lengthening or distraction osteogenesis as a technique for treating malunions of the distal radius. METHODS: Twenty patients with clinical and radiographic evidence of distal radius malunion were treated with osteotomy of the distal radius using distraction osteogenesis. At the follow-up evaluation each patient was evaluated for healing rates, complications, resolution of pain, and radiographic alignment. Surgical treatment consisted of an application of a nonbridging external fixator that could be distracted to correct the deformity. A loosely set screw that connected the distal pins to the fixator served as a hinge and allowed the distal radius to rotate into a corrected position. Gradual distraction via distraction osteogenesis was initiated 1 week after surgery. RESULTS: Seventeen osteotomies healed uneventfully in an average of 9 weeks. Complications included 9 pin track infections. Two nonunions required bone grafting. One patient inadvertently compressed rather than distracted the fixator, leading to premature healing of the osteotomy. One patient ruptured the extensor pollicis longus. All complications resolved with additional intervention. Overall the patients showed radiographic and symptomatic improvement. CONCLUSIONS: An external fixator and distraction lengthening through distraction osteogenesis is a viable alternative to plate fixation and bone grafting. In 18 of 20 of our patients, the technique eliminated the need for bone grafting and the need for a second surgical procedure to remove a dorsal plate.     

TGF-β1 als pathophysiologischer Faktor bei der Frakturheilung

AIM: TGF-beta1 is an important local and systemic regulatory molecule during fracture healing. Various authors have shown differences in the systemic levels of TGF-beta1 over the time taken for bone healing in distraction osteogenesis and osteotomies. Previous studies have shown characteristic differences in the physiological levels of growth factors between normal fracture healing and delayed fracture union. The aim of the present study was to evaluate possible differences in sera levels of patients with normal and delayed union fracture healing. METHODS: Patients with long bone shaft fractures were recruited prospectively. Peripheral blood samples were collected over a period of 1 year using a standardized time schedule. At the end of the individual's investigation period, TGF-beta1 levels were determined. To achieve a homogeneous collective of patients, only those with a maximum of two fractures were included in the study. After matching for four criteria, we compared patients with normal fracture healing to patients with delayed unions. The fact of delayed union was accepted in case of failed consolidation 4 months after trauma. RESULTS: During a prospective study period of 1 year, 15 patients with normal fracture healing could be compared to 15 patients suffering from delayed union. By determining the absolute sera levels we found a typical increase of TGF-beta1 up to 2 weeks after fracture in both groups, with a subsequent decrease up to the sixth week after fracture. However, a decline in serum concentration occurred earlier in patients with delayed union, causing significantly lower TGF-beta1 levels in the non-union group 4 weeks after trauma (P=0.00006). CONCLUSION: Even with a relatively small number of patients, we could show a significant difference in serum concentrations of TGF-beta1 between the investigated groups. If these results can be verified within a larger collective, TGF-beta1 could be used as a predictive cytokine for delayed fracture healing.     

转化生长因子-β超家族对新骨生成的调节作用

目的　对近年来国内外有关转化生长因子 - β(TGF- β)超家族对新骨形成作用的研究进行综述。方法　广泛查阅相关文献 ,对 TGF- β、骨形成蛋白 (BMPs)及激活素 (ACT)在新骨形成 ,尤其是牵张性新骨形成(DO)作用进行分析综合。结果　 TGF- β、BMPs及 ACT对新骨形成均有促进作用 ,其作用机制各有特点。BMPs启动间充质细胞向骨细胞系分化 ,TGF- β刺激骨形成前体细胞生长分化 ,ACT增强 BMPs成骨 ,本身也促进成骨细胞增殖和胶原合成。结论　 TGF- β超家族对新骨形成的调控 ,有助于缩短 DO的周期     

Rat model of distraction osteogenesis

Prior studies of distraction osteogenesis in dog and rabbit models have shown predominantly intramembranous bone formation. Other models of fracture healing normally display mixtures of both endochondral and intramembranous bone formation. We have established a rat model of tibial lengthening that reliably reproduces the pattern of zonal osteogenesis previously observed in dog and rabbit models. A distraction rate of 0.25 mm twice a day with a 0-day latency period produced intramembranous bone with zones of progressive mineralization from collagen. With this protocol, rats bridged the distraction gap with a 25% increase in the tibial bone length. After 20 days of distraction and 50 days of consolidation, the three-point bending stiffness, as a percentage of the contralateral control, reached a level equivalent to that measured in the canine model for a 15% lengthening (28-day distraction and 84-day consolidation). Radiodensitometric analysis of the regenerate bones measured 97% of the unaffected contralateral tibial densities, and mineral analyses demonstrated that calcium and phosphorus levels in the regenerate bone reached 78% of contralateral tibial levels by day 70. We concluded that a rat model of distraction ostegenesis will be useful for a wide range of studies involving rapid intramembranous bone formation.     

Injizierbare Trägersysteme für die Wachstumsfaktorapplikation zur minimal-invasiven Knochenheilungsstimulation

The characterization and cloning of growth factors for bone healing provide an enormous potential for minimally invasive treatment procedures for bone defects or fractures. However, the clinical application of injection vehicles for these growth factors must be made user-friendlier. In this study, two different injection vehicles were tested for their practicability and efficacy to enhance callus maturation during distraction osteogenesis. Calcium phosphate carriers showed a rather low user-friendliness and were less efficient in the animal model of distraction osteogenesis in sheep. Collagen carriers provided both a higher practicability for injection procedures and a higher efficacy.     

Growth factors in human serum during operative tibial lengthening with the ilizarov method

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 = 12), unilateral posttraumatic tibial shortening (n = 7), and healthy patients that underwent cosmetic bilateral tibial lengthening (n = 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‐α, TGF‐β1, and TGF‐β2 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. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1966–1970, 2013