Effect of electroporation-mediated transfecting recombinant plasmid pIRES-hBMP2-hVEGF165 on mandibular distraction osteogenesis

Distraction osteogenesis requires a long consolidation period and has a low but real failure rate. Bone morphogenetic proteins (BMPs) accelerate bone deposition in fractures and critical-sized bone defects. Vascular endothelial growth factor (VEGF) is a promising reagent for inducing angiogenesis, and is an essential coordinator of extracellular matrix remodeling, angiogenesis, and bone formation in the growth plate. However, their effects on mandibular distraction osteogenesis are unknown. We investigated the effect of local delivery of plasmid pIRES-hBMP-2-hVEGF165 into a distraction area by electroporation-mediated approach.A New Zealand rabbit model were used. Activation of the device was commenced after 3 days of latency period and proceeded at the rate of 0.8 mm per day for 7 days. After the completion of activation, the rabbits were randomly divided into 5 groups: group A: recombinant plasmid 2 μg (0.1 μg/μL) pIRES-hVEGF165-hBMP2 was injected into the distraction area after the completion of activation; group B: recombinant plasmid pIRES-hBMP2 was injected into the distraction area; group C: recombinant plasmid pIRES-hVEGF165 was injected into the distraction area; group D: pIRES was injected into the distraction area, and group E: normal saline was injected into the distraction area. After injection every group used electroporation. Subsequently, the rabbits were examined by quantitative computed tomography, mechanical testing, and histomorphometric analysis.BMD of newly formed bone of the distraction area in groups A, B, and C were remarkably higher than those of groups D and E at different times (P < 0.001). At 4 and 8 weeks of consolidation, the crushing strength of 3 points of the newly formed bone in group A was remarkably higher than those of groups B, C, D, and E (P < 0.01). The results demonstrated statistically remarkable increase in regenerated bone in the gene-transfected groups.Electroporation-mediated transfecting recombinant plasmid pIRES-hVEGF165-hBMP2 could produce a satisfactory proceeding of osteogenesis and calcification, which surpassed that of the control group. This finding indicates that a combination of VEGF and BMP may make osteogenesis and angiogenesis appear at the same time. Furthermore, it may magnify the effect of single growth factor, and promote growth and reparative process of bone.     

Expression of osteotropic growth factors and growth hormone receptor in a canine distraction osteogenesis model

Osteotropic growth factors play an important role in bone metabolism. Nevertheless, knowledge about their expression in relation to distraction osteogenesis remains limited. The aim of the present study was to determine the expression of growth hormone (GH), growth hormone receptor (GHR), insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), and bone morphogenetic protein 2 (BMP-2) in distraction-induced bone regeneration. Expression of these factors was assessed during the consolidation phase, comparing distraction osteogenesis with osteotomy-induced bone formation. Real-time PCR was performed as a semiquantitative measurement of mRNA, and the relative expression levels of these factors were determined. In addition, plasma GH profiles and plasma concentrations of IGF-I, IGF-II, and insulin-like growth factor-binding protein 4 and -6 (IGFBP-4 and -6) were measured to assess their potential systemic role during bone formation. Expression of GHR, IGF-I, and BMP-2 had significantly increased in comparison with the expression of these factors in mature bone. Expression of GHR was significantly higher in distraction-induced bone regenerate than in osteotomy-induced bone. No significant differences were found for the expression of IGF-I and BMP-2 between distraction and osteotomy. Plasma concentrations of GH, IGF-I, IGF-II, IGFBP-4, and IGFBP-6 did not demonstrate any significant differences between treatment groups and controls. Upregulation of GHR expression in distraction osteogenesis may enhance sensitivity to endogenous systemic GH and thus promote consolidation of the regenerated bone. Changes in the systemic osteotropic growth factors GH, IGF-I, IGF-II, IGFBP-4, and IGFBP-6 do not seem to be of importance during distraction osteogenesis.     

Expression of smooth muscle actin in cells involved in distraction osteogenesis in a rat model.

Distraction osteogenesis has proven to be of great value for the treatment of a variety of musculoskeletal problems. Little is still known, however, about the phenotypic changes in the cells participating in the bone formation process, induced by the procedure. Recent findings of the expression of a contractile muscle actin isoform, alpha-smooth muscle actin (SMA), in musculoskeletal connective tissue cells prompted this immunohistochemical study of the expression of SMA in cells participating in distraction osteogenesis in a rat model. The tissues within and adjacent to the distraction site could be distinguished histologically on the basis of cell morphology, density, and extracellular matrix make-up. The percentage of SMA-containing cells within each tissue zone was graded from 0 to 4. The majority of the cells in each of the zones stained positive for SMA within five days of the distraction period. The SMA-containing cells included those with elongated morphology in the center of the distraction site and the active osteoblasts on the surfaces of the newly forming bone.These finding warrant further investigation of the role of this contractile actin isoform in distraction osteogenesis and investigation of the effects of modulation of this actin isoform on the procedure.     

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。     

Characterizing the BMP pathway in a wild type mouse model of distraction osteogenesis

Distraction osteogenesis (DO) is a well established surgical technique for limb lengthening and replacement of bone loss due to trauma, infection or malignancies. Although the technique is widely used, one of its limitations is the long period of time required for the newly formed bone to consolidate. We have previously shown that exogenous application of bone morphogenetic proteins (BMPs) can increase bone formation during DO, however, exogenous BMPs have many drawbacks. An alternative method for accelerating the rate of bone formation may be to modulate the intrinsic BMP signaling pathway. The aim of the current study was to analyze the expression of various genes involved in the BMP pathway at various time periods during DO in order to identify potential targets for therapeutic manipulation. DO was applied to the right tibia of 80 adult wild type mice. Distraction began after a latency period of 5 days at a rate of 0.2 mm/12 h for 2 weeks. Mice were sacrificed in groups of 12 at the following times post surgery: day 5 (latency), days 11 and 17 (distraction) and days 34 and 51 (consolidation). Specimens were examined using radiology, microCT, histology, RT(2)PCR, immunohistochemistry and Western analysis. Genes involved in the BMP pathway including the BMP ligands, receptors, antagonists and downstream effectors were examined. A significant upregulation of BMPs 2, 4 and 6 was observed using both PCR and immunohistochemistry during the distraction phase. The expression of BMP7 remained constant throughout the distraction and consolidation process. Surprisingly, the only receptors which were upregulated significantly were the Activin Receptor Type 1 (ActR1) during distraction and Activin Receptor Type 2b (ActR2b) during consolidation. Most interestingly, simultaneously with the ligands, an increase in the expression of the antagonists, Noggin, Chordin, Inhibin and BMP3 was observed. This study provides a clearer understanding of expression patterns during DO, which is a valuable resource for finding therapeutic options to stimulate bone formation. The results suggest that blocking BMP inhibitors may be a possible method for increasing the function of intrinsic growth factors involved in bone regeneration.     

Locally applied nerve growth factor enhances bone consolidation in a rabbit model of mandibular distraction osteogenesis.

Distraction osteogenesis is widely used in treating deformities, defects, and fractures of both long bones and craniofacial bones. Demands for acceleration of bone consolidation are increased in distraction osteogenesis. Nerve growth factor (NGF) can enhance innervation and bone regeneration in a fracture model and stimulate differentiation of osteoblastic cells. In this study, we tested the ability of locally applied NGF to enhance bone regeneration in a rabbit model of mandibular distraction osteogenesis. Twenty rabbits underwent bilateral distraction osteogenesis with a rate of 0.5 mm per 12 h. Two times 0.04 mg human NGFbeta (hNGFbeta) in buffer was injected into the callus after distraction. The contralateral side received placebo injections. Rabbits were euthanized at consolidation times of 14 and 28 days. Specimens were subjected to radiography, callus dimensions measurement, mechanical testing, and bone histological and histomorphometric analysis. The maximum load, bone volume/total volume, mineral apposition rate of the 1st to 11th day, and mineralized bone percentage were significantly higher in the hNGFbeta side at 14 and 28 days (p<0.05). The data indicate that locally applied hNGFbeta can accelerate callus maturation and may be an option to shorten the consolidation period in distraction osteogenesis.     

Systemic regulation of distraction osteogenesis: a cascade of biochemical factors.

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] beta1, 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-beta1, 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-beta1 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-beta1, 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.     

富含血小板血浆对牵引成骨过程中新骨生成的影响

目的　探讨富含血小板血浆 (PRP)对成骨细胞以及牵引成骨过程中新骨生成的作用。方法　通过体外成骨细胞的培养及牵引成骨动物模型的制备 ,采用MTT法检测成骨细胞的增殖情况 ,采用组织化学、四环素标记等方法观察骨组织新骨生成情况。结果　PRP可以促进体外培养成骨细胞的增殖 ,促进牵引成骨过程新骨的生成。结论　PRP可以加快牵引成骨过程的新骨生成 ,临床应用可能缩短牵引成骨的治疗过程。     

Trigeminal Nitric Oxide Synthase Expression Correlates with New Bone Formation During Distraction Osteogenesis

Nitric oxide synthase (NOS) has been reported to be involved with both bone healing and bone metabolism. The aim of this study was to test the null hypothesis that there is no correlation between new bone formation during mandibular distraction osteogenesis and NOS expression in the trigeminal ganglion of rats. Newly formed tissue during distraction osteogenesis and trigeminal NOS expression measured by the NADPH-diaphorase (NADPH-d) reaction were evaluated in 72 male Wistar rats by histomorphometric and histochemical methods. In animals submitted to 0.5 mm/day distraction osteogenesis, the percentage of bone tissue was higher in the basal area of the mandibles compared with the center and significantly increased through the experimental periods (P < 0.05). At the sixth postoperative week, the difference in bone formation between the continuous and acute distraction osteogenesis groups was the highest. Significant correlation between new bone formation by distraction osteogenesis and NADPH-d-reactive neurons was found, varying according to neuronal cell size (r = −0.6, P = 0.005, small cells strongly stained; r = 0.5, P = 0.018, large cells moderately stained). The results suggest that NOS may play a role in the bone healing process via neurogenic pathways, and the phenomenon seems to be neuronal cell morphotype-dependent. Further studies are now warranted to investigate the mechanistic link between the expression of trigeminal NOS and mandibular new bone formation by distraction osteogenesis. R. F. de Albuquerque is also a visiting professor at Faculty of Dentistry, McGill University, Quebec, Canada. M. T. Moura de Oliveira and J. P. Mardegan Issa are graduate students.     

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.     

Bone lengthening (distraction osteogenesis): a literature review

Distraction osteogenesis (DO) is a surgical technique widely used in orthopedic surgery for the treatment of various pathological conditions such as leg length discrepancy, bone deformity or bone defects. The basic principle of the callotasis technique includes performing a transverse bone section before gradually distracting the two bone segments. New bone tissue is generated in the gap between the two segments. Bone regeneration during DO is believed to occur in response to the longitudinal mechanical strain applied to the callus during healing. One of the limitations of this technique is the long period of time required for the newly formed bone tissue to mineralize and consolidate. Various studies have reported that among growth factors, bone morphogenetic proteins (BMPs) may play a central role in the molecular signaling cascade leading to bone renegeration and remodeling in a DO procedure. Ongoing research is aimed at developing methods to accelerate bone consolidation in order to reduce the time required to obtain consolidation. One of these methods is to test the ability of exogenous BMPs to increase bone regeneration and accelerate bone consolidation.     

管状骨增宽牵引成骨组织形态学变化的实验研究

目的 探讨管状骨增宽牵引后新骨形成的变化。方法 在成年山羊的后肢放置2只增宽牵引器。实验组9只术后第8天牵引，对照组3只不牵引。牵引完毕后不同时期，各宰杀3只，评价管状骨增宽牵引后新骨形成的质和量。结果 实验组胫骨平均增宽7．83mm，X射线见牵引间隙逐渐变模糊，暴露侧有骨不连，非暴露侧成骨良好，牵引完毕3个月，未暴露侧新形成板层骨与原来胫骨融为一体，而暴露侧骨不连区为致密纤维组织。结论 管状骨增宽牵引成骨后，两侧牵引间隙成骨不一致，良好的血供，对新骨形成至关重要。     

Bone formation after distraction osteotomy of the radius in sheep

Gradual distraction by external fixation was performed one week after osteotomy of the radius in 12 sheep. Bone regeneration in the distraction area was studied by light and electron microscopy. One week after starting the distraction the gap was composed of hematopoietic cells and fibroblasts. The collagen production had already started and it continued actively throughout the distraction period. The collagen produced by the fibroblasts in the central inter-zone of the gap was organized according to the direction of the distraction. The osteoblasts were lined up along the collagen bundles and osteoid formation was most active around the capillaries. Mineralization started two weeks after the beginning of the distraction. After cessation of the distraction, separate groups of cartilaginous cells were found in some specimens among the newly formed bone. Our findings suggest that osteogenesis as a result of gradual distraction occurs through the whole distraction area with preceding formation of organized collagen matrix. The bone structure in the distracted segment represents an organized lamellar structure at an early stage of the osteogenesis.     

Distraction osteogenesis after irradiation in a rabbit model

Background The present study was performed to investigate the effects of preoperative irradiation on distraction osteogenesis, as little is known about how preoperative irradiation delays distraction osteogenesis. Methods A single dose of irradiation was applied to the right rear legs of rabbits. This was followed by tibial lengthening at a rate of 0.5 mm/day, which was continued for 4 weeks. Bone regeneration was examined radiographically and histologically. Results In the irradiation group, the radiographs showed little regeneration during the elongation phase. During the maturation phase, the callus appeared slowly, and its formation was spotty. Furthermore, regeneration was not completed until the fourth week of the maturation period. Histological examination at the end of distraction showed a gap in the distraction consisting of loose connective tissue, with part of the fibrous tissue oriented longitudinally. Four weeks after completion of distraction, the major part of the radiolucent region consisted of cartilage. The spotty osteogenesis was identified as enchondral ossification. Immunohistochemical examination of the regeneration area revealed that the blood vessels were extremely localized, and that the level of expression of vascular endothelial growth factor (VEGF) in the osteoblasts was high. Microangiography showed that vascularization at the distracted sites was poor. Distraction osteogenesis was decreased markedly by preoperative irradiation in terms of both rate and process. The results suggested that most of the osteoprogenitor cells were damaged immediately after irradiation. The high level of VEGF in the osteoblasts and the enchondral ossification also suggested a hypoxic state in the distracted region. Conclusions Preoperative irradiation interferes with distraction osteogenesis by inducing a state of poor angiogenesis.     

内置式山羊下颌骨二维牵张器的实验研究

目的：探讨用自行研制的内置式二维牵张器在山羊下颌骨上进行二维牵张的可能性。方法：进行离体实验预牵张;建立山羊下颌骨二维牵张的动物模型,牵引过程中拍摄X线片以监测牵张进度和牵张间隙内戍骨情况,牵张结束后和固定8周后进行HE染色做组织学观察。结果：离体实验可以完成整个牵张过程;4只山羊成功建立下颌骨二维牵张动物模型,牵张间隙内新骨形成确切,X线片和组织学切片均显示固定8周后新生骨已经接近正常骨质。结论：自行设计的内置式山羊下颌骨二维牵张器可以完成山羊下颌骨的二维牵张成骨并可能具有一定的临床应用价值。     

电穿孔介导的基因治疗对兔下颌骨牵引区新生骨骨密度与强度的影响

目的 探索电穿孔介导的基因治疗对下颌骨DO过程中牵引间隙新生骨密度与强度的影响,从而为促进下颌骨DO新骨生成,缩短牵引周期,减少并发症提供新思路.方法 以新西兰大白兔为实验动物模型,于术后3 d开始下颌骨牵引,每天0.8 mm,连续牵引7 d后,将实验动物分为5组.A组:在牵引区注射2 μg(O.1μg/μl)重组质粒pIRES-hVEGF165-hBMP2;B组:在牵引区注射2 μg(0.1μg/μl)重组质粒pIRES-hBMP2;C组:在牵引区注射2 μg(0.1μg/μl)重组质粒pIRES-hVEGF165;D组:在牵引区注射2 μg(0.1μg/μl)空质粒pIRES;E组:在牵引区注射相同剂量的生理盐水.5组实验动物均施加电穿孔刺激.各组分别于固定期第1、2、4、8周行X线及QCT检查.选整个牵张间隙新生骨痂部分为兴趣区,测定骨密度.然后处死动物.取材测量牵引区新生骨的三点抗压强度.结果 A、B、C组新生骨痂密度各时相点新生骨痂密度明显高于D、E组(P<0.01).固定2周,A组明显高于各组,但B、c组间比较差异无统计学意义.固定4周,A、B组明显高于C、D、E组(P<0.01).固定8周A组明显高于B、c、D、E组(P<0.01).B、C组间比较差异无统计学意义,但高于D、E组(P<0.01).固定4周,A组新生骨的三点抗压强度明显高于B、C、D、E组(P<0.01).固定8周,A组仍明显高于各组(P<0.01),且B组也明显高于c、D、E组(P<0.05).结论 电脉冲介导的pIRES-hVEGF165-hBMP2重组质粒体内转染可使牵引区获得较满意的骨再生和骨化成熟进程,其新骨骨化、改建过程均超过对照组.提示联合应用BMP与VEGF,可能会实现成骨与血供的联合重建,并且使单一生长因子的效应放大,使骨愈合的速度加快.     

Digital radiography. A predictor of regenerate bone stiffness in distraction osteogenesis

The newly formed regenerate bone during limb lengthening usually is assessed by observing standard plain radiographs. However, no objective data correlate the radiographic appearance with the quantitative strength of the newly formed bone. A noninvasive method was developed to assess the regenerate bone using digital radiography. The investigations were performed in a micropig animal model. The right tibia was distracted for 10 days at 2 mm per day. Digital radiography was performed after the distraction during a 10-day consolidation period. For quantitative analysis, an aluminum step wedge was placed on all images, and a region of interest was outlined in the regenerate bone. A calibration curve was calculated by measuring the corresponding gray values of the aluminum phantom. The calibration equation was used for determining the mean density in the region of interest. This method was sensitive in assessing day to day healing progress. The measurements taken the day before sacrifice were compared with torsional mechanical data using a material testing machine. The high correlation between the densimetric evaluations and the biomechanical data suggests this method is a useful tool for in vivo assessment of the regenerate bone during the consolidation period in distraction osteogenesis.     

BMSCs联合3D打印PLLA支架促进兔颅骨PDO的研究

目的探讨骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)促进骨膜牵张成骨(Periosteal distraction osteogenesis,PDO)的可行性。方法全骨髓贴壁培养法获取新西兰白兔BMSCs,体外扩增培养并进行成骨、成脂、成软骨诱导分化。用3D打印(Three-dimensional printing,3D printing)的左旋聚乳酸(Poly l-lactic acid,PLLA)支架牵张兔颅骨骨膜,在牵张期结束时,实验组于骨膜与颅骨间隙内注射兔BMSCs,对照组则注射等量生理盐水(Normal saline,NS)。分别在固定期后的4周和8周,对兔颅骨的牵张部位进行取材,行Micro-CT扫描和组织学染色,评价新骨形成情况。结果全骨髓培养法获取的兔BMSCs具有多向分化能力。Micro-CT显示实验组和对照组的牵张区域在4周和8周均有新骨生成,实验组在两个时间点的新生骨量(Bone volume,BV)、骨量/组织量(Bone volume/Tissue volume,BV/TV)、骨密度(Bone mineral density,BMD)、骨小梁数量(Trabecular number,Tb.N)和厚度(Trabecular thickness,Tb.Th)均明显高于对照组,骨小梁间隔(Trabecular separation,Tb.Sp)小于对照组。HE染色显示,实验组与对照组均有新骨生成,实验组新生骨小梁增厚增多,比对照组成熟,且与颅骨皮质的界线不明显。结论在兔颅骨PDO区域内注射自体BMSCs可以补充成骨细胞的不足,有利于骨膜牵张成骨。