Effects of recombinant human basic fibroblast growth factor on cell proliferation during mandibular fracture healing in rabbits

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A single local application of recombinant human basic fibroblast growth factor accelerates initial angiogenesis during wound healing in rabbit ear chamber

Local angiogenic therapy with recombinant human basic fibroblast growth factor (rhbFGF) has been used to promote wound healing. To obtain useful information for the development of optimal angiogenic therapy, we chronologically evaluated the effects of a single local application of rhbFGF on angiogenesis in a rabbit ear chamber model of wound healing by observing the subcutaneous vessel bed intravitally. New vessel formation during wound healing was macroscopically and microscopically evaluated for 5 wk. Each rabbit ear chamber received a single dose of 6 microg rhbFGF (treatment B1: n = 13), 18 microg rhbFGF (treatment B2: n = 16), or physiological saline as control (n = 13). At 1 wk the newly vascularized area was significantly larger in groups B1 and B2 than in control. At 2 wk, the vascularized areas in groups B1, B2, and control were similar. At 5 wk, the percentage of rabbits with complete vascularization was significantly larger in group B1 than in control. Capillary density at 5 wk was similar among the three groups. These results suggest that locally applied rhbFGF accelerated angiogenesis during early wound healing in rabbits; however, this effect was transient and no increase in capillary density occurred at the completion of vascularization.     

生骨再造散对兔骨折愈合影响的实验研究

目的探讨中成药生骨再造散对兔实验性骨折愈合的影响. 方法将30只青紫蓝家兔制成双侧桡骨骨膜下3 mm骨缺损模型,随机分为生骨再造散组(A组)、仙灵骨葆组(B组)及对照组(C组),每组10只,于术后14天和31天每组各处死5只兔,按规定时间进行四环素双标记(即14天处死的兔于术后4、5天行第1次标记,术后11、12天行第2次标记;31天处死的兔于术后20、21天行第1次标记,术后28、29天行第2次标记),并分别取材,进行骨组织形态学计量检测. 结果术后14天A组与C组比较,类骨质面积密度、矿化骨痂均宽及活性成骨面、矿化表面及动态参数等差异均有统计学意义(P＜0.01);术后31天,活性成骨面、矿化骨痂均宽及吸收陷窝均深等差异均有统计学意义(P＜0.01).B组与C组比较,结果与A组和C组比较类似;但与A组比较,术后14天破骨细胞指数、矿化表面及骨形成率[组织水平(tissue level)及BMU水平(basic multicellular unit level)]差异均有统计学意义(P＜0.05),术后31天类骨质表面、吸收陷窝均深、骨形成率(BMU水平)差异均有统计学意义(P＜0.05). 结论生骨再造散在兔桡骨骨折14天前可促进类骨质分泌,加快矿化沉积速度,提高骨形成率,缩短矿化延迟时间;14～31天后则可增加矿化骨痂宽度,提高破骨细胞活性,对骨折愈合有促进作用.     

Deleterious Effects of Intermittent Recombinant Parathyroid Hormone on Cartilage Formation in a Rabbit Microfracture Model: a Preliminary Study

Intermittent parathyroid hormone administration can enhance fracture healing in an animal model. Despite the success of exogenous parathyroid hormone on fracture healing and spine fusion, few studies have examined the role of parathyroid hormone on cartilage formation. We determined the effects of intermittent parathyroid hormone on cartilage formation in a rabbit microfracture model of cartilage regeneration. Twelve rabbits were divided into three equal groups: (1) microfracture alone, (2) microfracture + parathyroid hormone daily for 7 days, and (3) microfracture + parathyroid hormone for 28 days. Nonoperated contralateral knees were used as controls. The animals were sacrificed at 3 months and gross and histologic analysis was performed. The microfracture alone group demonstrated the most healing on gross and histologic analysis. Treatment with either 1 or 4 weeks of parathyroid hormone inhibited cartilage formation. Although discouraging from a cartilage repair point of view, this study suggests that the role parathyroid hormone administration has in clinical fracture healing must be examined carefully. Although parathyroid hormone is beneficial to promote healing in spine fusion and midshaft fractures, its deleterious effects on cartilage formation suggests that it may have adverse effects on the outcomes of periarticular fractures such as tibial plateau injuries that require cartilage healing for a successful clinical outcome.     

云南白药促进骨缺损修复及引导性骨再生的实验研究

目的：阐明云南白药促进骨缺损修复及引导性骨再生的愈合机制，为临床上治疗骨折提供理论依据。方法：36只新西兰兔制作桡骨缺损不愈合模型和引导性骨再生模型，随机均分用药组及对照组，手术后3d、1、3、5、10、12周取材，观察骨痂愈合程度的差异及骨痂组织学变化，结果：实验组有明显的促进骨缺损修复作用，并在引导性骨再生中的膜内成骨作用强于对照组，结论：云南白药对骨缺损修复及引导性骨再生有明显的促进作用。     

肌腱愈合过程中转化生长因子β1基因表达的变化

目的研究兔屈趾肌腱Ⅱ区伤口愈合过程中转化生长因子β1（transforming growth factorpβ1，TGF-β1）基因表达的变化。方法取成年新西兰大白兔60只，体重4．0～4．5kg，将左前中趾屈趾深肌腱切断并采用标准Kessler缝合法修复作为实验组，分别于术后1、7、14、21、28及56d获取肌腱及腱鞘进行观察，每个时间点取10只；同一动物的右前肢正常肌腱及腱鞘作为对照组。采用原位杂交和免疫组织化学染色分析TGF—β1的表达情况。结果原位杂交结果示：实验组TGF—β1mRNA在术后1d开始上调，14～21d达高峰，56d保持较高水平；对照组存在TGF—β1mRNA的表达，但表达水平较低；实验组各时间点TGF—β1mRNA表达与对照组比较，差异均有统计学意义（P〈O．05）。免疫组织化学染色：实验组术后1d，TGF—β1蛋白信号的表达增加，14～21d达高峰，56d仍保持一定水平；对照组术后各时间点存在TGF—β1蛋白信号表达，但表达水平较低。结论正常无损伤的肌腱和腱鞘能产生TGF—β1，当肌腱损伤后，细胞因子被激活，增加的细胞因子主要由肌腱细胞与腱鞘细胞产生，与肌腱的内、外源性愈合机制是一致的。     

海水浸泡开放性骨折伤愈合过程中组织病理学和血管内皮细胞生长因子(VEGF)表达

目的通过观察普通开放骨折、海水浸泡开放骨折愈合过程的组织学变化和骨痂中血管内皮细胞生长因子(VEGF)的表达,了解海水浸泡开放性骨折愈合过程VEGF的作用与机制。方法新西兰大白兔59只,随机分为普通开放骨折组(对照组)24只和海水浸泡开放骨折组(实验组)35只。造成桡骨横行1.5mm缺损完全开放骨折,普通开放骨折伤组旷置3h,海水浸泡开放骨折伤组海水浸泡伤口3h,之后依次缝合伤口。于第1、3、7、14、21、28、45天处死动物。观察海水浸泡开放骨折伤在骨折愈合中不同时间的病理过程。采用RT-PCR方法检测普通开放骨折、海水浸泡开放骨折不同阶段骨痂中的VEGF的表达及变化。结果海水浸泡开放骨折伤骨痂形成延迟,骨折后第28天,对照组断端间骨痂为骨性骨痂者8例,为软骨者4例,实验组断端间骨痂为骨性骨痂者6例,为软骨者14例。海水浸泡骨折伤愈合过程中新生骨痂的VEGF表达在骨折后逐渐升高,术后14d达到高峰,之后逐渐下降,但在28d时仍保持较高水平,与一般开放骨折愈合过程的VEGF表达无显著差异(P>0.05)。结论海水浸泡使骨折骨痂形成不良率增高,骨折愈合过程有延迟倾向;但骨折愈合过程中VEGF表达无明显变化。     

Impaired angiogenesis, early callus formation, and late stage remodeling in fracture healing of osteopontin-deficient mice.

OPN is an ECM protein with diverse localization and functionality. The role of OPN during fracture healing was examined using wildtype and OPN(-/-) mice. Results showed that OPN plays an important role in regulation of angiogenesis, callus formation, and mechanical strength in early stages of healing and facilitates late stage bone remodeling and ECM organization. INTRODUCTION: Osteopontin (OPN) is an extracellular matrix (ECM) protein with diverse localization and functionality that has been reported to play a regulatory role in both angiogenesis and osteoclastic bone remodeling, two vital processes for normal bone healing. MATERIALS AND METHODS: Bone repair in wildtype and OPN(-/-) mice was studied using a femoral fracture model. microCT was used for quantitative angiographic measurements at 7 and 14 days and to assess callus size and mineralization at 7, 14, 28, and 56 days. Biomechanical testing was performed on intact bones and on fracture specimens at 14, 28, and 56 days. Histology and quantitative RT-PCR were used to evaluate cellular functions related to ECM formation and bone remodeling. RESULTS: OPN deficiency was validated in the OPN(-/-) mice, which generally displayed normal levels of related ECM proteins. Intact OPN(-/-) bones displayed increased elastic modulus but decreased strength and ductility. Fracture neovascularization was reduced at 7 but not 14 days in OPN(-/-) mice. OPN(-/-) mice exhibited smaller fracture calluses at 7 and 14 days, as well as lower maximum torque and work to failure. At 28 days, OPN(-/-) mice had normal callus size but a persistent reduction in maximum torque and work to failure. Osteoclast differentiation occurred normally, but mature osteoclasts displayed reduced functionality, decreasing late stage remodeling in OPN(-/-) mice. Thus, at 56 days, OPN(-/-) fractures possessed increased callus volume, increased mechanical stiffness, and altered collagen fiber organization. CONCLUSIONS: This study showed multiple, stage-dependent roles of OPN during fracture healing. We conclude that OPN deficiency alters the functionality of multiple cell types, resulting in delayed early vascularization, altered matrix organization and late remodeling, and reduced biomechanical properties. These findings contribute to an improved understanding of the role of OPN in vivo and provide new insight into mechanistic control of vascularization and bone regeneration during fracture repair.     

表皮生长因子与碱性成纤维细胞生长因子促进创面修复的比较研究

目的　比较重组人表皮生长因子 (rhEGF)与重组人碱性成纤维细胞生长因子 (rhbFGF)促进创面修复的效应及其作用机制。　方法　选用 12只大耳白兔 2 4只耳上的 72个创面 ,随机分为rhEGF(10 μg/cm2 )治疗组、rhbFGF(10 0AU/cm2 )治疗组和对照组 (涂以 1%磺胺嘧啶银霜 )。观察创面的愈合情况 ,分别取不同时段的标本进行病理学、电镜检查 ,并通过原位杂交检测标本中整合素 β1mRNA的表达情况。　结果　两治疗组整合素 β1mRNA的表达明显高于对照组 ;创面修复的时间明显比对照组快 ,且修复质量较对照组高 (P <0 .0 5 ) ;成纤维细胞数和毛细血管胚芽数与对照组比较差异有显著性意义 (P <0 .0 5 )。　结论　rhEGF、rhbFGF均能提高创面修复质量 ,在修复早、中期使用rhbFGF能促进肉芽组织生长 ;中、晚期使用rhEGF能加速创面的再上皮化。根据时效 ,联合应用rhEGF、rhbFGF可获得最佳效价比。     

Expression of growth factors in the early phase of supraspinatus tendon healing in rabbits

Growth factors are known to appear during wound healing. We hypothesized that growth factors would also appear during the healing process of a rotator cuff tear. We determined the expression of various growth factors during healing of acute rotator cuff tears in the rabbit. We made a full-thickness defect in the supraspinatus tendon of 27 Japanese white rabbits. The shoulders were harvested on days 1, 3, 5, 7, 9, 11, 14, 21, and 28 postoperatively (n = 3 at each time point). We assessed the expression of basic fibroblast growth factor, insulin-like growth factor 1, platelet-derived growth factor, and transforming growth factor beta. Basic fibroblast growth factor appeared with its peak on days 7 and 9, insulin-like growth factor 1 appeared with its peak on day 5, platelet-derived growth factor appeared with a mild expression between days 7 and 14, and transforming growth factor beta appeared with constant mild expression throughout the observation period. It is likely that each of these growth factors plays a role in the early phase of healing of the supraspinatus tendon in rabbits.     

骨折愈合的应力适应性研究

目的 探讨骨折端受力、肌肉动力、骨痂密度与骨折愈合的关系。方法 通过传感器电测技术与X线灰度分析的方法，从三个方面对骨折愈合的应力适应性进行了研究：①分别对14只1岁龄山羊进行了断端受力与骨折愈合的关系的研究；②对10只健康成年家兔进行了肌肉动力与骨折愈合的关系的研究；③对56只健康成年家兔进行了骨痂密度与骨折愈合的关系的研究。结果与结论 ①理想的骨折愈合与最佳的应力状态相适应；②肌肉动力是应力适应的反馈调节因素；③骨痂密度是应力适应的反馈结果。     

上肢骨干骨折内固定实验动物模型的建立

目的：建屯内固定术治疗上肢骨干骨折的实验动物模型，为研究不同内固定下骨折愈合的机制提供实验对象。方法：新西兰大白兔25只，随机选择一侧前肢上臂，取外侧切口显露肱骨，以线锯在其中段截骨，复位后植入天鹅记忆接骨器(SMC组)；同法在对侧植入4孔动力加压接骨板(DCP组)。分别在术后l、2、48、12周摄片观察骨折愈合情况，并处死5只动物取材，HE染色，行病理学观察。结果：术后动物全部成活，前肢负重明显低于后肢。X线片显示：SMC组在术后8周骨折愈合，愈合过程中未见明显外骨痂及板下皮质骨疏松；DCP组骨折愈合速度较慢，术后12周时骨折端仍有骨痂，伴局部皮质骨疏松。，病理学观察：术后8周，SMC组骨折端直接由板层骨替代；术后12周，DCP组形成骨性连接，处于改建塑形期。结论：SMC固定下实验性兔肱骨干骨折愈合过程与临床相似，愈合速度和质量优于DCP固定。兔肱骨干骨折内固定模型是一种较为理想的上肢骨干骨折内固定实验模型。     

成骨细胞移植促进老龄鼠骨质疏松性骨折愈合过程中VEGF的动态变化

目的：动态观察在幼龄成骨细胞移植促进骨质疏松性骨折愈合过程中，VEGF在不同时相的表达及其生物学意义。方法：通过建立老龄骨质疏松SD大鼠骨折的动物模型，并将体外培养的SD雄性乳鼠成骨细胞移动到SD雌性鼠老年骨质疏松性骨缺损部位，利用免疫组化及原位杂交检测骨折愈合过程中不同时间相的移植标本VEGF、VEGFmRNA的表达，并作图像分析、绘出其动态变化图。结果：实验组VEGF、VEGFmRNA均在7d左右可见有阳性表达的细胞，14d有分泌,高峰其中以软骨细胞中阳性最强，21 d分泌量开始下降，56d后基本消失。而对照且未见明显分泌高峰。结论；成骨细胞细胞促进老龄鼠骨质疏松性骨折愈合，其机制可能是通过促进VEGF的转录和表达，从而促进骨折部位建立良好的血液循环，加速骨形成。     

Effect of stenting after laryngotracheal reconstruction in a subglottic stenosis model.

OBJECTIVES: This study addresses the impact of stenting on early wound healing after laryngotracheal reconstruction (LTR) in a rabbit model with established subglottic stenosis. METHODS: Subglottic stenosis was created in 42 New Zealand white rabbits through a transoral, endoscopic technique. Three weeks later, endoscopy and axial CT were performed to document and grade the degree of stenosis. Subsequently, LTR was performed in all animals, with half of the animals receiving an intraluminal stent. Four rabbits from each group were euthanized on postoperative days 6, 9, 14, 21, and 28. Measurements of graft vascularization were obtained with a computerized image measurement program, and a comparison was made regarding the rate of vascularization. RESULTS: There was a statistically significant increase in the rate of vascularization in the stented group (mean 75% +/- 5% vs 56% +/- 3% at day 14; P < 0. 05). However, clinical and radiographic comparisons of the stented and nonstented specimens revealed a trend toward increased mucosal edema and granulation tissue in the stented group at later time intervals (days 21 and 28). CONCLUSION: This analysis suggests that stenting does not inhibit graft vascularization in the early postoperative period after LTR; however, complications were seen in the stented group at longer time intervals.     

刺老苞根皮水提物对骨折愈合相关因子受体表达的影响

目的：研究刺老苞根皮水提物在骨愈合修复过程中对相关因子受体表达的干预作用。方法：采用SD大鼠胫骨打孔的方法建立单因素骨折模型,分别于实验造模第14、28天取材,用免疫组织化学方法检测刺老苞根皮水提物在骨愈合修复过程中对成纤维细胞生长因子受体2（fibroblastgrowthfactorreceptor2,FGFR2）、血管内皮生长因子受体1、受体2（fms-liketyrosinekinase,Flt-1;fetallicerkinase,Flk-1）表达的影响。结果：14d时,刺老苞根皮高剂量组增强了Flt-1、Flk-1的表达,以Flk-1更为显著,均高于模型组及刺老苞根皮低剂量组;刺老苞根皮高剂量组FGFR2有较高表达,与阳性对照药伤科接骨片组相近,高于模型组。28d时,各用药组FGFR2、Flk-1、Flt-1表达减弱,模型组表达均高于各用药组。结论：刺老苞根皮水提物在骨愈合过程中对血管形成有明显促进作用,对成纤维细胞、成骨细胞、软骨细胞的增值和活性有较大影响,加速软骨修复,提前进入骨化和塑形期,促进骨折愈合。     

Biological effects of extracorporeal shockwave in bone healing: a study in rabbits

INTRODUCTION: This study is an investigation of the biological effects of extracorporeal shockwave treatment (ESWT) on bone healing in a rabbit model. MATERIALS AND METHODS: Sixteen 12-month-old New Zealand white rabbits with body weight ranging from 2.5 to 3.5 kg were used in the study. An intra-medullary pin was inserted retrograde into the femur canal. A closed fracture of the femur was created with a three-point bend method. The animals were randomly divided into the study group and the control group with eight rabbits in each group. The study group received shockwave treatment, whereas the control group did not. The animals were killed at 12 weeks, and a 5-cm long femur bone including the callus was harvested. The specimens were subjected to biomechanical study, histomorphological examination, and immunohistochemical analysis. RESULTS: The shockwave group showed significantly better bone strength in biomechanical study, more cortical bone formation in histomorphological examination and higher number of neo-vessels and angiogenic and osteogenic growth markers including VEGF, eNOS, PCNA, and BMP-2 on immunohistochemical stains than the control group. CONCLUSION: ESWT significantly improved bone healing after fracture of the femur in rabbit. ESWT promoted the formation of cortical bone what might have been associated with increased biomechanical results. ESWT-promoted bone healing was associated with increased neovascularization and up-regulation of angiogenic and osteogenic growth factors.     

Histomorphometrical and radiological comparison of low-level laser therapy effects on distraction osteogenesis: experimental study

Distraction osteogenesis (DO) is the application of traction to the callus formed between bone segments and stimulation of bone formation by creating stress on the callus with this traction. Shorten the duration of DO and increasing the capacity of bone formation is important to prevent the possible complications of DO. For this reason, it was considered that low-level laser therapy (LLLT) may affect positively DO and it can decrease the complication range by shortening the period. Unilateral mandibular distractors were applied on 16 female white New Zealand rabbit to prove this hypothesis with micro CT, plain radiograph and histomorphometric analyses. Eight rabbits were applied LLLT with GaAlAs laser on the distraction area during the distraction period. On the post-distraction 28th day, four rabbits from study group and four rabbits from control groups were sacrificed. The rest of the rabbits were sacrificed on post-distraction 56th day. As a result of this study, significant positive effects of LLLT on post-distraction 28th day were revealed with all analyses. In histomorphometrical analyses, new bone formation was significantly higher in short-term laser applied group comparing to that of short-term control group (p?=?0.029). In both microCT and plain radiograph, the highest radioopacity values were observed in short-term laser group when compared with that of the controls (p?=?0.043 and p?=?0.025, respectively). Even though LLLT increased the healing capacity on short-term, it was not sufficient on long-term (post-distraction 56th day) healing. LLLT application on distraction period, activate healing on bone so it may decrease DO period. The result of this study should be supported with clinical studies and the most effective laser source, dose and application time should be revealed with experimental and clinical studies.     

Improved healing of experimental long bone fractures in rabbits by delayed internal fixation.

Experiments in rabbits were conducted to test the clinical propositions that a) the risk of nonunion in certain long bone fractures can be sharply reduced by open reduction and internal fixation, and b) that there is an optimum critical period following trauma in which this procedure should be carried out to facilitate healing. Bilateral standard open fractures of rabbit radii were internally fixed by intramedullary K-wires immediately or at 5, 10, and 17 days after trauma. These bones were subjected to stress and histometric analyses at 4-56 days after the initial fractures. The observations suggested that the pace of fracture healing could be optimized by time-delay surgery. The most exuberant and strongest calluses developed when the bones were fixed 10 days after fracture, and the rate of healing was improved. Delay times of 5 or 17 days did not provide better healing than that achieved by an immediate operation. Callus quality, in terms of the proportion of fiber bone, lamellar bone, and cartilage, was similar in all groups. The tensile strengths of the bones during callus formation were highly correlated with the ratio of callus/cortical bone areas (p less than 0.01).     

高能震波对骨痂中骨形成蛋白表达的影响

目的 观察高能震波（ＨＥＳＷ）对骨痂中骨形成蛋白（ＢＭＰ）表达的影响，探讨其促进骨折愈合的机理。方法 手术造成家兔双侧胫骨中段标准骨折模型，对一侧胫骨骨折部施以ＨＥＳＷ，另一侧为对照组，术后每周进行Ｘ线及组织学检查，并取骨痂组织切片用ＢＭＰ抗体进行免疫组化染色（ＳＡＢＣ法（）。结果 实验组骨痂中ＢＭＰ表达强度在第２－４周时明在于对照组（Ｐ〈０．０１），且Ｘ线摄片及组织学观察均显示实验组骨折愈合时间     

The effect of rhPTH on the healing of tendon to bone in a rat model

Successful rotator cuff tendon repair depends on secure tendon‐to‐bone healing. Recombinant human parathyroid hormone (rhPTH) has been shown in multiple studies to accelerate bone healing. Recent studies have also shown that rhPTH is chondrogenic by increasing chondrocyte recruitment and differentiation. We hypothesized that rhPTH would improve tendon‐to‐bone healing in a rat rotator cuff repair model. One hundred and fourteen Sprague Dawley rats underwent division and repair of the supraspinatus tendon. Fifty seven rats received daily subcutaneous injections of 10 µg/kg of rhPTH. Rats were sacrificed at 3, 7, 14, 28, and 56 days for histologic and immunohistochemical analysis. In addition, rats in each group were sacrificed at 14, 28, and 56 days for biomechanical testing and micro CT analysis. At 2 weeks the controls had a significantly higher load to failure than the rhPTH group. At 28 and 56 days there were no differences in load to failure. rhPTH specimens had significantly higher stiffness at 56 days. MicroCT analysis showed that the rhPTH group had significantly greater total mineral content at all time points, as well as significantly higher bone volume (BV) at 14 and 28 days. Histologically, the rhPTH specimens had more fibrocartilage, osteoblasts, and blood vessels at all timepoints, with significantly better collagen fiber orientation at 28 and 56 days. Although treatment with rhPTH resulted in an increase in bone and mineralized fibrocartilage formation, as well as better collagen fiber organization, this did not translate into improved biomechanical properties. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:769–774, 2012