组织工程周围神经血管化研究进展

目的对组织工程周围神经血管化的研究现状进行文献回顾,为寻找促进组织工程周围神经血管化的新方法提供理论依据。方法查阅近年来关于组织工程周围神经血管化方面的相关文献,围绕周围神经再生局部微环境及血供特点、支架材料修饰、种子细胞、自体血管束的植入、促血管因子等方面进行回顾及总结。结果组织工程周围神经为神经损伤的修复带来了新的希望,但对于大段神经缺损的修复效果欠佳,这主要与神经移植物的血管化程度相关,因此促进组织工程周围神经早期血管化尤为重要。既往研究多从支架材料修饰、种子细胞、自体血管束植入及促血管生成相关因子4个方面单一进行研究,近年研究表明将上述两种或多种因素共同作用于组织工程周围神经,能够更好地促进组织工程周围神经血管化。结论促进组织工程周围神经早期血管化,能够使支架上的种子细胞及时获得营养支持,促进轴突生长、神经再生,有利于修复大段周围神经缺损。     

长段同种异体骨与自体带血管组织组合治疗大段骨缺损的总结

目的 通过研究血管化及促进骨愈合的方法,解决大段异体骨移植骨吸收、骨不连及再骨折等并发症.方法 对四肢10 cm以上长段骨缺损21例,采用钢板、外固定架等常规方法将大段异体骨与自体骨固定,同时采用异体骨与自体血管束、带血供骨或骨膜组合的方法,使异体骨血管化,达到促进骨愈合、防止异体骨溶解、吸收等并发症作用.其中4例采用局部血管束植入一侧断端;4例采用带血管髂骨块嵌入长段骨中部;2例带血管髂骨植入一侧骨断端;2例带血管腓骨嵌入中部;6例带血管骨膜植入一侧断端;另外血管束或骨膜加骨块组合植入3例.结果 21例中经一次治疗骨性愈合14例,经二次手术痊愈6例,失败1例.出现骨吸收、不愈合及再骨折等并发症7例,合并感染4例.经随访功能、外观满意.结论 采用自体带血管骨(膜)与同种异体骨组合移植治疗长段骨缺损,可改善大段异体骨移植的骨吸收、骨不连等并发症,有利于骨折的早期愈合.为治疗长段骨缺损较为理想的方法.     

血管束、感觉神经束植入组织工程骨修复大段骨缺损的成骨研究

目的 观察血管束、感觉神经柬植入组织工程骨修复兔股骨大段骨缺损的成骨特点,探讨其对骨修复的影响. 方法 36只新西兰大白兔均制备左侧股骨干1.5 cm节段性骨缺损模型,随机分为三组(n=12),组织工程骨组(A组):自体骨髓基质干细胞复合β-磷酸三钙构建组织工程骨植入骨缺损;血管束植入组(B组):组织工程骨与血管束同时植入骨缺损;感觉神经束植入组(C组):组织工程骨与感觉神经束同时植入骨缺损.各组动物术后1、3、6个月行X线检查及影像学评分,同时每组各处死4只动物行大体及组织学观察.结果 影像学评分显示各时间点B组与C组的成骨优于A组,差异有统计学意义(P<0.05);B、C组间成骨差异无统计学意义(P>0.05).组织学观察显示新生骨多出现在血管周围,成骨方式以软骨内成骨为主. 结论血管束、感觉神经束植入组织工程骨的方法能更好地促进组织工程骨成骨及大段骨缺损修复.     

组织工程骨神经化构建的组织学研究

[目的]通过观察不同神经束植入组织工程骨后组织学的变化,探讨组织工程骨神经化构建的效果.[方法]将新西兰大白兔的骨髓基质干细胞(bone marrow stromal cells,BMSCs )进行诱导分化为成骨细胞,与β-磷酸三钙(β-tricalcium phosphate,β-TCP)复合制作组织工程骨,分别将不同类型神经束植入组织工程骨,进行以下分组:A组,组织工程骨组; B组,感觉神经束植入组; C组,运动神经束植入组; D组,血管束(含有自主神经)植入组; E组,感觉、运动神经束联合植入组.分别用于修复兔股骨1.5 cm大段骨缺损;各组分别在12周进行组织学的Masson染色,降钙素基因相关肽(calcitonin gene-related peptide,CGRP)的免疫组织化学检测,并进行统计学分析,以比较骨缺损修复情况.[结果]在观察期内,有感觉神经植入的组别和血管束植入的组别在组织学上具有明显骨化作用,而运动神经束植入却没有明显的促进作用.[结论]感觉神经束和血管束具有明显的促组织工程骨成骨作用,可以作为组织工程骨神经化构建的一种方法.     

神经植入对大段组织工程骨成骨效果的一年观察

目的 观察在组织工程骨内植入神经束后大段组织工程骨的长期成骨效果.方法 新西兰大白兔64只,随机分为四组:A组,单纯组织工程骨组;B组,运动神经束植入组(股神经肌支);C组,感觉神经束植入组(隐神经);D组,感觉、运动神经束联合植入组.每只动物均在左侧股骨制作长1.5 cm的段缺性骨与骨膜缺损,钢板固定后在骨缺损处分别植入四种方法制备的组织工程骨.术后1、3、6、12个月行大体观察、X线和组织学定量观察成骨情况. 结果 术后1、3、6个月时,在组织工程骨内植入感觉神经或联合植入两种神经后,比单纯组织工程骨和运动神经束植入的修复效果均有明显提高.术后12个月时,各组骨再生情况基本一致,但新生骨组织出现一定程度的吸收,其外观较正常股骨细,新生骨组织与兔股骨牢固愈合,并开始塑形且出现髓腔再通. 结论 在组织工程骨内植入感觉神经可促进成骨,而植入运动神经未见促进作用;组织工程骨可以修复兔大段骨缺损;该实验新生骨组织的吸收可能与模型的制作方法有关.     

神经植入对大段组织工程骨成骨影响的早期实验研究

目的 研究在组织工程骨内植入神经束对大段组织工程骨成骨的影响以及初步探讨促进成骨的机制.方法 48只新西兰大白兔,随机分为4组:A组,单纯组织工程骨组;B组,组织工程骨+运动神经束植入组(股神经肌支);C组,组织工程骨+感觉神经束植入组(隐神经);D组,组织工程骨+感觉、运动神经束联合植入组.每只动物均在右侧股骨制备长1.5 cm的节段性骨与骨膜缺损,钢板固定后在骨缺损处分别植入四种方法制备的组织工程骨.术后4、8、12周行大体观察、X线观察、新生骨组织定量观察成骨效果,降钙素基因相关肽(CGRP)及S-100蛋白的免疫组化染色观察其在组织工程骨内的分布,并行半定量分析.结果 在组织工程骨内植入感觉神经后,比单纯组织工程骨和运动神经束植入的修复效果均有明显提高.而在组织工程骨中植入运动神经与单纯组织工程骨修复效果无明显差异,感觉神经植入与感觉、运动联合植入的成骨效果无明显差异.免疫组化染色显示神经肽类物质CGRP、S-100在C组与D组中的表达高于A组与B组,差异有统计学意义.结论 在组织工程骨内植入感觉神经在早期可促进成骨,而植入运动神经未见促进作用, 其机制可能是植入的感觉神经在组织工程骨内分泌神经肽所致.     

复合骨形态蛋白-2的聚乳酸-乙醇酸共聚物/磷酸三钙人工骨结合带血供组织修复羊大段骨缺损的试验研究

目的研究复合骨形态蛋白-2的聚乳酸-乙醇酸共聚物/磷酸三钙(PLGA-TCP-BMP-2)人工骨结合尺骨骨膜及长段自体骨移植修复大段骨缺损的效果。方法手术造成30 mm绵羊桡骨骨缺损,A组植入人工骨及带血运的长段尺骨,B组植入人工骨及带血运的尺骨骨膜,C组仅植入人工骨,D组不植入任何材料。四组均以钢板固定桡骨缺损区。术后行X线摄片,24周处死行CT及组织学检查,进行系统评价。结果 X线检查示术后放射学评分A组高于其它各组。组织学结果显示,A组新生骨完全修复骨缺损区;B组新生骨与断端皮质骨融合,新生骨痂较为纤细;C组新生板层骨及骨陷窝排列较为紊乱;D组无骨连接表现。A、B、C组均未见人工骨材料残留。A组组织学评分最高,D组最低,差异有显著性。结论PLGA-TCP-BMP-2人工骨结合长段自体骨或带血供尺骨骨膜移植能够很好的修复绵羊桡骨大段骨缺损。     

复合骨形态蛋白-2的聚乳酸-乙醇酸共聚物/磷酸三钙人工骨结合带血供骨膜修复羊大段骨缺损

目的 研究复合骨形态蛋白-2的聚乳酸-乙醇酸共聚物/磷酸三钙(PLGA-TCP-BMP-2)人工骨结合自体带血供尺骨骨膜移植修复大段骨缺损的效果. 方法 手术造成30 mm绵羊桡骨骨缺损,A组植入PLGA-TCP-BMP-2人工骨及带血运的尺骨骨膜,B组仅植入PLGA-TCP-BMP-2人工骨,C组不植入任何材料.3组均以钢板固定骨缺损区.术后24周拍摄X线片并处死动物,进行组织学观察评价. 结果 X线检查示A组桡骨缺损处完全成骨修复,皮质骨与髓腔的轮廓清晰;B组亦完全修复,但新生骨密度及髓腔轮廓清晰度均不如A组;C组无有效骨痂形成.组织学检查示A、B组骨痂外层形成为板层骨,C组缺损区可见大量纤维组织填充. 结论 PLGA-TCP-BMP-2人工骨结合自体带血供尺骨骨膜移植能够很好的修复绵羊桡骨30 mm的骨缺损.     

低强度脉冲超声波促进组织工程骨血管化重建的研究进展

组织工程骨修复骨缺损过程中血管化构建一直是骨科领域的一个难题,随着对骨折修复分子生物学机制的深入研究,以及对骨折愈合机制认识的不断加深,发现血管化是制约组织工程骨修复骨缺损速度与程度的重要因素[1].构建组织工程骨血管化的方法很多,如血管束植入、血管蒂筋膜瓣包裹术或肌肉瓣预制术等均不十分令人满意.     

血管铸型方法检测组织工程骨修复羊胫骨缺损的远期血管化

目的采用血管铸型方法对组织工程技术修复中国青山羊胫骨大段骨缺损的远期血管化情况进行检测。方法中国青山羊3只，制备单侧胫骨20mm的骨与骨膜缺损，缺损部位内植入组织工程骨-珊瑚羟基磷灰石／骨髓基质干细胞（coral hydroxyapatite／bone marrow stem cells，CHAP／BMSCs）。术后3年分别行双侧后肢股动脉甲基丙烯酸甲酯血管铸型灌注，灌注后采用大体解剖、未脱钙骨片、扫描电镜等方法检测组织工程骨的远期血管化，并与健侧相同部位正常骨对照，观察其血管化效果。结果血管灌注后大体解剖观察示组织工程骨血管来源于周围软组织、髓腔血管及两端正常骨皮质。灌注后未脱钙骨磨片显示组织工程骨内微血管沿哈弗管和伏克曼管分布交织形成网状。采用图像分析仪比较组织工程骨与正常骨横切面磨片上血管相对面积，分别为（763．89±47．38）U／10万U与（788．00±65．83）U／10万U，差异无统计学意义（t=0．390，P〉0．05）。结论采用CHAP／BMSCs修复山羊胫骨大段骨缺损远期观察结果表明，新生骨远期血管化效果与正常骨无异，为组织工程骨下一步的临床应用提供可靠的依据。     

Die vaskularisierte Knochentransplantation

Vascularized pedicled bone grafts are known since the beginning of the last century. Microvascular bone grafts (free vascularized bone transfer) are known since the beginning of the seventies. In many experimental and clinical studies vascularized bone grafts were compared to their non-vascularized analogues. Because of their own non-interrupted blood supply and thus nearly normal vitality vascularized bone grafts show more rapid fracture healing, more pronounced adaptation to the new mechanical loadings (e. g. graft hypertrophy), higher survival and consolidation rate in poor or bad recipient bed conditions (infection, bad vascularization) and some neovascularization potential on the surrounding tissue. Because of those properties, it became possible to successfully treat a large segmental bone defect by only few operations. As the treatment of complicated combined soft tissue/bone defects led to high complication rate up to 40 to 60% the indications of this method were altered.     

Vaskularisierte Knochentransplantate

Although pedicled bone grafts (local vascularized bone transplantation) have been used since the beginning of the 19th century, microvascular bone grafts (free vascularized bone transplantation) have been known since the early 1970s. In many experimental and clinical studies, vascularized bone grafts were compared to their nonvascularized analogs. Because of their own noninterrupted blood supply and, thus, nearly normal vitality, vascularized bone grafts show, compared to their nonvascularized analogs at the recipient site (1) more rapid fracture healing, (2) more pronounced adaptation to the new mechanical loadings (e.g., graft hypertrophy), (3) higher survival and consolidation rates in poor or bad recipient bed conditions (infection, poor vascularization), and (4) some neovascularization potential to the surrounding tissue. Because of these properties, it became possible to successfully treat a large segmental bone defect in only a few operations. Severely damaged extremities which would have previously been amputated could be functionally reconstructed. The initial enthusiasm to treat all complicated, combined soft tissue/bone defects by a free vascularized composite flap subsided because of a high complication rate of 40?C60% during the 1980s. During the subsequent years, the various indications were increasingly defined as a result of large clinical studies and the complication rate was lowered to approximately 10%. With increasing knowledge, it became possible to choose the best suited vascularized bone graft for the individual case. Because of the long operation times and the absolute need to master microsurgical techniques, this type of graft was reserved to only a few well-specialized centers. Being very often described as competing with reconstruction techniques, cancellous bone grafting, callus distraction, and free vascularized bone grafts are actually increasingly understood as complementary procedures with every technique having a so-called main indication, e.g., situation in which the use of a distinct technique will lead to the best functional and cosmetic result with the lowest risk for the patient. In fact, even for vascularized bone grafts, the functional state of the surrounding soft tissue plays a major role in successful bone healing; thus, bone reconstruction must never precede soft tissue repair. The aim of this overview is to define the value of a (free) vascularized bone graft within the decision making in case of a large segmental bone defect at the mandible and the extremities, as well as in the treatment of avascular necrosis. In order to avoid relying on subjective impressions as the base of therapeutic decision making, objective criteria in favor or against vascularized bone grafts are described based on a literature review.     

Healing of cortical bone grafts in athymic rats.

We studied healing of allogeneic and syngeneic cortical tibial segment grafts in athymic and normal rats. After 3, 6, and 12 weeks, the weight, circulation, and mineralization rate of the healing segment, and mechanical strength and stiffness of the healing tibia were measured. There were no differences between allogeneic and syngeneic grafts in athymic and normal animals at 3 or 6 weeks. After 12 weeks, the vascularization and mineralization of the grafts, but not of the surrounding callus, were smaller in the allogeneic grafts in the normal recipients than in the other groups. Also after 12 weeks, the stiffness of the healing tibiae was less in allogeneic grafts in normal recipients than in the other groups. The strength of the allogeneic grafts was less than the strength of the syngeneic grafts in both athymic and normal recipients. This suggests that T-cell-mediated rejection is responsible for decreased vascularization and mineralization of allogeneic bone and that the difference in strength between allogeneic and syngeneic grafts is not due to T-lymphocyte graft rejection.     

On Injuries to the Acromio-Clavicular Joint: Treatment and Results

A study was made of bone ingrowth into fiber metal composite prostheses used to replace large segments of the femur in baboons. Bone grafts of two different types were used to cover the segment: chips of bone with large particle size and ground bone with a smaller particle size. The prosthetic segment was bridged by bone at 3 and 6 months in all cases irrespective of the structure of the transplant. In animals sacrificed at 6 months bone ingrowth occurred, with a marked difference between specimens with the two different grafts. In the ground bone specimens ingrowth occurred over the total surface area, and bone penetrated deep into the composite. With the chip grafts ingrowth was more irregular occurring only in some areas and it was always superficial. The difference is believed to be due to the improved contact between the fiber metal surface and the transplant. The lesser bulk of the ground transplant is advantageous when the soft tissue cover of the bone is thin.     

Segmental Replacement of the Femur in Baboons with Fiber Metal Implants and Autologous Bone Grafts of Different Particle Size

A study was made of bone ingrowth into fiber metal composite prostheses used to replace large segments of the femur in baboons. Bone grafts of two different types were used to cover the segment: chips of bone with large particle size and ground bone with a smaller particle size. The prosthetic segment was bridged by bone at 3 and 6 months in all cases irrespective of the structure of the transplant. In animals sacrificed at 6 months bone ingrowth occurred, with a marked difference between specimens with the two different grafts. In the ground bone specimens ingrowth occurred over the total surface area, and bone penetrated deep into the composite. With the chip grafts ingrowth was more irregular occurring only in some areas and it was always superficial. The difference is believed to be due to the improved contact between the fiber metal surface and the transplant. The lesser bulk of the ground transplant is advantageous when the soft tissue cover of the bone is thin.     

Tissue-engineered vascularized bone grafts: basic science and clinical relevance to trauma and reconstructive microsurgery

Bone grafts are an important part of orthopaedic surgeon's armamentarium. Despite well-established bone-grafting techniques, large bone defects still represent a challenge. Efforts have therefore been made to develop osteoconductive, osteoinductive, and osteogenic bone-replacement systems. The long-term clinical goal in bone tissue engineering is to reconstruct bony tissue in an anatomically functional three-dimensional morphology. Current bone tissue engineering strategies take into account that bone is known for its ability to regenerate following injury, and for its intrinsic capability to re-establish a complex hierarchical structure during regeneration. Although the tissue engineering of bone for the reconstruction of small to moderate sized bone defects technically feasible, the reconstruction of large defects remains a daunting challenge. The essential steps towards optimized clinical application of tissue-engineered bone are dependent upon recent advances in the area of neovascularization of the engineered construct. Despite these recent advances, however, a gap from bench to bedside remains; this may ultimately be bridged by a closer collaboration between basic scientists and reconstructive surgeons. The aim of this review is to introduce the basic principles of tissue engineering of bone, outline the relevant bone physiology, and discuss the recent concepts for the induction of vascularization in engineered bone tissue.     

Immediate mandibular reconstruction: human experience with autogenous frozen mandibular grafts

Autogenous mandibular grafts that have been cryosurgically devitalized and reimplanted have been studied in the canine. Initial investigations have lent impetus to further application in human cancer surgery. Six patients underwent immediate autogenous mandibular graft reconstruction. All failed because of intraoral complications and decreased vascularization in the midportion of the bone graft. None evidenced recurrence of tumor in bone, although two patient's grafts failed on the basis of tumor recurrence in the adjacent soft tissue.     

De novo generation of axially vascularized tissue in a large animal model

De novo generation of axially vascularized tissue with clinically relevant dimensions in a large animal model and implementation of clinically established imaging modalities for in vivo evaluation of vascularization. To be used for reconstruction of tissue defects, engineered grafts need to be axially vascularized to enable transplantation without graft loss due to hypoxia. Limitations to dimensions in small animal models had not yet been overcome, which is necessary to yield clinical relevance. Anatomical studies of groin and axillary regions in eight merino sheep were followed by microsurgical creation of an arteriovenous loop (AV-loop), embedded in an isolation chamber filled with fibrin matrix. Constructs were implanted in the groin of six sheep for up to 6 weeks. Course of vascularization in de novo forming tissue was assessed by sequential computed tomography angiography (CTA) and magnetic resonance angiography (MRA) in vivo, as well as by postexplantational micro-computed tomography and histology. A vascular axis was constantly found epifascially at the medial aspect of all sheep's thighs, which was used for AV-loop creation. Patency of AV-loop could be visualized by CTA and MRA scans during 1-6 weeks. Complex 3D-vessel-reconstruction revealed increasing axial vascularization of the fibrin matrix and growing connective tissue within the isolation chamber, which was confirmed by micro-computed tomography and histology postexplantation. De novo formation of axially vascularized tissue was demonstrated for the first time ever in a large animal model, paving the way for the first application of tissue engineering vascularized grafts with clinically relevant dimensions.     

Neo-osseous flaps using demineralized allogeneic bone in a rat model

Surgical reconstruction with revascularized bone grafts can be compromised by donor tissue limitations and may be refined by prefabrication of compound neoflaps using bone substitutes. The principal suitability of demineralized allogeneic bone (DALB) slabs in fabricating neo-osseous flaps based on the inferior epigastric vascular system was studied and compared with neoflaps with autologous bone (AUB). In 45 rats, the histological pattern of bone formation in response to angiogenesis induced by vessel implantation was assessed, and characteristics of vascularization of the neoflap were studied microangiographically at 2, 4, 6, and 8 weeks. Histological techniques included decalcified and nondecalcified sections, as well as intravital polyfluorochrome labeling. Blood flow of the neoflap was also assessed quantitatively using 15-microm microspheres labeled with technetium 99-methylene diphosphate (99-MDP) 8 weeks after flap fabrication. Although the DALB neoflaps showed consistent bone formation and neovascularization, the bone regeneration process was delayed distinctly in comparison with AUB. Microangiographically, however, no differences between the two types of grafts became apparent during all time periods tested. Furthermore, the radioactivity of the DALB neoflap, which means bone blood flow per dry weight, was significantly higher than in AUB grafts and even more than that of intact iliac bone (p = 0.001). The exact meaning of elevated blood flow in DALB and similar degrees of vascularization corresponding to native AUB grafts remains to be determined, but may be a sign of ongoing bone formation resulting in a suitable DALB-containing neo-osseous flap in the long term. The authors findings support that allogeneic bone could be a potential substitute for AUB in creating a prefabricated neo-osseous flap.     

Accelerated endothelialization of interpositional 1-mm vascular grafts

BACKGROUND: There is an increased need for alternative, synthetic, small-diameter vascular grafts due to a growing segment of the population who suffer from ischemic heart disease and lack suitable autologous vein grafts for use in coronary artery bypass grafting (CABG). We hypothesized that a cell-mediated extracellular matrix (ECM) modification of ePTFE would stimulate increased vascularization within the graft and thus promote lumenal endothelialization in a 1-mm rat abdomenal aortic implant model. METHODS AND RESULTS: Expanded polytetrafluoroethylene (ePTFE) vascular grafts (1 mm i.d.) were modified on the ablumenal surface with ECM deposited by the HaCaT or II-4 cell lines and implanted intrapositionally into the descending aorta of rats. Five weeks after implantation, all samples were patent and examination of the grafts demonstrated that the ECM modified samples exhibited extensive ablumenal vascularization and tissue incorporation compared to nonmodified samples. Also, ECM modified grafts had a cellular lining, while the nonmodified grafts were void of a cellular lining except for a limited pannus ingrowth. CONCLUSION: HaCaT and II-4 cell ECM modifications of ePTFE increase new blood vessel growth in association with the graft, and the II-4 cell modification results in formation of an endothelial monlayer on the lumenal surface of the graft.