肌腱组织工程研究新进展

组织工程化肌腱原理是将分离的高浓度有活力的肌腱种子细胞种植于生物相容性好，可生物降解的细胞载体中，体外培养后植到缺损部位，形成新的、自身的，具有功能的肌腱组织，最终达到生物学意义上的完全修复。现就肌腱组织工程中肌腱细胞的特征、种子细胞来源、支架材料的制备、临床应用和面临的问题和展望等做一综述。     

构建组织工程肌腱膜与腹壁缺损修复

<正>随着外科技术手段的进步,腹壁缺损的治疗已由传统的单纯直接缝合修补,转变为以材料学为基础、利用各种补片进行的无张力修复。传统的手术治疗主要是将腹壁缺损周围的肌肉或韧带强行缝合,术后病人疼痛明显,恢复时间长,复     

组织工程肌腱修复陈旧性跟腱断裂伴缺损的疗效观察

目的探讨应用组织工程肌腱修复陈旧性跟腱断裂伴缺损的手术方法及临床效果。方法1999年8月～2002年6月,采用同种异体肌腱来源的成纤维细胞,以5×106/ml细胞密度接种在医用碳纤维与聚羟基乙酸纤维制作的编织带上,体外培养5d后,修复跟腱缺损7例,缺损长度为5～7cm。术后踝跖屈外固定4～6周后开始功能锻炼。结果7例均获随访22～56个月,平均46.9个月。除1例术后伤口延迟愈合外,其余6例均期愈合。无全身及局部不良反应,无跟腱粘连再手术患者。按尹庆水疗效评定标准,优5例,良1例,可1例。结论组织工程肌腱修复跟腱缺损可获较好临床效果,是一种可选择的新治疗方法。     

自体肌腱细胞介导修复肌腱缺损的实验研究

目的　探讨以自体肌腱细胞为种子细胞的组织工程化肌腱体内形成。方法　取自体肌腱细胞经体外培养、扩增 ,与可吸收生物材料聚羟基乙酸 (polyglycolicacid ,PGA)混合培养形成细胞 生物材料复合物 ,将细胞 生物材料复合物移植于手术缺损的家猪趾浅屈肌腱处 ,并设单纯PGA组作为对照组。术后 6周取材 ,对标本进行大体观察、组织学和生物力学检测和评价再生组织。结果　实验组新生组织在大体、组织学和胶原排列等方面与正常肌腱相似 ,对照组新生组织细胞、胶原排列较为紊乱。生物力学显示其最大拉力、最大应力和弹性模量 ,实验组比对照组分别提高 3 6.1% (t =3 5 6、P <0 .0 5 )、2 7.4%(t =2 94、P <0 .0 5 )和 15 .0 % (t =2 62、P <0 .0 5 )。结论　应用组织工程技术以自体肌腱细胞可以修复肌腱缺损     

组织工程化肌腱研究进展

对组织工程化肌腱领域中目前研究的主要成果进行综述，着重阐述了肌腱细胞外基质替代物、肌腱细胞生物学性质及肌腱细胞与细胞外基质材料复合研究中的主要问题。认为，研制适于肌腱细胞生长、粘附和发挥功能的细胞外基质材料；建立生长、增殖可调控的肌腱细胞系；在模拟体内力学条件下，进行肌腱细胞三维培养，将是研究具有特定修复功能的组织工程化肌腱的重要问题。     

肌腱组织工程的研究进展

肌腱缺损一直是整形外科关注的重要问题之一，这是因为自体肌腱移植来源有限，异体肌腱移植存在免疫排斥反应，而很多替代物在肌腱修复应用中无法获得满意的远期疗效。随着组织工程技术的发展，组织工程化肌腱为修复肌腱缺损开辟了一条新途径。     

应用猪小肠黏膜下层与肌腱细胞构建组织工程支架修复大鼠腹壁缺损的实验研究

目的:应用猪小肠黏膜下层(small intestinal submucosa,SIS)与肌腱细胞构建组织工程支架,研究其在修复腹壁缺损时的生物力学特性。方法:制作SD大鼠腹壁缺损模型,应用所构建的组织工程支架修补缺损,术后4周取样进行大体观察,检测组织学及力学性能。结果:组织工程支架修补术后的SD大鼠无腹部裂开及疝发生,支架与腹腔内脏器有轻微粘连;HE及Masson染色发现支架与肌肉组织交界区有显著新生血管出现及肌肉组织长入。力学性能检测显示组织工程支架的力学强度显著大于SD大鼠腹壁肌肉强度。结论:构建组织工程支架可有效修补大鼠的腹壁缺损。     

组织工程神经修复大鼠坐骨神经缺损的研究

目的观察组织工程神经修复SD大鼠1．5cm长坐骨神经缺损的效果。方法用甘油处理10只SD大鼠2．0cm长坐骨神经，制备成同种异体脱细胞基质，备用。取SD乳鼠10只，分离坐骨神经，去神经外膜后，剪成小碎块，在DMEM中培养3周，扩增后的细胞鉴定、备用。3个月龄的SD雌性大鼠40只，单纯随机分成4个神经移植组（A、B、C、D），每组10只。A组：用扩增的雪旺细胞加同种异体脱细胞基质桥接，即组织工程化人工神经组。B组：用元雪旺细胞但具有内部支架结构的同种异体脱细胞基质桥接。C组：自体神经移植组。D组；空白对照组。术后12周，进行一般情况、小腿三头肌湿重、再生神经的组织学观察。结果完成对40只大鼠（每组10只）的实验评估。所有大鼠伤口瑚愈合，元死亡。A、B、C组大鼠足部元溃疡形成，D组7只足部有溃疡形成，所有组实验侧小腿三头肌较健侧萎缩，但以D组最明显。小腿三头肌湿重、神经电生理监测A组、C组差异无统计学意义（P〉O．05），A、C组与B、D组差异有统计学意义（P〈O．05），B组与D组差异有统计学意义（P〈0．05）。A组和C组的胫前肌中均能诱发出波幅明显的神经肌肉复合动作电位（CMAP），B组、D组中则仅录到波幅很低的CMAP。A组和C组再生轴突已通过移植段神经全长，远端肌肉轻度萎缩。B组部分通过移植段神经；D组不能通过移植段神经，6例形成神经瘤。结论组织工程人工神经可用来修复大鼠长段神经缺损。     

Tissue‐engineered tendon constructs for rotator cuff repair in sheep

Current rotator cuff repair commonly involves the use of single or double row suture techniques, and despite successful outcomes, failure rates continue to range from 20 to 95%. Failure to regenerate native biomechanical properties at the enthesis is thought to contribute to failure rates. Thus, the need for technologies that improve structural healing of the enthesis after rotator cuff repair is imperative. To address this issue, our lab has previously demonstrated enthesis regeneration using a tissue‐engineered graft approach in a sheep anterior cruciate ligament (ACL) repair model. We hypothesized that our tissue‐engineered graft designed for ACL repair also will be effective in rotator cuff repair. The goal of this study was to test the efficacy of our Engineered Tissue Graft for Rotator Cuff (ETG‐RC) in a rotator cuff tear model in sheep and compare this novel graft technology to the commonly used double row suture repair technique. Following a 6‐month recovery, the grafted and contralateral shoulders were removed, imaged using X‐ray, and tested biomechanically. Additionally, the infraspinatus muscle, myotendinous junction, enthesis, and humeral head were preserved for histological analysis of muscle, tendon, and enthesis structure. Our results showed that our ETC‐RCs reached 31% of the native tendon tangent modulus, which was a modest, non‐significant, 11% increase over that of the suture‐only repairs. However, the histological analysis showed the regeneration of a native‐like enthesis in the ETG‐RC‐repaired animals. This advanced structural healing may improve over longer times and may diminish recurrence rates of rotator cuff tears and lead to better clinical outcomes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:289–299, 2018.     

肌腱和韧带组织工程支架材料研究进展

肌腱和韧带损伤的修复治疗方法包括自体移植、异体移植和合成假体替代物的植入.组织工程肌腱和韧带作为生物性替代治疗具有其独特的优点,支架材料作为绀织工程的重要部分对肌腱和韧带的成功构建起着重要的作用.本文就近年来用于肌腱和韧带组织工程的天然和人工合成的支架材料进行综述.     

藻酸钙凝胶-成骨细胞-骨粉构建工程化骨修复兔颅骨缺损的实验研究

目的探讨藻酸钙凝胶、成骨细胞、骨粉复合构建的可塑形组织工程骨修补兔颅骨缺损后的形态学变化和成骨效果.方法28只日本大耳白兔,随机分为A(n=20)、B(n=8)两组,手术在兔颅顶骨矢状缝两侧分别各建立一个直径1cm的圆形全层缺损,用两种方法藻酸钙凝胶、成骨细胞、骨粉和藻酸钙凝胶、骨粉分别构建组成可塑形的组织工程骨复合材料,分别填补修复A组兔颅骨左右两侧的缺损,B组为空白对照组,通过大体、组织学、X线观察材料的形态变化、成骨情况,并对X线片和组织学切片进行评分.结果材料植入后,局部未见红肿、积液、渗出等异常反应.①藻酸钙凝胶-成骨细胞-骨粉组修补后12周颅骨缺损基本被硬性组织所修复,镜下见修复材料大多被骨组织替代,骨粉基本被吸收,有块状凝胶残留其中,组织学评分为(5.50±1.00).X线片见兔颅骨缺损处有高密度骨痂影存在,布满整个缺损区,X线片评分为(3.25±0.95).②藻酸钙凝胶-骨粉组修补后12周部分颅骨缺损被硬性组织所修复,镜下见修复材料部分转变成骨组织,骨粉基本被吸收,有凝胶残留其中,组织学评分为(3.25±1.50).X线片见兔颅骨缺损处有高密度骨痂影存在,主要分布在缺损区的边缘部位,X线片评分(2.25±0.25).③空白对照组骨缺损主要被膜样纤维组织修复,在紧邻骨缺损边缘处有硬性组织形成,镜下见修复组织边缘有骨组织存在,中央大部为膜状致密纤维组织,组织学评分为(1.50±0.50),X线片仅见靠近骨缺损边缘的部位存在致密骨痂影,X线片评分为(1.00±0.57).结论藻酸钙凝胶、成骨细胞、骨粉构建的可塑形组织工程骨可根据颅骨缺损的形态进行塑形填补,在体内有良好的成骨能力,可达到对兔颅骨缺损的骨性修复,但部分藻酸盐凝胶吸收缓慢,手术后12周仍不能满意吸收.     

封闭负压引流结合掌背动脉穿支皮瓣、掌长肌腱移植修复手背复合组织缺损

目的探讨封闭负压引流(VSD)技术结合逆行掌背动脉穿支岛状皮瓣、掌长肌腱移植修复手背复合组织缺损的临床疗效。方法应用VSD技术结合逆行掌背动脉穿支岛状皮瓣、掌长肌腱移植修复11例手背复合组织缺损患者。组织缺损面积1.8 cm×2.0 cm~3.0 cm×6.0 cm,切取皮瓣面积2.0 cm×2.5 cm~3.5 cm×7.0 cm。结果切取第2掌背动脉穿支岛状皮瓣7例,第3掌背动脉穿支岛状皮瓣2例,第4掌背动脉皮瓣皮支岛状皮瓣2例。皮瓣全部成活,无皮瓣感染病例。患者均获得随访,时间7~25个月。伤指关节功能、皮瓣外形较满意。末次随访时按手指总主动活动度(TAM)标准评估伤指功能:优6例,良3例,中2例。结论 VSD可有效控制创面炎症,结合逆行掌背动脉穿支岛状皮瓣、掌长肌腱移植修复手背复合组织缺损,外形及功能恢复满意、血运可靠,是一种较好的修复方法。     

组织工程化人工骨血管化研究

目的 比较三种促进组织工程化人工骨体内血管化方法的效果及研究血管化与成骨的相关关系。方法 将HA/β-TCP与PDLLA形成复合支架材料，再复合Ⅰ型胶原、rhBMP-2，与成骨细胞联合培养，仿生制备成组织工程化人工骨，采用包裹带血管蒂筋膜，复合血管内皮细胞或两者联合的促血管化手段。将人工骨修复兔桡骨干骨膜-骨完全缺损，手术后4、8、12周，观察移植物组织学，体视学方法观察血管化，成骨作用及其两者的关系。结果 3种方法均有促血管作用。其优劣依次为；材料包裹带血管蒂筋膜及复合血管内皮细胞大于材料单纯复合血管内皮细胞，后者又大于材料单纯包裹带血管蒂筋膜，术后4周为快速血管化阶段。4-8周间血管化有平缓发展，12周完全血管化，血管化与新骨生成数量成正相关。结论 促血管化手术对组织工程化人工骨移植修复骨缺损的效果起重要促进作用。     

Tendon-derived stem cells (TDSCs) promote tendon repair in a rat patellar tendon window defect model

Injured tendons heal slowly and often result in the formation of mechanically and functionally inferior fibrotic scar tissue or fibrous adhesions. This study investigated the use of tendon-derived stem cells (TDSCs) for tendon repair in a rat patellar tendon window defect model. Fibrin glue constructs with or without GFP-TDSCs were transplanted into the window defect. The patellar tendons were harvested for histology, ex vivo fluorescent imaging and biomechanical test at various time points up to week 4. Our results showed that TDSCs significantly enhanced tendon healing as indicated by the increase in collagen production as shown by hematolxylin stain-ability of the tissue, improvement of cell alignment, collagen fiber alignment and collagen birefringence typical of tendon. The labeled cells were observed at weeks 1 and 2 and became almost undetectable at week 4. Both the ultimate stress and Young's modulus were significantly higher in the TDSCs group compared to those in the fibrin glue group at week 4. In conclusion, TDSCs promoted earlier and better repair in a rat patellar tendon window defect model.     

Increase in cell migration and angiogenesis in a composite silk scaffold for tissue‐engineered ligaments

The purpose of this study was to evaluate the biocompatibility of silk and collagen‐hyaluronan (HA) in vitro by assessing anterior cruciate ligament (ACL) cell and T‐lymphocyte cultures on scaffolds. The use of composite scaffolds as artificial ligaments in ACL reconstruction and their effects on angiogenesis were evaluated in vivo. The silk scaffold was knitted by hand and dry coated with collagen‐HA, whereas the composite silk scaffold was made by covering a silk scaffold with a lyophilized collagen‐HA substrate. The initial attachment and proliferation of human ACL cells on the composite silk scaffold was superior to the attachment and proliferation observed on the silk scaffold. The immune response was higher in both scaffolds after 72 h (p < 0.05) compared with the control culture condition without scaffolding, as assessed by T‐lymphocyte cultures in vitro. There was no significant difference in the immune response in vitro between the silk and composite silk scaffolds. Silk and composite silk scaffolds were implanted as artificial ligaments in ACLs removed from the knees of dogs, and they were harvested 6 weeks after implantation. On gross examination, the onset of an inflammatory tissue reaction, such as synovitis, was seen in both the silk scaffold and the composite silk scaffold groups. An histological evaluation of the artificial ligament implants revealed the presence of monocytes in the silk composite scaffold and the absence of giant cells in all cases. MT staining in the composite silk scaffold‐grafted group showed granulation tissue consisting of fibroblasts, lymphocytes, monocytes, and collagen fibers. In addition, CD31 staining revealed the formation of new blood vessels. On the other hand, no reparative tissues, such as blood vessels, collagen, and cells, were observed in the silk scaffold‐grafted group. These results suggest that the lyophilized collagen‐HA substrate is biocompatible in vitro and enhances new blood vessel and cell migration in vivo. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 495–503, 2009     

组织工程骨联合外固定修复大鼠股骨节段性骨缺损

目的 观察以外固定器固定,骨髓间充质干细胞 (BMSCs) 联合双相磷酸钙(BCP)修复大鼠股骨节段性骨缺损的效果.方法 A组:BMSCs与BCP复合植入缺损区;B组:BCP植入缺损区;C组:空白组.定期摄X线片,术后12周取材.结果 A组随时间延长X线评分递增,12周时平均为4.17分,B组为1.18分,C组为1.08分,差异有统计学意义(P<0.05).组织学检查见A组缺损区有大量的新生骨生成,而B、C组无新生骨生成.A组的抗压刚度和扭转刚度分别为(8.09±2.42)N/mm、(1.89±0.72)Nmm/deg;B组为(1.75±0.90)N/mm、(0.40±0.21)Nmm/deg,差异有统计学意义(P<0.05).结论 组织工程骨联合外固定可以修复节段性骨缺损.

Abstract：

Objective To evaluate the efficacy of bone mesenchymal stem cells (BMSCs) combined with biphasic calcium phosphate (BCP) repair of segmental bone defect, which was stabilized with an adaptable external fixation system.Methods In group A, the femoral defect was filled with BCP combined with BMSCs; In group B, the femoral defect was filled with BCP, and in group C, defects were left empty. Animals were sacrificed 12 weeks post-operation.Results In group A, radiographic scores were average 4.17, significantly (P<0.05) greater than in group B (1.18) and group C (1.08). Histological evaluations displayed the bridging of the defect in group A, with remarkable new bone formation. In contrast, group B and group C showed no formation of new bone. The mechanical testing revealed that axial stiffness was (8.09±2.42) N/mm and torsional stiffness was (1.89±0.72) Nmm/deg in group A, and those in group B were (1.75±0.90) N/mm and (0.40±0.21) Nmm/deg respectively. There was significant difference in biomechanical tests between group A and group B (P<0.05).Conclusion External fixator combined with tissue engineered bone can repair segmental bone defect.     

Studies in flexor tendon reconstruction: biomolecular modulation of tendon repair and tissue engineering

The Andrew J. Weiland Medal is presented each year by the American Society for Surgery of the Hand and the American Foundation for Surgery of the Hand for a body of work related to hand surgery research. This essay, awarded the Weiland Medal in 2011, focuses on the clinical need for flexor tendon reconstruction and on investigations into flexor tendon biology. Reconstruction of the upper extremity is limited by 2 major problems after injury or degeneration of the flexor tendons. First, adhesions formed after flexor tendon repair can cause decreased postoperative range of motion and hand function. Second, tendon losses can result from trauma and degenerative diseases, necessitating additional tendon graft material. Tendon adhesions are even more prevalent after tendon grafting; therefore these 2 problems are interrelated and lead to considerable disability. The total costs in terms of disability and inability to return to work are enormous. In this essay, published work from the past 12 years in our basic science laboratory is summarized and presented with the common theme of using molecular techniques to understand the cellular process of flexor tendon wound healing and to create substances and materials to improve tendon repair and regeneration. These are efforts to address 2 interrelated and clinically relevant problems that all hand surgeons face in their practice.