显微外科技术修复肌腱的实验研究 (四)切除纤维鞘管及腱纽对肌腱愈合的影响

36只新西兰大白兔双前爪腱鞘区肌腱,在切除纤维鞘管及腱纽的基础上切断,采用显微外科技术修复。从组织学方面研究其愈合方式。结果显示修复肌腱均发生粘连,且随术后时间延长,逐渐加重。肌腱缝合处术后7天内以血细胞及纤维素充填,术后7天及以后,来源于腱外膜的纤维母细胞增生明显,向腱端延伸。术后21天出现胶原纤维。28天,腱缝合处结缔组织有重建倾向。腱端组织无明显反应。本实验结果表明,肌腱以外源形式愈合,这与腱鞘区肌腱营养系统破坏有关。     

表皮生长因子复合可降解胶原膜防止鸡鞘管区肌腱粘连的实验研究

目的观察和探索表皮生长因子（epidermal growth factor，EGF）复合胶原膜在预防鞘管区肌腱粘连、促进肌腱内源性愈合中的作用。方法将30只10个月龄的雄性leghorn鸡随机分为A、B、C三组，每组10只。将鸡的左侧第三趾造成挤压撕脱伤模型，用改良Kessler法进行缝接。A组：肌腱缝合口包裹EGF复合胶原膜；B组：缝合口包裹单纯的胶原膜；C组：缝合口不做任何处理。4周后取材，右侧第三趾为对照组。对标本进行肉眼观察、生物力学测定、光镜和电镜等观察。结果A组肌腱粘连程度较轻，肌腱缝合段内的胶原纤维数量多，以粗大的Ⅰ型胶原为主，成纤维细胞数量少，腱细胞成熟。B组肌腱粘连程度较轻，但肌腱缝合段内的胶原纤维数量少，排列稀疏，以纤细的Ⅲ型胶原为主。C组肌腱与周围组织粘连较严重，肌腱缝合段内的胶原纤维数量较多，排列紊乱，Ⅰ、Ⅲ型胶原交错排列。结论1.EGF复合胶原膜能起到屏障肌腱外源性愈合、防止肌腱粘连的作用，但同时也明显减慢了肌腱的愈合速度。2、EGF复合可降解胶原膜，同时也加快了肌腱的内源性愈合速度，起到了较理想的防止粘连的效果。     

肌腱粘连预防的研究进展

肌腱损伤的修复由肌腱外愈合和内愈合共同完成。肌腱外愈合时，腱周组织的成纤维细胞随增生的毛细血管长入肌腱断端，伴随修复形成疲痕粘连。肌腱粘连的预防需要调控细胞的增殖，抑制腱周组织细胞的增生；建立肌腱和周围组织间的屏障，恢复鞘内肌腱的滑液营养，促进肌腱的内愈合；改进缝合方法；术后早期活动。     

大隐静脉修复鞘管防止屈肌腱粘连的实验研究与临床应用

目的：观察大隐静脉修复鞘管防止屈肌腱粘连的效果和临床应用的可行性。方法：将３０只鸡的双足中趾造成相同的肌腱和腱鞘损伤，一侧用大隐静脉修复鞘管为实验趾；一侧不修复鞘管直接缝合切口为对照趾。随机分组，分别在术后１、２、４、６和１２周进行双侧趾的屈曲功能、大体解剖和光镜观察。结果：实验趾的屈曲功能明显优于对照趾。大体解剖见用大隐静脉修复的鞘管近似正常鞘管结构，可完全阻止肌腱粘连，不影响肌腱愈合。对照趾肌腱与周围组织粘连紧密，影响肌腱滑动。光镜下见移植的静脉外膜与周围组织愈合好，内膜光滑与肌腱无粘连。临床应用２１例３３指，平均随访１８个月，优良率达９０％以上。结论：大隐静脉修复鞘管是防止屈肌腱粘连较有效的方法。     

带蒂肌腱移植修复鞘内腱缺损的实验研究

目的：观察带蒂移植腱在鞘内的愈合情况,为临床应用提供依据。方法：９８只来亨鸡按左、右足分为实验组和对照组,通过大体、光镜、电镜观察移植腱愈合过程中的大体形态、细胞形态及组织学变化,测定移植腱在不同愈合时期的羟脯氨基酸含量及吻合口部抗张强度。结果：实验组腱内、外膜细胞增殖早、且活跃,内源性愈合倾向明显,粘连轻而局限,术后早中期羟脯氨酸含量及吻合口部抗张强度均优于对照组。结论：移植腱在鞘内可从滑液中获取营养而成活并保持一定的细胞增殖能力,但单纯依靠滑液营养难以满足全部内源性愈合过程,带蒂腱移植可改善移植腱营养状态,减少粘连,有利于术后早期功能锻炼及肌腱功能恢复     

鸡屈趾肌腱鞘内损伤修复中转化生长因子-β1的表达

目的了解鸡屈趾肌腱鞘内损伤愈合过程中转化生长因子-β1(TGF-β1)表达的变化。方法42只来亨鸡随机平均分成6个实验组和1个正常肌腱对照组,切断右第3趾屈趾长肌腱后缝合并重新覆以腱鞘。分别于术后1、3、7、14、28、56d切取实验组及对照组标本,用逆转录聚合酶链反应(RT-PCR)和免疫组化方法测定腱鞘、腱外膜和腱实质中TGF-β1的表达,将实验组分别与正常对照组比较。结果TGF-β1在正常对照组肌腱仅出现低水平表达,而在实验组肌腱的各个时间点均明显上调,术后第7天最高。免疫组化染色显示腱鞘、腱外膜的TGF-β1蛋白表达比腱实质的明显。结论TGF-β1参与鸡屈趾肌腱损伤修复的愈合过程,适当调控其在不同部位的表达程度可望既促进肌腱愈合又防止肌腱粘连。     

聚乳酸凝胶预防肌腱粘连的实验研究

目的：聚乳酸凝胶(PLA—G)置于肌腱吻合部，观察防粘连作用。方法：将16只兔左后肢比目鱼肌腱切断一半后吻合，鞘内涂PLA—G为实验组；兔的右后肢同样操作，不涂PLA—G为对照组。术后2、4、6、10周随机取动物每组2只共4只的手术段肌腱和鞘管做大体、组织及超微观察。结果：(1)大体：实验组2周时PLA—G隔离好无粘连；4周开始降解，肌腱滑动良好；6、10周瘢痕小，吻合口生长及滑动良好。对照组瘢痕大、广泛粘连；(2)组织：实验组纤维排列由不规则向规则变化，成纤维细胞少。对照组腱与鞘无分界，成纤维细胞增生、胶原分泌活跃；(3)超微：实验组成纤维细胞粗面内质网较对照组明显稀疏、胶原纤维少。结论：PLA—G具防粘连作用，不影响肌腱愈合。     

滑液营养条件下肌腱愈合的实验研究

为了探索单一滑液营养条件下肌腱的愈合能力，选用５０只新西兰大白兔，采用随机、单盲、自身对照实验设计。取动物前爪的屈趾深肌腱一段从中分切断，应用显微外科技术吻合后，置入该动物膝关节腔内。通过大体、扫描电镜、光镜、透射电镜观察及羟脯氨酸、氨基己糖测定，动态观察术后第１，２，３，４，６周实验样品吻合口及其附近细胞的形态、组织学改变以及胶原和基质代谢变化。结果发现，单一滑液营养条件下不仅能提供肌腱成活的全部营养，而且能满足肌腱愈合的需要。肌腱愈合由腱段本身的腱外膜细胞和腱内细胞共同完成。证明吻合后的肌腱在单一滑液营养条件下能够愈合。     

透明质酸钠防治肌腱粘连的实验研究

作者用透明质酸钠进行了兔肌腱愈合与术后粘连影响的实验研究，大体观察、光镜观察、成纤细胞胞计数、胶原纤维含量测定以及抗张强度测定结果证实透明质酸钠能减少术后腱周成纤维细胞、胶原纤维的含量，减少腿周瘢痕纤维的形成，透明质酸钠，对于防止术后肌腱粘连，改善肌腱滑移功能有良好效果，且不影响肌腱愈合。     

防止屈指肌腱损伤后粘连的研究进展

屈指肌腱损伤是手外科常见的病症之一,由于其修复后常有粘连形成,使手指的功能受到影响,因此,半个多世纪以来,国内外学者进行了大量的临床与动物实验研究,力求找到一种既能保证肌腱愈合,又无粘连形成的方法,木文对此作一简要综述.一、腱鞘的作用早在1916年,Mayer即指出恢复腱与鞘的正常解剖关系可减少肌腱术后的粘连,主张尽可能保留或修复腱鞘.他报告了在腱转移手术中利用已瘫痪腱的鞘的方法.Peacock则用新鲜尸体完整的腱与鞘的复合体修复病人严重损伤的屈指肌腱,获得满意的效果.但由于受到腱愈合必须有周围血管及细胞长入观点的影响,完整的腱鞘被认为有碍于腱的愈合,加重疤痕性粘连,因而许多人主张尽可能     

Flexor tendon wound healing in vitro: the effect of lactate on tendon cell proliferation and collagen production

Flexor tendon repair in zone II is complicated by adhesions to the surrounding fibro-osseous sheath. Lactate is an early mediator of wound healing known to play an important role in stimulation of collagen production after cellular injury. Little attention has been paid to the role of lactate in flexor tendon wound healing. In this study tendon and tendon sheath were excised from rabbit forepaws. We examined proliferation of tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes; collagen production by each of these 3 cell types; and effects of lactate on cell proliferation and collagen production. Three cell lines, tendon sheath, epitenon, and endotenon, were isolated and cultured. Tendon sheath fibroblasts showed the greatest proliferation. All 3 cell lines produced collagen I, II, and III. Lactate significantly increased collagen production by all 3 cell lines. We show that cells of the tendon sheath, epitenon, and endotenon produce collagen in vitro. Modulation of lactate levels may provide a means to modulate collagen production.     

Participation of human superficialis flexor tendon segments in repair in vitro

We analyzed the healing capability of the human superficialis (sublimus) flexor tendon in response to injury in vitro by performing a morphologic study of tendon repair. Tendon segments were cultured in cell-free medium for 2, 4, or 8 weeks, and the cut-end repair sites were compared with those in fresh control segments on light and electron microscopy. Tendon encapsulation had occurred at 2 weeks, by the proliferation and migration of elongated fibroblasts from the epitenon and from collagen bundles of the endotenon to cover the surface of the cut tendon ends. As migrating epitenon cells approached the cut margin, they consistently shifted from a parallel to a circumferential orientation with respect to the tendon axis. By 4 weeks, the encapsulating surface cells had lost their membranous ruffles and had become flattened. Within the capsule, phagocytic cells engulfed collagen debris, and fibroblasts containing extensive rough endoplasmic reticulum profiles and secretory vesicles participated in protein synthesis. The surface of the capsule was smooth and, at 8 weeks, was almost indistinguishable from the epitenon surface of uncultured controls. The capsule now had a collagen-synthesizing layer of fibroblasts superimposed on layers of maturing collagen bundles that separated resting-phase fibroblasts. These findings demonstrate that segments of human superficialis flexor tendons participate in an intrinsic repair response without contributions from extrinsic cells.     

Genetic expression for type I procollagen in the early stages of flexor tendon healing.

To determine the precise mechanism by which contact tendon healing occurs at the cellular level, the production of pro alpha (I) collagen messenger RNA (mRNA) produced by fibroblasts of healing intrasynovial flexor tendons was determined by an in situ hybridization technique. The repair site and the proximal and distal tendon stumps of repaired tendons treated with early controlled passive mobilization were fixed and buffered in formalin, 3, 7, 10, and 17 days after repair. A complimentary DNA (cDNA) probe corresponding to alpha (I) procollagen mRNA was labeled with [32P]d-CTP. After hybridization, autoradiography, and staining of the sections, the level of procollagen mRNA was assessed by microscopic examination. Rising levels of procollagen mRNA, indicating progressively increasing levels of synthetic collagen activity, were detected in the healing tendons through 10 days. A moderate decrease in procollagen mRNA was seen at 17 days. Genetic expression for procollagen mRNA was localized specifically to the epitenon cells on the tendon surface overlying the repair site and to cells in the gap between the tendon stumps. No detectable expression was noted in endotenon fibroblasts. The finding of high levels of expression for procollagen type I mRNA in the surface layer of healing tendons demonstrates that cells intrinsic to tendon epitenon contribute the greatest quantity of native tendon collagen to the repair site during these important early intervals after tendon suture.     

Tendon healing in vivo. An experimental model

Flexor tendon segments were incubated in a diffusion chamber in the subcutis of rabbits. Tendons incubated up to 6 weeks in the diffusion chamber showed proliferating and migrating cells from the epitenon cell layer as well as viable endotenon cells. Explants frozen in liquid nitrogen prior to incubation showed no signs of extrinsic cell contamination and remained non-viable indicating that no cell penetration occurred through the Millipore filter and that cell division seen in non-frozen and incubated tendons was an expression of intrinsic cellular proliferative capacity of the tendon. In tendon segments incubated in chambers for three weeks, collagen synthesis was reduced by 50% and the rate of cell proliferation measured as 3H-thymidine incorporation, was 15 times that of native tendons. Frozen and incubated tendons showed only traces of remaining matrix synthesis or cell proliferation. With this experimental model we have histologically and biochemically shown that tendons may survive and heal while the nutrition exclusively could be based on diffusion and the tendons have an intrinsic capacity of healing. The described model enables further studies on tendon healing and its regulation.     

Flexor tendon healing and restoration of the gliding surface. An ultrastructural study in dogs

Healing canine flexor tendons were treated with either total immobilization and were studied by light, scanning, and transmission electron microscopy at ten, twenty-one , and forty-two days. The immobilized tendons healed by ingrowth of connective tissue from the digital sheath and cellular proliferation of the endotenon. The ingrowth of reparative tissue from the digital sheath overwhelmed the epitenon response. At the ultrastructural level, collagen resorption was prominent whereas protein synthesis was limited. This was observed at all study-intervals. In contrast, the mobilized tendons healed by proliferation and migration of cells from the epitenon. Ingrowth of reparative tissue from the tendon sheath was notably lacking in this group. The epitenon cells exhibited greater cellular activity and collagen production at each interval compared with cells of the immobilized repairs.     

Collagen synthesis during primate flexor tendon repair in vitro

The concept that flexor tendons have the intrinsic capacity to participate actively in the repair process following laceration has been developed in recent years as the result of experimental studies from numerous laboratories. However, the role of the outer/epitenon and inner/endotenon cell populations with regard to protein synthesis is still controversial. The purpose of this study was to investigate the respective participation of these tendon fibroblast populations in the synthesis of the collagen matrix during in vitro repair of the flexor tendon from nonhuman primates, utilizing immunohistochemical techniques and a Type I procollagen antibody. Zone II profundus flexor tendon segments were obtained from young adult Macaca nemestrina monkeys. One centimeter segments were cultured, either with or without a transverse laceration across 90% of the midsection of the tendon segment. Frozen sections of the cultured tendon segments were reacted with the (mouse monoclonal) Type I procollagen antisera. At all times of culture of the nonlacerated tendon segment, only a few of the epitenon cells along the surface of the tendon and distant from the cut end stained positively for active collagen synthesis. In the lacerated segments, and as early as 9 days of culture and repair, the procollagen reaction product was starting to appear in those cells of the outer epitenon cell layer. These studies support the concept that the inner fibroblasts do actively participate in collagen production. However, it now also appears that a significant degree of collagen synthesis during tendon repair resides in the outer/epitenon layer of cells enveloping the tendon segment, and that the repair response of the flexor tendon in vitro is proportional to the degree of injury.     

Effects of chitosan on cell proliferation and collagen production of tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes

Flexor tendon repair in zone II is often complicatedby adhesions to the surrounding fibro-osseoustendon sheath. Adhesion between the tendon andtendon sheath will impair the gliding function oftendons and result in marked impairment of handfunction. Experimental strategies to decrease adhesionformation, with the long-term goal of improving clinicaloutcome after tendon injury and repair, have been thesubject of numerous studies over the past 20 years.Biochemical agents such as antihistamines, s…     

Fibronectin in the tendon-synovial complex: Quantitation In vivo and In vitro by elisa and relative mRNA levels by polymerase chain reaction and northern blot

An enzyme-linked immunosorbent assay was used to quantitate fibronectin (Fn) levels in the outer synovia (epitenon) and internal fibrous portion (endotenon) of chicken flexor tendon and sheath. Primary cell cultures from these tissues and their secretions also were assayed for Fn levels. The polymerase chain reaction (PCR) was used to determine relative steady-state levels of Fn mRNA in primary cultures of synovial and internal fibroblasts from chicken tendon, and Northern blot analysis was performed to verify relative levels of the Fn message. The epitenon contained 3.8-fold more Fn than did the endotenon, and the sheath synovium contained 21-fold more Fn than did the internal fibrous portion of sheath. Cells cultured from the epitenon produced 9.3 and 13-fold more cell-associated and secreted Fn, respectively, than did cultured endotenon fibroblasts. Sheath synovial cells produced 17 and 3.2-fold more cell-associated and secreted Fn, respectively, than did sheath internal fibroblasts. Levels of Fn mRNA, as measured by PCR and Northern blot, were 1.6 and 1.8-fold greater, respectively, in tendon synovial cells compared with tendon internal fibroblasts. The biologic reason for increased Fn in tendon synovium is not known. We theorize that Fn may stabilize tendon synovium to shear stress and may play a role in the modulation of synovial rheology in the normal tendon. In the injured tendon, Fn may be involved in the organization of collagen deposition or may act through association with growth factors to aid healing.     

Flexor tendon repair in vitro: a comparative histologic study of the rabbit, chicken, dog, and monkey

Flexor tendon healing in four different animal species was explored in a tissue culture system. Ninety percent transverse lacerations were made in 88 tendon segments obtained from rabbits, chickens, dogs, and monkeys. The tendons were removed from culture and studied by light and electron microscopy at intervals of 3, 6, 9, and 12 weeks. A characteristic sequence of repair including epitenon thickening, cellular differentiation, cell migration, and phagocytosis was seen in each of the repaired tendons. The endotenon cells of several animal tendons appeared to be synthesizing collagen. There was a consistent difference in the rate of healing between the four species. The rabbit tendons demonstrated nearly complete closure of the repair site by 12 weeks. A lesser response was seen in the chicken, followed by the dog and monkey. The differences in healing rate appeared to be due to the non-species-specific in vitro culture media. The in vitro flexor tendon culture system is particularly useful in studying the tendon repair responses of various species with the contributions of vascularity and synovial cells excluded.     

Intrinsic healing of the laceration site in human superficialis flexor tendons in vitro

The intrinsic capability of the human superficialis flexor tendon to heal a reapproximated laceration site in vitro was examined. Segments of lacerated and sutured human superficialis flexor tendons from zone II were cultured for two, four, or eight weeks in vitro and analyzed by use of light and electron microscopy. We noted a specific pattern of intrinsic healing at the repair site during the incubation period. After two weeks, the cells of the epitenon from both tendon stumps proliferated and appeared to migrate into the repair site. Elongated cells of the thickened epitenon bridged the laceration and synthesized new collagen fibers. By eight weeks, a smooth contiguous tendon surface was restored and the internal collagen bundles were brought into intimate contact. The fibroblasts bridging the laceration, both in the epitenon and centrally in the repair site, were oriented perpendicular to the cut surfaces. The cells in the repair site were active in protein synthesis, and new collagen fibers were present. No cellular necrosis was observed in the internal repair site. Our results indicate that human superficialis tendons possess an intrinsic capacity to heal with diffusion and without dependence on extratendinous cells or on adhesions.