Ya Fang Wu

Tendon Healing In Vivo: Gene Expression and Production of Multiple Growth Factors in Early Tendon Healing Period

PURPOSEnThe actions of growth factors during healing of injured flexor tendons are not well characterized, although information pertinent to some individual growth factors is available. We studied gene expression and protein production of a number of growth factors at several time points during the early healing period in a chicken model.nnnMETHODSnSeventy-four long toes of 37 white Leghorn chickens were used. The flexor digitorum profundus tendons of 60 toes were surgically repaired after complete transection and were harvested for analysis 3, 5, 7, 9, 14, and 21 days after surgery. The expression of 6 growth factors was studied at 4 time points after surgery with real-time quantitative polymerase chain reactions, and production and distribution of 3 growth factors at all 6 time points were studied by immunohistochemical staining with antibodies. Fourteen tendons that had no surgery served as day 0 controls. Tendon healing status was also assessed histologically.nnnRESULTSnThroughout the early tendon healing period, connective tissue growth factor (CTGF) and transforming growth factor beta (TGF-beta) showed high levels of gene expression. Levels of gene expression of vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1) were high or moderately high. Expression of the TGF-beta gene was upregulated after injury, whereas the basic fibroblast growth factor (bFGF) gene was downregulated at all postsurgical time points and expressed at the lowest levels among 6 growth factor genes 2 to 3 weeks after surgery. The platelet-derived growth factor B (PDGF-B) gene was also minimally expressed. Findings of immunohistochemistry corresponded to TGF-beta, bFGF, and IGF-1 gene expression.nnnCONCLUSIONSnIn this model, up to 3 weeks after surgery, gene expression and production of TGF-beta are high and are upregulated in this healing period. However, expression of the bFGF gene and protein is low and decreases in the healing tendon. The CTGF, VEGF, and IGF-1 genes are expressed at high or moderately high levels, but PDGF-B is minimally expressed.

Recent developments in flexor tendon repair techniques and factors influencing strength of the tendon repair

Over the last decade, both basic researchers and surgeons have sought to identify the most appropriate techniques to be applied in flexor tendon repairs. Recent developments in experimental tendon repairs and clinical outcomes of newer repair techniques have been reviewed in an attempt to comprehensively summarize the most critical mechanical factors affecting the performance of tendon repairs and the surgical factors influencing clinical outcomes. Among them, attention to annular pulleys, the purchase and tension of the core suture, and the direction and curvature of the path of tendon motion have been found to be determining factors in the results of tendon repair.

Effects of tension across the tendon repair site on tendon gap and ultimate strength.

PURPOSEnTendons repaired with varying amounts of tension result in different degrees of shortening of the tendon segment within core sutures, which may affect tensile strengths. We aimed to investigate the effects of tension across the repair site on gap formation forces and ultimate strength.nnnMETHODSnFifty-seven porcine flexor tendons were repaired with a 2-strand modified Kessler repair or a 4-strand cross-lock repair. For each type of repair, the tendons were divided into 3 groups: by 0%, 10%, or 20% shortening of tendon segment encompassed within core sutures. The repaired tendons had a single load-to-failure test in a materials testing machine. The forces required for initial and 2-mm gap formation and ultimate failure were recorded and statistically compared for each group.nnnRESULTSnWith either 2-strand or 4-strand repair, the tendons in the 10% tendon-segment shortening group withstood significantly higher initial and 2-mm gap formation forces than those in the group with no shortening, with average increases of 5 to 10 N after 10% shortening. Increasing the tendon-segment shortening to 20% produced a slight increase in the initial and 2-mm gap forces compared with those with 10% shortening, which was statistically significant only in the tendons with the 4-strand repair. The ultimate strengths were not significantly different among the tendons with either 2-strand or 4-strand repair of any degrees of shortening.nnnCONCLUSIONSnTensioning the core suture to shorten its encompassed tendon segment by 10% substantially increases resistance to postoperative gapping. Further tensioning to produce 20% shortening of the tendon segment increased the gapping forces by a much smaller amount. This study suggests that a slightly tensioned surgical repair, shortening the encompassed tendon segment by approximately 10%, is appropriate.nnnCLINICAL RELEVANCEnSlightly tensioning core sutures across the tendon repair site, such as adding tension to cause 10% tendon-segment shortening, would greatly increase the gap resistance of the surgical repair.

Effectiveness of MicroRNA in Down-Regulation of TGF-β Gene Expression in Digital Flexor Tendons of Chickens: In Vitro and In Vivo Study

PURPOSEnTransforming growth factor (TGF)-beta is considered to be responsible for the formation of scars such as adhesions around healing digital flexor tendons. We proposed to deliver microRNAs (miRNAs) to silence expression of the TGF-beta1 gene and to investigate the effectiveness of miRNAs in down-regulation of the TGF-beta1 gene in vitro and in vivo.nnnMETHODSnWe designed and engineered 4 miRNAs according to genetic sequences of chicken TGF-beta1. Four plasmid vectors harboring the respective engineered miRNAs and 1 control vector were constructed. We transfected 30 wells of cultured tenocytes with these vectors and harvested them 48 hours later. The gene expression levels were quantified using real-time polymerase chain reactions. Subsequently, the miRNA that most effectively silenced TGF-beta gene in vitro was tested on 25 chickens in vivo. The miRNA and control vectors were injected into the injured tendons, respectively. At 1 and 6 weeks after surgery, the tendons were analyzed for gene expression and protein production.nnnRESULTSnIn both in vitro and in vivo settings, delivery of miRNA to the tendon substantially down-regulated expression of the TGF-beta gene but did not affect expression of the collagen I gene. In the healing tendon, TGF-beta gene expression was significantly down-regulated by 50% to 60% at 1 and 6 weeks. At 6 weeks, the collagen III gene expression was significantly down-regulated by 55% at 6 weeks and the connective tissue growth factor gene was significantly down-regulated by 25%. At 6 weeks, TGF-beta protein was substantially decreased.nnnCONCLUSIONSnMicroRNA significantly down-regulates expression of the TGF-beta in vitro and in vivo. Application of miRNA did not down-regulate expression of the collagen I, but downregulated the collagen III gene. Application of miRNA treatment to modulate TGF-beta expression holds great promise in preventing tendon adhesion formation.

Digital Oedema, Adhesion Formation and Resistance to Digital Motion after Primary Flexor Tendon Repair:

The development of digital oedema, adhesion formation, and resistance to digital motion at days 0, 3, 5, 7, 9 and 14 after primary flexor tendon repairs using 102 long toes of 51 Leghorn chickens was studied. Oedema presented as tissue swelling from days 3 to 7, which peaked at day 3. After day 7, oedema was manifest as hardening of subcutaneous tissue. The degree of digital swelling correlated with the resistance to tendon motion between days 3 and 7. At day 9, granulation tissues were observed around the tendon and loose adhesions were observed at day 14. Resistance to digital motion increased significantly from day 0 to day 3, but did not increase between days 3 and 9. The early postoperative changes appear to have three stages: initial (days 0–3, increasing resistance with development of oedema), delayed (days 4–7, higher resistance with continuing oedema) and late (after day 7–9, hardening of subcutaneous tissue with development of adhesions).

Molecular Events of Cellular Apoptosis and Proliferation in the Early Tendon Healing Period

PURPOSEnCellular proliferation is accompanied by cellular apoptosis. In the healing digital flexor tendon, molecular events concerning cellular apoptosis have not been investigated. This study aimed to investigate the relationship between cellular apoptosis and proliferation in early tendon healing.nnnMETHODSnThe flexor digitorum profundus tendons of 50 long toes in 25 chickens were transected and were repaired surgically. On postoperative days 3, 7, 14, 21, and 28, we subjected tendons to in situ terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay to detect apoptotic cells, immunofluorescence staining with antibodies to proliferating cell nuclear antigen to assess proliferation, and Bcl-2, an anti-apoptotic protein, to assess responses suppressive to apoptosis. The positively labeled tenocytes were counted microscopically and compared statistically. We also stained sections with hematoxylin and eosin to observe their healing status. An additional 12 tendons (6 chickens) served as day 0 controls.nnnRESULTSnCompared with tendons at day 0, the healing tendons had notably greater cellularity in both epitenon and endotenon areas. The total number of cells and number of TUNEL-positive cells peaked at day 3. At days 7 to 21, the number of proliferating cell nuclear antigen-positive cells peaked. At days 7 and 14, the cells positively stained with Bcl-2 peaked. At days 14 to 28, the total number of cells and TUNEL-positive cells decreased significantly compared with those at days 3 and 7, yet the numbers remained greater than those on day 0.nnnCONCLUSIONSnApoptosis in the healing tendons peaks at day 3, followed about 10 days later by the peak proliferation period. Because Bcl-2 serves to inhibit apoptosis, a later increase in Bcl-2-positive cells indicates that tendon apoptosis is inhibited. These findings indicate that tenocyte apoptosis is accelerated within several days after injury, followed by increases in cellular proliferation and activation of molecular events to inhibit apoptosis in 2 to 4 weeks.

BIOMECHANICAL COMPARISONS OF FOUR-STRAND TENDON REPAIRS WITH DOUBLE-STRANDED SUTURES: EFFECTS OF DIFFERENT LOCKS AND SUTURE GEOMETRY

Multi-strand repairs are often used in flexor tendon surgery. We evaluated the strength of four four-strand tendon repairs with different locks and suture geometry. Forty-eight pig flexor tendons were repaired with the following methods: a cross-lock four-strand repair; a U-shaped repair with circle-locks; a modified Kessler repair with Pennington locks (with longitudinal sutures located more centrally; and another modified Kessler (with longitudinal sutures located more laterally). The tendons were loaded to complete failure of the repairs. The two Kessler repairs showed a 35% lower 2u2009mm gap force, and 15% lower ultimate strength compared with the other two repairs; the differences were of statistical significance. The failure pattern was breakage of sutures in almost all tendons. These four-strand repairs differed in gapping and ultimate strengths. The Kessler-type repairs with different geometry in the longitudinal sutures produced identical strengths. The Kessler-type repairs with Pennington locks were weaker than the two repairs with either cross-locks or circle-locks.

Effect of A2 Pulley Release on Repaired Tendon Gliding Resistance and Rupture in a Chicken Model

PURPOSEnRelease of part of the A2 pulley has been suggested to improve function after zone II primary flexor tendon repairs. The purpose of this study was to measure the effect of A2 pulley release compared with pulley preservation on repaired tendon gliding resistance and rupture in the early postoperative period in a chicken model.nnnMETHODSnWe divided 104 long toes from 52 Leghorn chickens into 2 experimental groups. In group 1, the flexor digitorum profundus tendons were cut completely and repaired surgically with either release or preservation of the A2 pulley. The tendon laceration was positioned so that the repair had to glide through the pulley with toe flexion. The toes were subjected to simulated active flexion at the end of the 2nd and 4th postsurgical weeks. The rupture rates of the tendons were recorded. In group 2, the volar two thirds of the tendons were cut and repaired, also in the region of the A2 pulley, with the pulley released or preserved. The force resisting tendon motion and the work of digital flexion with simulated active digital flexion were tested 3, 5, 7, and 14 days after surgery.nnnRESULTSnAt the end of the 2nd and 4th weeks, the tendon repairs showed significantly higher rates of rupture in the presence of an intact A2 pulley compared with those with the pulley released. Tendon gliding resistance was significantly increased with preservation of the pulley on postsurgical days 3, 5, 7, and 14; these toes required a greater number of motion cycles to reach steady levels of resistance and showed significantly higher steady resistance after digital motion.nnnCONCLUSIONSnRelease of the A2 pulley decreases the rate of repair rupture and the resistance to tendon gliding when the site of tendon repair has to glide through the pulley in the early postsurgical period. The findings of this study support partial release of the A2 pulley to favor successful tendon repair.

A study of the anatomy and repair strengths of porcine flexor and extensor tendons: are they appropriate experimental models?

Although both porcine flexor and extensor tendons have been used in tendon repair research, no studies have specifically studied the anatomical differences and repair strengths in both types of tendons. We used 12 pig trotters to observe the anatomy of these tendons and compared the 2 mm gap and ultimate strengths of flexor and extensor tendons. There were four annular (A1, A2, A3, and A4) pulleys and one oblique pulley, which form a fibro-osseous tunnel for the flexor tendons, but the anatomy of the porcine extensor tendons was markedly different from the human flexor or extensor tendons. The diameter of flexor tendons was significantly greater than that of the extensors. The 2 mm gap and ultimate strengths of the flexor tendon with either two-strand or four-strand repairs were significantly greater than those of the extensor tendon. We conclude that the porcine flexor tendon systems are similar to those in the human, but the extensor tendons are not similar to either the flexor or extensor tendons in humans. Flexor and extensor tendons have different repair strengths which should be taken into account when interpreting findings from investigations using these tendons.

Relative contribution of tissue oedema and the presence of an A2 pulley to resistance to flexor tendon movement: an in vitro and in vivo study.

Increases in the resistance to tendon during movement may lead to rupture or gapping of the repaired tendon. The relative contribution of these factors—tissue oedema, tendon oedema, and the intact A2 pulley—to resistance to tendon gliding is unclear. In in vitro chicken models, we created oedema in subcutaneous tissue or tendon. The work of digital flexion (WOF) increased significantly after creation of oedema in those tissues. WOF decreased significantly after division of the A2 pulley. Preservation of the integrity of the A2 pulley significantly increased WOF at post-operative weeks 1 and 2 in the in vivo chicken model; increases in WOF were greater than those due to the presence of oedematous subcutaneous tissue. Pulley division at the time of surgery reduced WOF more drastically than the removal of volar subcutaneous tissue. Presence of an intact A2 pulley adds greater resistance to the movement of the repaired flexor tendon than volar oedematous subcutaneous tissue.