Nanoparticle-mediated delivery of TGF-β1 miRNA plasmid for preventing flexor tendon adhesion formation

Treatment of the disrupted digital flexor tendon is troublesome because of the lack of sufficient healing capacity and the formation of adhesions. Sustained gene delivery may be a promising approach of modulating gene expression in enhancing tendon healing and decreasing adhesions. In this study, a microRNA-based RNAi plasmid was used to specifically silence the expression of TGF-β1 gene associated with scar and adhesion formation in the flexor tendons. The miRNA plasmids were complexed with polylactic-co-glycolic acid (PLGA) nanoparticles to form nanoparticle/TGF-β1 miRNA plasmid (nanoparticle/plasmid) complexes. In vitro and in vivo transfection efficiencies experiments against tenocytes revealed that nanoparticle/plasmid complexes have significantly superior transfection efficiency over the lipofectamine/plasmid complexes. The gene and protein expression associated with adhesion of tendon treated with nanoparticle/plasmid complexes were evaluated by real-time PCR and immunoblotting. The grading of adhesions for tendons treated with nanoparticle/plasmid complexes was less severe than that treated with the nanoparticle/mock plasmid complexes. However, the ultimate strength of repaired tendons treated with nanoparticle/plasmid complexes was significantly lower than that of tendons treated with the nanoparticle/mock plasmid complexes.     

Morphological and histochemical analysis of a case of superficial digital flexor tendon injury in the horse

This report compares the morphology and the concentrations of glycos-aminoglycans (GAGs) in an injured superficial digital flexor tendon (SDFT) of a horse with those of a normal tendon. An injured 6-year-old male Thoroughbred exhibited heat and swelling around the SDFT of the right forelimb. On histopathological examination, exuberant granulation was observed in the affected tendon, with activated tenocytes, angiogenesis, haemorrhage, and infiltration of small numbers of leucocytes. The collagen fibres were loosely packed and irregularly arranged. The diameter of control collagen fibrils was 20-360 nm and that of affected collagen fibrils 20-240 nm. In the analysis of GAGs in the matrix, hyaluronic acid (HA), dermatan sulphate (DS), and chondroitin sulphate (CS) were found to be major components in both control and affected tendons. Increases in DS in the affected tendon were striking. Our observations suggest that fibrillogenesis was activated by increases in DS and decreases in HA and CS. It is also assumed that absence of collagen fibrils of normal thickness and in a parallel arrangement reflected the morphological and biochemical characteristics of fibrillogenesis in the injured tendon. If the inflammatory features of an injured tendon could be altered, it might return eventually to its normal structure.     

Overexpression of synoviolin facilitates the formation of a functional synovial biomembrane

Digital flexor tendon repair poses a significant challenge for hand surgeons. Currently, extrasynovial tendon grafts are frequently used in clinical settings to bridge flexor tendon defects. However, the healing process is always accompanied by postoperative adhesion. This is mostly due to the fact that no synovial membrane covers the extrasynovial tendon surface, in contrast to the intrasynovial tendon. In this study, we present an efficient method of developing a functional synovial biomembrane on the surface of the extrasynovial tendon. Synoviocytes were isolated from the knee joint of a Japanese white rabbit. After being infected with lentivirus, the over-expression of synoviolin in these synoviocytes was confirmed by semi-quantitative RT-PCR and western blotting. Cellular proliferation and increased hyaluronic acid secretion were confirmed in the synoviolin over-expressing synoviocytes by MTT-based method, cell cycle assays and ELISA. Furthermore, the synoviolin over-expressing synoviocytes were co-cultured with extrasynovial tendons that were harvested from the hind leg of rabbits. After being co-cultured in vitro for 3 and 7 days, these infected synoviocytes were found to accelerate the formation of a biomembrane on the tendon surface compared to the control group. More importantly, Alcian blue staining confirmed the ability of this cultured biomembrane to produce specific matrices containing acidic carboxyl mucopolysaccharides (mainly hyaluronic acid). All these results demonstrate that the over-expression of synoviolin stimulates the proliferation and HA secretion of synoviocytes and facilitates the formation of a functional synovial biomembrane.     

Synovial cell culture and tissue engineering of a tendon synovial cell biomembrane

Restrictive adhesions are a common complication of tendon injury and repair in the hand, resulting in severe dysfunction. Creating a barrier between the repair sites and surrounding tissue layers may prevent adhesions. We present the first stage in the process of developing a synovial biomembrane for this purpose. Synovial cells harvested from the Achilles tendon sheath and the knee joint of a Wistar albino rat were cultured for 2 weeks in culture medium, and then impregnated into a collagen type 1 matrix for another 2 weeks. Cells originating from both tendon and synovium demonstrated cell growth and layer formation on the surfaces of the matrix 2 weeks after impregnation. Alcian blue staining using Scott's method demonstrated the presence of acidic mucopolysaccharide, indicating hyaluronic acid (HA) production. This provides indirect evidence of functioning synovial cells on the membrane. It is possible to culture synovial cells and engineer a synoviocyte-collagen membrane that synthesizes endogenous HA. Application of this biomembrane to tendon repair sites may help to prevent adhesions after tendon repairs. Evaluation of this method on in vivo models is required.     

Gap junction protein expression and cellularity: comparison of immature and adult equine digital tendons

Injury to the energy-storing superficial digital flexor tendon is common in equine athletes and is age-related. Tenocytes in the superficial digital flexor tendon of adult horses appear to have limited ability to respond adaptively to exercise or prevent the accumulation of strain-induced microdamage. It has been suggested that conditioning exercise should be introduced during the growth period, when tenocytes may be more responsive to increased quantities or intensities of mechanical strain. Tenocytes are linked into networks by gap junctions that allow coordination of synthetic activity and facilitate strain-induced collagen synthesis. We hypothesised that there are reductions in cellular expression of the gap junction proteins connexin (Cx) 43 and 32 during maturation and ageing of the superficial digital flexor tendon that do not occur in the non-injury-prone common digital extensor tendon. Cryosections from the superficial digital flexor tendon and common digital extensor tendon of 5 fetuses, 5 foals (1-6 months), 5 young adults (2-7 years) and 5 old horses (18-33 years) were immunofluorescently labelled and quantitative confocal laser microscopy was performed. Expression of Cx43 and Cx32 protein per tenocyte was significantly higher in the fetal group compared with all other age groups in both tendons. The density of tenocytes was found to be highest in immature tissue. Higher levels of cellularity and connexin protein expression in immature tendons are likely to relate to requirements for tissue remodelling and growth. However, if further studies demonstrate that this correlates with greater gap junctional communication efficiency and synthetic responsiveness to mechanical strain in immature compared with adult tendons, it could support the concept of early introduction of controlled exercise as a means of increasing resistance to later injury.     

组织工程化肌腱修复鸡趾屈肌腱缺损的生物力学特性研究

组织工程化肌腱植入体内修复的肌腱其抗拉强度达不到正常肌腱的数值。为探讨这一问题的原因，我们选择罗曼雏鸡足趾屈肌腱细胞与可降解聚羟基乙酸筛网体外复合培养构建组织工程化肌腱。用此工程化肌腱修复20只罗曼鸡第二趾深屈肌腱0．5～0．8cm缺损。术后第2、4、6、8周取材，测定样品中材料的重量、羟脯氨酸含量及抗拉强度等力学特性指标。结果显示，植入2、4、6、8周，支架材料重量下降很快，至第8周基本降解；修复的肌腱中代表胶原合成总量的羟脯氨酸含量随时间增加，但变化不明显；修复的肌腱断裂能量和抗拉强度均随时间呈一先降低后逐渐增大的变化，抗拉强度在第8周才达到正常肌腱的23％。结果提示，植入的组织工程化肌腱在其材料迅速降解的同时，胶原生成量并不多，二者出现明显的不匹配，导致修复的肌腱抗拉强度低。     

Surface modification of extrasynovial tendon by chemically modified hyaluronic acid coating.

We investigated a method for chemically binding hyaluronic acid (HA) to extrasynovial tendon and the effect of chemically modified HA on the gliding resistance of tendon. Canine peroneus longus (PL) tendons were immersed into one of three different solutions (saline, 1% HA, or 1% chemically modified HA) for 2 h. The gliding resistance of treated PL tendons was measured at 1, 5, 10, 20, 50, and 100 cycles in a saline bath. After treatment with unmodified HA and chemically modified HA, the gliding resistance of the PL tendons decreased significantly compared with the saline-treated tendons (p < 0.05), and this effect of the two HA treatments persisted through 10 cycles. For cycles 20-100, the gliding resistance of PL tendons treated with chemically modified HA remained significantly lower than that of tendons treated either with saline or unmodified HA (p < 0.01). The effect of paratenon removal on gliding resistance was neither statistically significant for repetitions beyond 10 cycles, nor was it an independent predictor of gliding resistance, as the effect (higher resistance after paratenon removal) was mainly seen in the saline-treated tendons. Chemically modified HA-treated extrasynovial tendons may improve gliding of tendon graft and reduce adhesion postoperatively, compared with traditional grafts.     

Effect of beta-aminopropionitrile and hyaluronic acid on repair of collagenase-induced injury of the rabbit Achilles tendon

Collagenase was injected into the Achilles tendon of both hind legs of 10 clinically normal adult male New Zealand white rabbits. One month after induction of the injury, beta-aminoproprionitrile (BAPN) or hyaluronic acid (HA) was injected into the tendon core of the right hind leg of each rabbit, the left hind leg being left untreated. The treatment effects were evaluated by electron microscopy and analysis of the glycosaminoglycan (GAG) content of samples at 2 and 6 months post-treatment. At 2 months, collagen fibrils in tendons from both hind legs were relatively small in diameter, irregularly arranged, and interspersed with abundant active tenocytes as compared with those in normal tendon uninjured by collagenase. In the matrix, the amount of HA increased, but chondroitin-6-sulphate was eliminated. At 6 months, BAPN-treated tendons had small-diameter, regularly arranged collagen fibrils. HA-treated tendons, on the other hand, had large diameters, as well as regularly arranged collagen fibrils by comparison with non-treated tendon. The results suggest that HA, unlike BAPN, promoted healing.     

An experimental study of healing of the partially severed flexor tendon in chickens

There is a lack of clinical and experimental studies of the treatment of incompletely transected tendons. The controversy concerning the source of flexor tendon nutrients is of important clinical concern in healing of the injured tendon; thus, the flexor tendon blood supply has cited as a reason for using specific tendon suture techniques, and as a rationale for preserving the superficialis tendon and its vincula during tendon repair surgery. Our knowledge of the normal physiology of digital flexor tendons and the mechanism of their healing process is deficient. The aim of this study was to investigate the relative importance of the synovial fluid and the blood supply respectively for the healing of partially severed flexor tendons. We observed the sequential histological and vascular changes which occur in healing of the partial lacerations in the dorsal and plantar aspects of the tendons. We observed the vascularities of the two partially severed tendon groups after injection of microfil and india ink through the femoral artery. In the healing process there was no sequential histological difference between the dorsal and the plantar severed tendons. The vascularity patterns of the healing tendons were significantly increased and the hypervascularity of dorsal severed tendons was greater than that of plantar severed tendons. Partially severed tendons were completely healed without surgical repair with dense collagen fibers without adhesion in most cases. We concluded from this study that the blood vessels appeared to play a significant role in the healing of the severed flexor tendons. An intact synovial environment did not seem to be required for healing of the severed tendon. It is not necessary to surgically repair the partially severed tendon for prevention of rupture and adhesion.     

Histopathology of tenosynovium in trigger fingers

Stenosing flexor tenosynovitis, trigger finger, is a common clinical disorder causing painful locking or contracture of the involved digits, and most instances are idiopathic. This problem is generally caused by a size mismatch between the swollen flexor tendon and the thickened first annular pulley. Although hypertrophic pulleys have been histologically and ultrasonographically detected, little is known about the histopathology of the tenosynovium covering the tendons of trigger fingers. We identified chondrocytoid cells that produced hyaluronic acid in 23 (61%) fingers and hypocellular collagen matrix in 32 (84%) fingers around the tenosynovium among 38 specimens of tenosynovium from patients with trigger fingers. These chondrocytoid cells expressed the synovial B cell marker CD44, but not the chondrocyte marker S‐100 protein. The incidence of these findings was much higher than that of conventional findings of synovitis, such as inflammatory infiltrate (37%), increased vascularity (37%), hyperplasia of synovial lining cells (21%), or fibrin exudation (5%). We discovered the following distinctive histopathological features of trigger finger: hyaluronic acid‐producing chondrocytoid cells originated from fibroblastic synovial B cells, and a hypocellular collagen matrix surrounding the tenosynovium. Thus, an edematous extracellular matrix with active hyaluronic acid synthesis might increase pressure under the pulley and contribute to the progression of stenosis.     

Effect of curing time and concentration for a chemical treatment that improves surface gliding for extrasynovial tendon grafts in vitro

The purpose of this study was to evaluate whether treatment time and concentration of these reagents have an effect on the resulting gliding resistance. Forty peroneus longus (PL) tendons were used, from 20 adult mongrel dogs, along with the A2 pulley obtained from the ipsilateral hind paw. After the baseline gliding resistance was measured, the PL tendons were treated with one of three concentrations of hyaluronic acid (HA) and 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide (NHS) mixed with 10% gelatin for various times (5, 30, and 60 min). Tendon friction was measured over 1000 cycles of simulated flexion/extension motion. Gliding resistance of the untreated PL tendons had no significant difference among the groups. After surface treatment with low concentration of HA and EDC/NHS for 5-min cure, the gliding resistance was similar to that of the untreated PL tendon and significantly higher than its 30- and 60-min treatment. For the rest of high concentration of HA and EDC/NHS groups, the gliding resistance was lower than that of untreated PL tendon. However, there was no significant difference among the timing points. It is possible to optimize the effect of surface treatment on friction and durability by regulating cure time and concentration of reagents in a canine extrasynovial tendon in vitro.     

An Age-Related Study of Morphology and Cross-Link Composition of Collagen Fibrils in the Digital Flexor Tendons of Young Thoroughbred Horses

The superficial digital flexor tendon is the most commonly injured tendon in the racing Thoroughbred. Despite the clinical significance of this structure, only limited data exist regarding normal age-related morphology of the tensile units, the collagen fibrils. The age at which these collagen fibrils become mature in composition and structure may be of importance. Consequently, the association of age and collagen fibril crosslink composition, diameter distribution and crimp morphology in the superficial and deep digital flexor tendons of Thoroughbreds up to and including three years of age has been studied. Replacement of immature crosslinks, peaking of the collagen fibril mass-average diameter and collagen fibril index, and stabilization of collagen crimp morphology changes supported the hypothesis that both digital flexor tendons become mature in structure by two years of age.     

High stiffness of human digital flexor tendons is suited for precise finger positional control

The objective of this study was to define the biomechanical properties of the human digital flexor tendons and to compare these biomechanical properties to other muscle-tendon units in the forearm. Mechanical measurements were performed on fresh-frozen tendons under physiological load and temperature conditions. Loads were determined by first measuring the physiological cross-sectional area of each digital belly of the flexor digitorum superficialis (FDS) and flexor digitorum profundus (FDP) and estimating maximum tension (P(o)) of that specific muscle head. Loading each tendon to the appropriate P(o) resulted in no significant difference in tendon strain among any of the tendons within each muscle (P > 0.05; digits 2-5) or between muscle types (FDP vs. FDS). The one exception to this finding was that a significantly higher strain at Po was observed in the FDP tendon to the small finger (P < 0.05). Average absolute strains observed for the FDP and FDS tendons (1.20 +/- 0.38%, mean +/- SD; n = 39) were significantly lower than those observed previously in a study of the prime movers of the wrist. The measured strain of approximately 1.5% was less than half of that predicted to occur in muscles of this architectural design. Modeling sarcomere shortening magnitudes during FDP or FDS contraction yielded a value of only 0.10 microm, which would have a negligible effect on the force generating capacity of these muscles. Thus the high stiffness of the digital flexor tendons suits them well for fine positional control and would render their muscle spindles quite sensitive to length perturbations at the fingertips.     

工程化肌腱修复肌腱缺损后力学特性的组织学基础

探讨组织工程化肌腱修复肌腱缺损后体内愈合过程中力学特性的组织学基础。取罗曼鸡肌腱细胞 ,经体外培养、扩增 ,与可降解生物材料聚羟基乙酸筛网构建工程化肌腱 ;将其植入修复 2 0只罗曼鸡第二趾深屈肌腱0 .5～ 0 .8cm缺损。术后第 2、4、6、8周取材 ,对标本进行大体、组织学及生物力学测定。植入 2、4、6、8周 ,新生肌腱在大体形态、细胞及胶原纤维排列方式上与正常肌腱相似 ,但新生肌腱的胶原纤维束并未形成较多的沿肌腱长度方向的致密结构 (“塑形”) ,导致其最大张力增加缓慢 ,到 8周时为 15 .4 0± 10 .6 3N,仅达正常肌腱的 2 3 ;8周时最大张应变为 2 2 .4 9± 10 .2 1 ,比正常肌腱大 10。结果表明 ,单纯聚羟基乙酸作支架 ,材料降解过快 ,新生肌腱失去了正常的力学刺激 ,“塑形”能力差 ,其生物力学强度低。提示 ,保持新生肌腱形成过程中正常的力学刺激对新生肌腱的“塑形”可能是至关重要的。     

Delivery of a vector encoding mouse hyaluronan synthase 2 via a crosslinked hyaluronan film

We have developed a crosslinked hyaluronic acid (HA) film with DNA incorporated within its structure and have characterized this system for its efficacy in sustained transferring of a vector encoding mouse hyaluronan synthase 2 (Has2). Analysis of the DNA release kinetics indicated that the HA films degraded when treated with hyaluronidase and that they released DNA over a prolonged period of time. Gel electrophoresis revealed that this DNA was intact and immunohistochemical analysis verified the transfection capabilities of DNA release samples. The ability of released DNA encoding Has2 to promote HA synthesis was confirmed by quantifying the amount of HA produced by COS-1 cells that were transfected with release samples. The intended future application of the HA films is in prevention of post-operative peritoneal adhesions. In addition to serving as a physical barrier, the film would function as a vehicle for sustained delivery of DNA encoding Has2, which would promote the synthesis of HA in transfected tissues.     

Distribution of proteins within different compartments of tendon varies according to tendon type

Although the predominant function of all tendons is to transfer force from muscle to bone and position the limbs, some tendons additionally function as energy stores, reducing the energetic cost of locomotion. To maximise energy storage and return, energy‐storing tendons need to be more extensible and elastic than tendons with a purely positional function. These properties are conferred in part by a specialisation of a specific compartment of the tendon, the interfascicular matrix, which enables sliding and recoil between adjacent fascicles. However, the composition of the interfascicular matrix is poorly characterised and we therefore tested the hypothesis that the distribution of elastin and proteoglycans differs between energy‐storing and positional tendons, and that protein distribution varies between the fascicular matrix and the interfascicular matrix, with localisation of elastin and lubricin to the interfascicular matrix. Protein distribution in the energy‐storing equine superficial digital flexor tendon and positional common digital extensor tendon was assessed using histology and immunohistochemistry. The results support the hypothesis, demonstrating enrichment of lubricin in the interfascicular matrix in both tendon types, where it is likely to facilitate interfascicular sliding. Elastin was also localised to the interfascicular matrix, specifically in the energy‐storing superficial digital flexor tendon, which may account for the greater elasticity of the interfascicular matrix in this tendon. A differential distribution of proteoglycans was identified between tendon types and regions, which may indicate a distinct role for each of these proteins in tendon. These data provide important advances into fully characterising structure–function relationships within tendon.     

Repair of tendon defect with dermal fibroblast engineered tendon in a porcine model

Harvesting autologous tenocytes for tendon engineering may cause secondary tendon defect at the donor site. Dermal fibroblasts are an easily accessible cell source and do not cause major donor site defect. This study aims to explore the possibility of tendon engineering using dermal fibroblasts. A total of 45 hybrid pigs were randomly divided into three groups: experimental group (n = 15)--repair of tendon defect with a dermal fibroblast engineered tendon; control group 1 (n = 15)--repair of defect with a tenocyte engineered tendon; and control group 2 (n = 15)-repair of defect with a scaffold alone. Both autologous dermal fibroblasts and tenocytes were seeded on polyglycolic acid (PGA) unwoven fibers to form a cell-scaffold construct and cultured in vitro for 7 days before in vivo implantation to repair a defect of flexor digital superficial tendon. Specimens were harvested at weeks 6, 14, and 26 for gross, histological, and mechanical analyses. Microscopy revealed good attachment of both dermal fibroblasts and tenocytes on PGA fibers and matrix production. In vivo results showed that fibroblast and tenocyte engineered tendons were similar to each other in their gross view, histology, and tensile strength. At 6 weeks, parallel collagen alignment was observed at both ends, but not in the middle in histology, with more cellular components than natural tendons. At weeks 14 and 26, both engineered tendons exhibited histology similar to that of natural tendon. Collagens became parallel throughout the tendon structure, and PGA fibers were completely degraded. Interestingly, dermal fibroblast and tenocyte engineered tendons did not express type III collagen at 26 weeks, which remained observable in normal pig skin and control group 2 tissue using polarized microscopy, suggesting a possible phenotype change of implanted dermal fibroblasts. Furthermore, both fibroblast and tenocyte engineered tendons shared similar tensile strength, about 75% of natural tendon strength. At 6 weeks in control group 2, neo-tissue was formed only at the peripheral area by host cells. A cord-like tissue was formed at weeks 14 and 26. However, the formed tissue was histologically disorganized and mechanically weaker than both cell-engineered tendons (p < 0.05). These results suggest that dermal fibroblasts may have the potential as seed cells for tendon engineering.     

Ultrastructural evidence for telocytes in equine tendon

The three-dimensional ultrastructure of the tendon is complex. Two main cell types are classically supported: elongated tenocytes and ovoid tenoblasts. The existence of resident stem/progenitor cells in human and equine tendons has been demonstrated, but their location and relationship to tenoblasts and tenocytes remain unclear. Hence, in this work, we carried out an ultrastructural study of the equine superficial digital flexor tendon. Although the fine structure of tendons has been previously studied using electron microscopy, the presence of telocytes, a specific type of interstitial cell, has not been described thus far. We show the presence of telocytes in the equine inter-fascicular tendon matrix near blood vessels. These telocytes have characteristic telopodes, which are composed of alternating dilated portions (podoms) and thin segments (podomers). Additionally, we demonstrate the presence of the primary cilium in telocytes and its ability to release exosomes. The location of telocytes is similar to that of tendon stem cells. The telocyte–blood vessel proximity, the presence of primary immotile cilia and the release of exosomes could have special significance for tendon homeostasis.     

Functional ability of tendinocytes to take up Fe substances in an inflamed tendon

The fate of iron (Fe) after bleeding has been analyzed in various connective tissues, but there have been only a few inconclusive studies on Fe in the tendon. In this study, energy dispersive X-ray microanalysis and an iron staining method were used to determine the localization of Fe in cells of the equine superficial digital flexor tendon. In inflamed tendons, Fe was detected in tendinocytes as well as macrophages. In contrast, negative or weak reactions were observed in many cells in the normal tendon. Although the accepted theory states that the reticuloendothelial system (macrophages and reticuloendothelial cells) is mainly responsible for the uptake and decomposition of erythrocytes, and tendon cells under conditions of inflammation show a functional ability to take up Fe substances. It has been reported that tendinocytes have multiple functions, including synthesis and secretion of collagen, cytokines, and matrix metalloproteinases. Taking these functions into consideration, indicates that the tendinocyte is not only an active cell but also a multi-functional cell.     

An experimental study on the role of the reciprocal tendinous apparatus of the horse at walk

Summary The locomotor pattern of the hind limb of seven horses has been studied in intact animals and after transection of the following structures: the peroneus tertius tendon, the cranial tibial muscle, both cranial tibial muscle and peroneus tertius tendon, and the superficial digital flexor tendon. The investigation was carried out by high speed cinematography and electromyography. It is concluded that (1) the muscles and tendons over the cranial aspect of the tibia play an important role during the support phase; (2) the movements of the hind limb may be performed without the action of the cranial tibial and gastrocnemius muscles; (3) the tendons in the shank store elastic energy during the support phase; (4) the gastrocnemius and cranial tibial muscles may centre the force of the load through the long axis of the tibia.