Tendon and ligament engineering: from cell biology to in vivo application

Tendons and ligaments are related connective tissues that join muscle to bone and bone to bone, respectively. Tendon and ligament injuries are widely distributed clinical problems in society and while healing of such disorders can occur, the original biological properties of the tissue do not return to normal. In this review, recent work on tendon and ligament development and the use of growth factors for successful cellular therapy of tendon and ligament disorders are discussed. In addition, anti-inflammatory concepts for the treatment of tendon and ligament injuries and recent developments in stem cell engineering for tendon and ligament tissues are examined. Lastly, gene transfer strategies for therapeutic applications to heal tendon and ligament disorders are reviewed.     

Compact platelet-rich fibrin scaffold to improve healing of patellar tendon defects and for medial collateral ligament reconstruction

BackgroundPlatelets are one of the most biocompatible and cost-effective sources of growth factors. Attention is being paid to autologous platelets and platelet-rich plasma. We developed a novel compact platelet-rich fibrin scaffold (CPFS) that was produced from blood and calcium gluconate only. The objective of this study was to investigate the potential of CPFS as a provisional scaffold in two rabbit models.MethodsIn the first rabbit model, the central half of the patellar tendon was resected bilaterally. Allogenic CPFS was attached to the defect in the right knee, while the left knee was untreated. In the other model, the medial collateral ligament was removed bilaterally. The ligament of the right knee was reconstructed with allogenic CPFS, whereas the left knee was untreated.ResultsAfter 12 weeks, the ultimate failure load and stiffness were higher for the right patellar tendon than for the left patellar tendon in the former model. It was found that CPFS promoted ligament repair tissue in contrast with that on the untreated side in the latter model. The ultimate failure load of the CPFS repair tissue at 20 weeks was 78% of that in healthy controls of the same age.ConclusionsCPFS enhanced the healing of tendons and ligaments.Clinical relevanceCPFS has the potential to accelerate healing of tendons and ligaments as a provisional bioscaffold or a material for graft augmentation.     

Mechanical Actuation Systems for the Phenotype Commitment of Stem Cell-Based Tendon and Ligament Tissue Substitutes

High tensile forces transmitted by tendons and ligaments make them susceptible to tearing or complete rupture. The present standard reparative technique is the surgical implantation of auto- or allografts, which often undergo failure.Currently, different cell types and biomaterials are used to design tissue engineered substitutes. Mechanical stimulation driven by dedicated devices can precondition these constructs to a remarkable degree, mimicking the local in vivo environment. A large number of dynamic culture instruments have been developed and many appealing results collected. Of the cells that have been used, tendon stem cells are the most promising for a reliable stretch-induced tenogenesis, but their reduced availability represents a serious limitation to upscaled production. Biomaterials used for scaffold fabrication include both biological molecules and synthetic polymers, the latter being improved by nanotechnologies which reproduce the architecture of native tendons. In addition to cell type and scaffold material, other variables which must be defined in mechanostimulation protocols are the amplitude, frequency, duration and direction of the applied strain. The ideal conditions seem to be those producing intermittent tension rather than continuous loading. In any case, all physical parameters must be adapted to the specific response of the cells used and the tensile properties of the scaffold. Tendon/ligament grafts in animals usually have the advantage of mechanical preconditioning, especially when uniaxial cyclic forces are applied to cells engineered into natural or decellularized scaffolds. However, due to the scarcity of in vivo research, standard protocols still need to be defined for clinical applications.     

Ligament repair: a molecular and immunohistological characterization

The anterior cruciate ligament (ACL) is the most commonly injured tissue of the human knee. Its poor ability to regenerate after injury represents a challenge to ligament tissue engineering. An understanding of the molecular composition of the structures used for its repair is essential for clinical assessments and for the implementation of tissue engineering strategies. The objective of this study was to evaluate, both at gene and protein levels, the expression of characteristic molecules in human ACL, patellar, semitendinosus and gracilis tendons and in the ligament reconstructed with patellar or semitendinosus and gracilis tendons. We demonstrated that primary ACL and tendon tissues all express collagen I, II, Sox-9, tenascin-C and aggrecan. Collagen X expression was detected at very low levels or undetectable. Cathepsin B, MMP-1 and MMP-13 were expressed at higher levels in the ACL reconstructed by the two tendons, showing that a remodeling process occurs during "ligamentization". Both our molecular and immunohistochemical evaluations did not reveal significative differences between the tendons and ligaments analyzed. However, ACL reconstructed with semitendinosus and gracilis tendon seems to present a higher expression of collagen type II when compared to that reconstructed with patellar tendon. This study could give a reasonable identification of genetic and protein markers specific to tendon/ligament tissues and be helpful in testing tissue engineering approaches for ACL reconstruction.     

Variations in internal structure,composition and protein distribution between intra‐ and extra‐articular knee ligaments and tendons

Tendons and ligaments play key roles in the musculoskeletal system in both man and animals. Both tissues can undergo traumatic injury, age‐related degeneration and chronic disease, causing discomfort, pain and increased susceptibility to wider degenerative joint disease. To date, tendon and ligament ultrastructural biology is relatively under‐studied in healthy, non‐diseased tissues. This information is essential to understand the pathology of these tissues with regard to function‐related injury and to assist with the future development of tissue‐engineered tendon and ligament structures. This study investigated the morphological, compositional and extracellular matrix protein distribution differences between tendons and ligaments around the non‐diseased canine stifle joint. The morphological, structural characteristics of different regions of the periarticular tendons and ligaments (the intra‐articular anterior cruciate ligament, the extra‐articular medial collateral ligament, the positional long digital extensor tendon and energy‐storing superficial digital flexor tendons) were identified using a novel semi‐objective histological scoring analysis and by determining their biochemical composition. Protein distribution of extracellular matrix collagens, proteoglycans and elastic fibre proteins in anterior cruciate ligament and long digital extensor tendon were also determined using immunostaining techniques. The anterior cruciate ligament was found to have significant morphological differences in comparison with the other three tissues, including less compact collagen architecture, differences in cell nuclei phenotype and increased glycosaminoglycan and elastin content. Intra‐ and interobserver differences of histology scoring resulted in an average score 0.7, indicative of good agreement between observers. Statistically significant differences were also found in the extracellular matrix composition in terms of glycosaminoglycan and elastin content, being more prominent in the anterior cruciate ligament than in the other three tissues. A different distribution of several extracellular matrix proteins was also found between long digital extensor tendon and anterior cruciate ligament, with a significantly increased immunostaining of aggrecan and versican in the anterior cruciate ligament. These findings directly relate to the different functions of tendon and ligament and indicate that the intra‐articular anterior cruciate ligament is subjected to more compressive forces, reflecting an adaptive response to normal or increased loads and resulting in different extracellular matrix composition and arrangement to protect the tissue from damage.     

Elastic properties of Thiel‐embalmed human ankle tendon and ligament

Thiel embalming is recommended as an alternative to formalin‐based embalming because it preserves tissue elasticity, color, and flexibility in the long term, with low infection and toxicity risk. The degree to which Thiel embalming preserves elasticity has so far been assessed mainly by subjective scoring, with little quantitative verification. The aim of this study is to quantify the effect of Thiel embalming on the elastic properties of human ankle tendons and ligament. Biomechanical tensile tests were carried out on six Thiel‐embalmed samples each of the peroneus longus, peroneus brevis, and calcaneal tendons, and the calcaneofibular ligament, with strain rates of 0.25%s^?1, 2%s^?1, and 8%s^?1. The stress?strain relationship was calculated from the force‐extension response with cross‐sectional area and gauge length. Young's modulus was determined from the stress?strain curve. The results showed that the tendon and ligament elasticity were lower after Thiel embalming than the literature values for fresh nonembalmed tendons and ligament. The biomechanical tensile test showed that the measured elasticity of Thiel‐embalmed tendons and ligaments increased with the strain rate. The Thiel embalming method is useful for preserving human ankle tendons and ligaments for anatomy and surgery teaching and research, but users need to be aware of its softening effects. The method retains the mechanical strain rate effect on tendons and ligament. Clin. Anat. 28:917–924, 2015. © 2015 Wiley Periodicals, Inc.     

Quantification of collagen organization using fractal dimensions and Fourier transforms

Collagen fibers and fibrils that comprise tendons and ligaments are disrupted or damaged during injury. Fibrillogenesis during healing produces a matrix that is initially quite disorganized, but remodels over time to resemble, but not replicate, the original roughly parallel microstructure. Quantification of these changes is traditionally a laborious and subjective task. In this work we applied two automated techniques, fast Fourier transformation (FFT) and fractal dimension analysis (FA) to quantify the organization of collagen fibers or fibrils. Using multi-photon images of collagen fibers obtained from rat ligament we showed that for healing ligaments, FA differentiates more clearly between the different time-points during healing. Using scanning electron microscopy images of overstretched porcine flexor tendon, we showed that combining FFT and FA measures distinguishes the damaged and undamaged groups more clearly than either method separately.     

Primary sarcoma of the anterior cruciate ligament - a case report

Clear cell sarcoma of tendons and aponeuroses (CCSTA) is a rare, aggressive soft tissue malignancy, which is found in intimate association with tendon, aponeurosis or fascia. It has not previously been reported in association with intraarticular ligaments. We report the first case of an intraarticular CCSTA, in this case of the anterior cruciate ligament and describe the diagnostic and treatment challenges of intraarticular tumours of the knee.     

喙肱韧带的应用解剖

目的:探讨喙肱韧带(coracohumeral ligament,CHL)的解剖学特点。方法:观察20侧正常成人肩关节标本的CHL位置、形态、起止点及与盂肱上韧带的关系;CHL在肩关节各种活动中所处的状态;CHL与关节囊融合处、CHL根部组织、关节囊及喙肩韧带组织学特点。结果:20侧CHL全部起于喙突基底部的外侧缘;9侧止于冈上肌腱,7侧止于肩袖间隙,3侧止于冈上肌腱和肩胛下肌腱,1侧止于肩胛下肌腱;其中11侧可见CHL与盂肱上韧带存在纤维组织连接;CHL于肩关节旋外、前屈、后伸、内收、肱骨头前后平动时紧张,于旋内、中立位、外展位时松弛;CHL与关节囊融合处、CHL根部组织与关节囊组织学特点相似,均呈现关节囊组织的典型特点,喙肩韧带呈现出韧带的特有表现。结论:CHL位置与形态相对固定,起点一致,但止点呈多样性;CHL与盂肱上韧带复合体不固定;CHL在不同运动状态其紧张度不一;CHL与关节囊组织形态相似,是关节囊增厚部。     

Myostatin in tendon maintenance and repair

Myostatin, a negative regulator of muscle growth, has recently been found to be expressed in tendons. Myostatin-deficient mice have weak and brittle tendons, which suggest that myostatin could be important for tendon maintenance. Follistatin expression in the callus tissue after tendon transection is influenced by loading. We found that follistatin antagonises myostatin, but not GDF-5 or OP-1 in vitro. To study if myostatin might play a physiological role in soft tissue, we transected 64 rat Achilles tendons and studied the gene expression for myostatin and its receptors at four different time-points during healing. Intact tendons were also studied. All samples were studied with or without mechanical loading. Unloading was achieved with botulinum toxin injections in the calf muscles. The expression of the myostatin gene was more than 40 times higher in intact tendons than in the callus tissue during tendon healing. The expression of myostatin was also influenced by loading status in both intact and healing tendons. Thereafter, we measured the mechanical properties of healing tendons after local myostatin administration. This treatment increased the volume and the contraction of the callus after 8 days, but did not improve its strength. Our results indicate that myostatin plays a positive role in tendon maintenance and that exogenous protein administration stimulates proliferation and growth of early repair tissue. However, no effect on further development towards connective tissue formation was found.     

Total flavones of Hippophae rhamnoides promotes early restoration of ultimate stress of healing patellar tendon in a rat model

Traditional Chinese herbal medicine has long been used for treatment of tendon injuries. Comparing to the modern way of treatments, Traditional Chinese medicine also stresses on strategies to promote the inherent healing capacity of tendons. Hippophae rhamnoides, known as Shaji, is one of Chinese herbal drugs that are traditionally used to promote tendon and ligament injuries. The total flavones of H. rhamnoides (TFH), with major constituents including quercetin, isorhamnetin and kaempferol, have been demonstrated with most of the bioactive properties of Shaji. In the present study, we evaluated the potential effect of TFH in the restoration of ultimate stress of healing patellar tendon in a well-established gap wound model in rats. A 0.1 mg TFH was injected to wound 1 day after the injury, and the ultimate stress of the healing tendon was measured at day 14 post-injury. The results showed that the ultimate stress of the healing tendon was significantly promoted by injection of TFH, increasing from 30 to 50% as compared to saline control. Excessive fibrotic response was not found in TFH-treated animals, but an enhanced collagen deposition and a better fibre alignment were observed. The results suggest that TFH may improve the ultimate stress of healing tendons at early stages, which implies possible earlier rehabilitation programme and better recovery.     

带血供大收肌腱转位修复膝关节韧带的应用解剖及临床 …

目的：报道以膝上内侧血管为蒂大收肌腱转位重建膝关节韧带断裂缺损的应用解剖及术式设计。方法：４０侧经动脉灌注红色乳胶成人标本,解剖观测大收肌腱的形态及其血供,２侧新鲜下肢标本模拟手术设计。以膝上内侧血管为蒂大收肌腱转位修复膝关节韧带断裂缺损。结果：应用带血供大收肌腱转位修复胫侧副韧带３０例,胫侧副韧带和前交叉韧带８例,胫侧副韧带和后交叉韧带４例,多韧带损伤１例,经随访疗效为满意。结论：大收肌腱与膝关     

The tensile properties of collagen fascicles harvested from regenerated and residual tissues in the patellar tendon after removal of the central third

The central one-third portion of the patellar tendon (PT) is commonly used for the reconstruction of the anterior cruciate ligament. For better understanding of the healing mechanisms of the PT, tensile properties of collagen fascicles harvested from the healing PT were studied. A rectangular defect was made at the central third portion in each right PT in the skeletally mature rabbit. At 6 and 12 weeks, tensile tests were performed on fascicles from the tissue regenerated in the defect and the non-resected, residual tissue. The elastic modulus and tensile strength of fascicles from the regenerated tissue gradually increased in a fashion similar to the bulk regenerated tissue. The properties of fascicles from the residual tissue were similar to those from normal tendons, which was very much different from those of the bulk residual tissue that were greatly deteriorated by the removal of the central portion.     

An in vitro scratch tendon tissue injury model: effects of high frequency low magnitude loading

The healing process of ruptured tendons is suboptimal, taking months to achieve tissue with inferior properties to healthy tendon. Mechanical loading has been shown to positively influence tendon healing. However, high frequency low magnitude (HFLM) loads, which have shown promise in maintaining healthy tendon properties, have not been studied with in vitro injury models. Here, we present and validate an in vitro scratch tendon tissue injury model to investigate effects of HFLM loading on the properties of injured rat tail tendon fascicles (RTTFs). A longitudinal tendon tear was simulated using a needle aseptically to scratch a defined length along individual RTTFs. Tissue viability, biomechanical, and biochemical parameters were investigated before and 7 days after culture . The effects of static, HFLM (20 Hz), and low frequency (1 Hz) cyclic loading or no load were also investigated. Tendon viability was confirmed in damaged RTTFs after 7 days of culture, and the effects of a 0.77 ± 0.06 cm scratch on the mechanical property (tangent modulus) and tissue metabolism in damaged tendons were consistent, showing significant damage severity compared with intact tendons. Damaged tendon fascicles receiving HFLM (20 Hz) loads displayed significantly higher mean tangent modulus than unloaded damaged tendons (212.7 ± 14.94 v 92.7 ± 15.59 MPa), and damaged tendons receiving static loading (117.9 ± 10.65 MPa). HFLM stimulation maintained metabolic activity in 7-day cultured damaged tendons at similar levels to fresh tendons immediately following damage. Only damaged tendons receiving HFLM loads showed significantly higher metabolism than unloaded damaged tendons (relative fluorescence units —7021 ± 635.9 v 3745.1 ± 641.7). These validation data support the use of the custom-made in vitro injury model for investigating the potential of HFLM loading interventions in treating damaged tendons.     

Enhanced Medial Collateral Ligament Healing Using Mesenchymal Stem Cells: Dosage Effects on Cellular Response and Cytokine Profile

Mesenchymal stem cells (MSCs) have potential therapeutic applications for musculoskeletal injuries due to their ability to differentiate into several tissue cell types and modulate immune and inflammatory responses. These immune-modulatory properties were examined in vivo during early stage rat medial collateral ligament healing. Two different cell doses (low dose 1?×?10⁶ or high dose 4?×?10⁶ MSCs) were administered at the time of injury and compared with normal ligament healing at days 5 and 14 post-injury. At both times, the high dose MSC group demonstrated a significant decrease in M2 macrophages compared to controls. At day 14, fewer M1 macrophages were detected in the low dose group compared to the high dose group. These results, along with significant changes in procollagen I, proliferating cells, and endothelialization suggest that MSCs can alter the cellular response during healing in a dose-dependent manner. The higher dose ligaments also had increased expression of several pro-inflammatory cytokines at day 5 (IL-1β, IFNγ, IL-2) and increased expression of IL-12 at day 14. Mechanical testing at day 14 revealed increased failure strength and stiffness in low dose ligaments compared to controls. Based on these improved mechanical properties, MSCs enhanced functional healing when applied at a lower dose. Different doses of MSCs uniquely affected the cellular response and cytokine expression in healing ligaments. Interestingly, the lower dose of cells proved to be most effective in improving functional properties.     

Tendon and ligament fibrillar crimps give rise to left-handed helices of collagen fibrils in both planar and helical crimps

Collagen fibres in tendons and ligaments run straight but in some regions they show crimps which disappear or appear more flattened during the initial elongation of tissues. Each crimp is formed of collagen fibrils showing knots or fibrillar crimps at the crimp top angle. The present study analyzes by polarized light microscopy, scanning electron microscopy, transmission electron microscopy the 3D morphology of fibrillar crimp in tendons and ligaments of rat demonstrating that each fibril in the fibrillar region always twists leftwards changing the plane of running and sharply bends modifying the course on a new plane. The morphology of fibrillar crimp in stretched tendons fulfills the mechanical role of the fibrillar crimp acting as a particular knot/biological hinge in absorbing tension forces during fibril strengthening and recoiling collagen fibres when stretching is removed. The left‐handed path of fibrils in the fibrillar crimp region gives rise to left‐handed fibril helices observed both in isolated fibrils and sections of different tendons and ligaments (flexor digitorum profundus muscle tendon, Achilles tendon, tail tendon, patellar ligament and medial collateral ligament of the knee). The left‐handed path of fibrils represents a new final suprafibrillar level of the alternating handedness which was previously described only from the molecular to the microfibrillar level. When the width of the twisting angle in the fibrillar crimp is nearly 180° the fibrils appear as left‐handed flattened helices forming crimped collagen fibres previously described as planar crimps. When fibrils twist with different subsequent rotational angles (< 180°) they always assume a left‐helical course but, running in many different nonplanar planes, they form wider helical crimped fibres.