Ligament tissue engineering using synthetic biodegradable fiber scaffolds

Tissue engineering offers the possibility of replacing damaged human ligaments with engineered ligament tissues. Hence, we attempted to culture in vitro ligament tissues by seeding human anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells onto synthetic biodegradable polymer fiber scaffolds. The ACL and MCL cells readily attached to the scaffold fibers. These cells and their secreted matrix soon surrounded the scaffold fibers and bridged the gaps in between. Beginning at 2 weeks, portions of the scaffolds were completely filled with tissue matrix. By 5 weeks, the scaffolds became single bundles of tissue. Thus the cell/fiber system appears to be a viable system for culturing ligament tissues. Additionally, cell proliferation under mechanical and biochemical stimuli was studied for up to 4 days. Whereas mechanical stimulus and transforming growth factor enhanced proliferation, inflammatory agents (lipopolysaccharide and complement C5a) had a negative effect. This work can thus contribute to a sound strategy for culturing replacement ligament tissues in vitro.     

The effect of growth factors on both collagen synthesis and tensile strength of engineered human ligaments

Growth factors play a central role in the development and remodelling of musculoskeletal tissues. To determine which growth factors optimized in vitro ligament formation and mechanics, a Box-Behnken designed array of varying concentrations of growth factors and ascorbic acid were applied to engineered ligaments and the collagen content and mechanics of the grafts were determined. Increasing the amount of transforming growth factor (TGF) β1 and insulin-like growth factor (IGF)-1 led to an additive effect on ligament collagen and maximal tensile load (MTL). In contrast, epidermal growth factor (EGF) had a negative effect on both collagen content and MTL. The predicted optimal growth media (50 μg/ml TGFβ, IGF-1, and GDF-7 and 200 μM ascorbic acid) was then validated in two separate trials: showing a 5.7-fold greater MTL and 5.2-fold more collagen than a minimal media. Notably, the effect of the maximized growth media was scalable such that larger constructs developed the same material properties, but larger MTL. These results show that optimizing the interactions between growth factors and engineered ligament volume results in an engineered ligament of clinically relevant function.     

Direct-, fibroblast- and myoblast-mediated gene transfer to the anterior cruciate ligament

The anterior cruciate ligament (ACL) has poor capabilities of healing. Maturation or "ligamentization" of the ACL following autograft or allograft reconstruction has been found slow and remains under investigation. In vitro and in vivo studies have shown that platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), and epidermal growth factor (EGF) have the potential to improve ligament healing. Gene therapy approaches may represent a new alternative in delivering these specific growth factors to the ACL. The aim of this study was to investigate the feasibility of three different gene therapy approaches (direct-, fibroblast-, and myoblast-mediated gene transfer) to the ACL. Rabbit myoblasts and ACL-fibroblasts were transduced with 5 x 10(7) recombinant adenoviral particles carrying the LacZ reporter gene (MOI = 50). Myoblasts and fibroblasts (1 x 10(6)) were each injected into the right ACL of 10 adult rabbits; direct injection of 5 x 10(7) adenoviral particles was performed in 10 other rabbits. The left side was used as sham. The beta-galactosidase production was revealed using the LacZ histochemical technique. The transduced fibroblasts and myoblasts were found in the ligament tissue and in the synovial tissue surrounding the ACL at 4, 7, 14, and 21 days postinjection. The myoblasts fused and formed myotubes in the ligament. The direct approach also allowed the transfer of the marker gene in the ligament at 4, 7, 21, and 42 days postinjection. X-gal staining revealed no expression of beta-galactosidase in the sham ligament. The presence of cells expressing the marker gene in the ACL opens up the possibility of delivering proteins (i.e., PDGF, TGF-beta, and EGF) capable of improving ACL healing and graft maturation. Furthermore, engineered myoblasts may mediate and accelerate the intraligament neovascularization. This new technology based on gene therapy and tissue engineering may allow a persistent expression of selected growth factors to enhance ACL healing following injury.     

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.     

Porcine anterior cruciate ligament fibroblasts are similar to cells derived from the ligamentum teres, another non-healing intra-articular ligament

Porcine ligament fibroblasts were cultured from the anterior cruciate (ACL), medial collateral (MCL), and ligamentum teres (LT). There were no apparent differences in confluent cellular morphology among the ligament cell types as evaluated by phase contrast microscopy. The proliferation rate of MCL cells from 24-120 h was significantly higher (p < 0.05) than that of cells from either the LT or the ACL. MCL cells produced more collagen and less non-collagenous protein than the LT and ACL as determined by [3H]proline incorporation. This resulted in MCL cells producing a higher percentage (37%, p < 0.05) of collagen relative to total protein than either the ACL (28%) or the LT (32%). The MCL cells produced a significantly higher percentage (34.7%, p < 0.05) of type-III collagen relative to total type-I and III collagen than either the ACL (29.2%) or the LT (29.5%). The LT and MCL cells had similar and significantly greater coverage of in vitro wounds than the ACL. This study provides the first in vitro study of the LT and demonstrates that fibroblasts from the LT and ACL, two ligaments that heal poorly, have similar in vitro characteristics, with the exception of wound healing.     

Factors Affecting the Longevity and Strength in an <Emphasis Type="Italic">In Vitro</Emphasis> Model of the Bone–Ligament Interface

The interfaces between musculoskeletal tissues with contrasting moduli are morphologically and biochemically adapted to allow the transmission of force with minimal injury. Current methods of tissue engineering ligaments and tendons do not include the interface and this may limit the future clinical success of engineered musculoskeletal tissues. This study aimed to use solid brushite cement anchors to engineer intact ligaments from bone-to-bone, creating a functional musculoskeletal interface in vitro. We show here that modifying anchor shape and cement composition can alter both the longevity and the strength of an in vitro model of the bone–ligament interface: with values reaching 23 days and 21.6 kPa, respectively. These results validate the use of brushite bone cement to engineer the bone–ligament interface in vitro and raise the potential for future use in ligament replacement surgery.     

Collagenase production by rabbit ligaments and tendon

Three periarticular connective tissues from normal rabbits were examined for collagenolytic activity. Enzyme activity was secreted by cultures of anterior cruciate ligament (ACL), medial collateral ligament (MCL) and patellar tendon (PT). A lag period of six days or more was often observed prior to the detection of active collagenase. We attributed this to the presence of an excess of inhibitor in the early days of culture. We quantitated the amount of enzyme and inhibitor produced in 13 days. The levels of collagenase in the ACL and MCL were comparable. The PT, however, consistently secreted more enzyme than the two periarticular (ACL and MCL) ligaments. The reaction products were analyzed for all three collagenases and compared to those generated by the rabbit skin enzyme. We observed the characteristic TCA and TCB collagen fragments for MCL and PT enzymes. Collagen cleavage by the ACL cultures resulted in a product with a molecular weight intermediate between the alpha 2 chain and the TCA piece. These data suggest that quantitative and qualitative differences exist in the ability of these similar connective tissues to degrade collagen.     

Ligament strain on the iliofemoral,pubofemoral, and ischiofemoral ligaments in cadaver specimens: Biomechanical measurement and anatomical observation

The iliofemoral, pubofemoral, and ischiofemoral ligaments are major structures that stabilize the hip joint. We have sought evidence on which to base more effective hip stretching positions. The purpose of this study was to measure strains on these ligaments and to observe them. Eight fresh/frozen translumbar cadaver specimens were used. Clinically available stretching positions for these ligaments were adopted. Strain on each ligament was measured by a displacement sensor during passive torque to the hip joint. Hip motion was measured using an electromagnetic tracking device. The strained ligaments were captured on clear photographs. Significantly, high strains were imposed on the superior iliofemoral ligament by external rotation of the hip (3.48%); on the inferior iliofemoral ligament by maximal extension and 10° or 20° of external rotation with maximal extension (1.86%, 1.46%, 1.25%); on the pubofemoral ligament by maximal abduction and 10°, 20°, or 30° of external rotation with maximal abduction (3.18%, 3.28%, 3.11%, 2.99%); and on the ischiofemoral ligament by 10° or 20° of abduction with maximal internal rotation (7.11%, 7.83%). Fiber direction in each ligament was clearly identified. Significantly, high strains on hip ligaments corresponded with the anatomical direction of the ligament fibers. Positions were identified for each ligament that imposed maximal increase in strain on it. Clin. Anat. 27:1068–1075, 2014. © 2014 Wiley Periodicals, Inc.     

Biomechanical and neuromuscular comparison of single- and multi-planar jump tests and a side-cutting maneuver: Implications for ACL injury risk assessment

BackgroundNon-contact anterior cruciate ligament (ACL) injuries are a major problem among adolescent female soccer and handball players. Therefore, the aim of this study was to examine if known biomechanical and neuromuscular ACL injury risk factors obtained from single-planar jump-landings and multi-planar side-jumps can resemble the demands of side-cutting maneuvers, a known high-risk ACL injury movement for this population.MethodsTwenty-four female soccer and handball players (mean ± SD: age: 17 ± 1 year; height: 172 ± 66 cm; mass: 67 ± 9 kg) performed a series of functional tasks including two single-planar jump-landings, two multi-planar side-jumps and a sports-specific side-cutting maneuver on their dominant leg. Frontal and sagittal plane knee and hip joint kinematics and kinetics were calculated from three-dimensional motion analysis, whereas hamstring and quadriceps muscle pre-activity levels were measured with surface electromyography.ResultsThe sports-specific side-cut was distinguished by more knee flexion at initial contact, greater abduction angles and external knee abduction moments, higher biceps femoris and semitendinosus muscle pre-activity levels than both the single-planar jump-landings and multi-planar side-jumps (p < .05). Whilst, poor-to-strong spearman rank correlation coefficients inconsistently were found for the biomechanical and neuromuscular ACL injury risk factors explored between the side-cut and the single-planar jump-landings (r_s = 0.01–0.78) and multi-planar side-jumps (r_s = 0.03–0.88) respectively.ConclusionSingle-planar jump-landings and multi-planar side-jumps should be used with caution to test for non-contact ACL injury risk factors in adolescent female soccer and handball players, because they do not mimic the biomechanical nor neuromuscular demands of the most frequent injury situation.     

Effect of isolated hip abductor fatigue on single-leg landing mechanics and simulated ACL loading

BackgroundAltered movement biomechanics are a risk factor for ACL injury. While hip abductor weakness has been shown to negatively impact landing biomechanics, the role of this musculature and injury risk is not clear. The aim of this musculoskeletal simulation study was to determine the effect of hip abductor fatigue-induced weakness on ACL loading, force production of lower extremity muscles, and lower extremity biomechanics during single-leg landing.MethodsBiomechanical data from ten healthy adults were collected before and after a fatigue protocol and used to derive subject-specific estimates of muscle forces and ACL loading using a 5-degree of freedom (DOF) model.ResultsThere were no significant differences in knee joint angles and ACL loading between pre and post-fatigue. However, there were significant differences, due to fatigue, in lateral trunk flexion angle, total excursion of trunk, muscle forces, and joint moments.ConclusionAltered landing mechanics, due to hip abductor fatigue-induced weakness, may be associated with increased risk of ACL injury during single-leg landings. Clinical assessment or screening of ACL injury risk will benefit from subject-specific musculoskeletal models during dynamic movements. Future study considering the type of the fatigue protocols, cognitive loads, and various tasks is needed to further identify the effect of hip abductor weakness on lower extremity landing biomechanics.     

髂股韧带与股骨头韧带黏弹性实验研究

目的 研究新鲜成人尸体髋关节髂股韧带和股骨头韧带的黏弹性力学性质,为临床提供生物力学参数.方法 取正常国人髂股韧带和股骨头韧带各10个试样进行应力松弛、蠕变实验.结果 得出了应力松弛、蠕变实验曲线,以回归分析的方法处理实验数据,得出了回归系数.结论 髂股韧带7200s应力松弛蠕变量显著大于股骨头韧带.     

Regenerative tendon and ligament healing: opportunities with recombinant human platelet-derived growth factor BB-homodimer

Intrinsic tendon healing in response to injury is a reparative process that often results in formation of scar tissue with functional and mechanical properties inferior to those of the native tendon. Development of therapies that can promote regenerative, rather than reparative, healing hold the promise of improving patient recovery from tendon and ligament injuries by producing tissue that is morphologically and functionally equivalent to the native tissue. One therapeutic approach that has been a frequent topic of investigation in the preclinical literature is the use of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) to augment tendon and ligament repair. The chemotactic, mitogenic, and pro-angiogenic properties of rhPDGF-BB have been shown to result in recruitment and proliferation of tenogenic cells and a commensurate boost in extracellular matrix deposition and organization, improving the morphological and biomechanical properties of healing tendons and ligaments. The outcomes of the preclinical studies reviewed here strongly suggest that rhPDGF-BB will provide a new therapeutic opportunity to improve the treatment of injured tendons and ligaments.     

Mapping Lubricin in Canine Musculoskeletal Tissues

Lubricin, also known as superficial zone protein or PRG4, has many distinct biological functions, including lubrication, antiadhesion, and as a regulator of cell growth. This study investigated lubricin in canine musculoskeletal tissues using RT-PCR and immunohistochemistry. One or more variants were noted in canine flexor digitorum profundus (FDP) tendon, Achilles tendon, patellar tendon, A2 pulley, anterior cruciate ligament (ACL), knee lateral collateral ligament (LCL), articular cartilage, meniscus, muscle, and skin. We found 6 N-terminal lubricin splicing variants. The variants with larger sizes were identified in FDP tendon, ACL, LCL, A2 pulley, and cartilage. Lubricin was distributed both on the tissue surfaces and at the interface of fiber bundles within tissues, but this distribution varied by tissue type. We conclude that lubricin is present in many tissues; variations in splicing and physical distribution suggest that the variants of lubricin may play different roles in different locations.     

In situ IGF-1 gene delivery to cells emerging from the injured anterior cruciate ligament

Ruptures of the anterior cruciate ligament (ACL) are common knee injuries that do not heal, even with surgical repair. Our research is directed towards developing novel, biological approaches that enable suture repair of this ligament. One promising strategy involves the insertion of a collagen hydrogel between the severed ends of the ACL. Cells migrate from the damaged ligament into the hydrogel and produce repair tissue. Here we have investigated the potential for augmenting this process by the transfer of insulin like growth factor (IGF) 1 cDNA to the repair cells using an adenovirus vector. The goal is to achieve direct, in situ gene delivery by loading the hydrogel with vector prior to its insertion into the defect. In a step-wise approach towards evaluating this process, we confirmed that monolayers of ACL fibroblasts were efficiently transduced by adenovirus vectors and continued to express transgenes when subsequently incorporated into the hydrogel; indeed, transgene expression persisted longer within collagen gels than in monolayer culture. Transfer of IGF-1 cDNA increased the cellularity of the gels and led to the synthesis and deposition of increased amounts of types I and III collagen, elastin, tenascin, and vimentin. The cells remained viable, even when subjected to high viral loads. Similar results were obtained when collagen hydrogels were preloaded with adenovirus prior to insertion into an experimental ACL lesion in vitro. These data confirm the promise of using vector-laden hydrogels for the in situ delivery of genes to cells within damaged ligaments and suggest novel possibilities for the biological repair of the ACL.     

Biochemical study of collagen and its crosslinks in the anterior cruciate ligament and the tissues used as a graft for reconstruction of the anterior cruciate ligament

Among tissue grafts used for reconstruction of the anterior cruciate ligament (ACL), the pateller tendon (PT) and semitendinosus tendon (ST) are commonly used. It was thought that there were differences in the biochemical composition and process of healing between PT and ST. The aim of this study was to investigate the biochemical difference between ACL and the graft tissues used for reconstruction of the ACL. Hydroxyproline and crosslinks of collagen and elastin were measured from samples of 29 knees from cadavers preserved in formalin solutions. The results of measurements were hydroxyproline: ACL 0.522, PT 0.577, ST 0.463 (micromol/mg dry weight); pyridinoline/collagen: ACL 0.381, PT 0.272, ST 0.244 (mol/mol); and pentosidine/collagen: ACL 0.0434, PT 0.0558, ST 0.0799 (mol/mol). The biochemical properties of PT was not so different from ST. Pentosidine also was measured in the present study to aid in the comparison of the ligament and tendons of the knee joint.     

Mapping lubricin in canine musculoskeletal tissues

Lubricin, also known as superficial zone protein or PRG4, has many distinct biological functions, including lubrication, antiadhesion, and as a regulator of cell growth. This study investigated lubricin in canine musculoskeletal tissues using RT-PCR and immunohistochemistry. One or more variants were noted in canine flexor digitorum profundus (FDP) tendon, Achilles tendon, patellar tendon, A2 pulley, anterior cruciate ligament (ACL), knee lateral collateral ligament (LCL), articular cartilage, meniscus, muscle, and skin. We found 6 N-terminal lubricin splicing variants. The variants with larger sizes were identified in FDP tendon, ACL, LCL, A2 pulley, and cartilage. Lubricin was distributed both on the tissue surfaces and at the interface of fiber bundles within tissues, but this distribution varied by tissue type. We conclude that lubricin is present in many tissues; variations in splicing and physical distribution suggest that the variants of lubricin may play different roles in different locations.     

Assessment of Ligament Viscoelastic Properties Using Raman Spectroscopy

Injuries to the ligaments of the knee commonly impact vulnerable and physically active individuals. These injuries can lead to the development of degenerative diseases such as post-traumatic osteoarthritis (PTOA). Non-invasive optical modalities, such as infrared and Raman spectroscopy, provide means for quantitative evaluation of knee joint tissues and have been proposed as potential quantitative diagnostic tools for arthroscopy. In this study, we evaluate Raman spectroscopy as a viable tool for estimating functional properties of collateral ligaments. Artificial trauma was induced by anterior cruciate ligament transection (ACLT) in the left or right knee joint of skeletally mature New Zealand rabbits. The corresponding contralateral (CL) samples were extracted from healthy unoperated joints along with a separate group of control (CNTRL) animals. The rabbits were sacrificed at 8 weeks after ACLT. The ligaments were then harvested and measured using Raman spectroscopy. A uniaxial tensile stress-relaxation testing protocol was adopted for determining several biomechanical properties of the samples. Partial least squares (PLS) regression models were then employed to correlate the spectral data with the biomechanical properties. Results show that the capacity of Raman spectroscopy for estimating the biomechanical properties of the ligament samples varies depending on the target property, with prediction error ranging from 15.78% for tissue cross-sectional area to 30.39% for stiffness. The hysteresis under cyclic loading at 2 Hz (RMSE = 6.22%, Normalized RMSE = 22.24%) can be accurately estimated from the Raman data which describes the viscous damping properties of the tissue. We conclude that Raman spectroscopy has the potential for non-destructively estimating ligament biomechanical properties in health and disease, thus enhancing the diagnostic value of optical arthroscopic evaluations of ligament integrity.

     

膝关节损伤修复后的生物力学分析

背景：对膝关节及膝关节韧带进行生物力学分析是防止膝关节损伤和治疗膝关节疾病的基础。 目的：总结膝关节损伤重建术中修复材料的应用进展及其修复后膝关节的生物力学特征。 方法：以“膝关节、韧带、半月板、生物材料、生物力学”为中文关键词,以“ tissue enginneering,articular cartilage,scaffold material,biomechanics” 为英文关键词,采用计算机检索中国期刊全文数据库、PubMed数据库相关文章。重点对修复后膝关节的生物力学特征进行了讨论。 结果与结论：生物力学研究证明膝关节后外侧结构在限制膝关节内翻,胫骨外旋、前移、后移有着重要作用。膝关节内韧带部分或完全断裂对股骨内髁生物力学特性均有不良影响,膝关节每条韧带又与其他韧带和组织协同完成某些方面的功能达到力学平衡,其中一条韧带断裂,平衡就会遭到破坏,各条韧带的应力分布将发生改变。膝关节的损伤类型与部位各有不同,修复与重建的材料与重建手段也各有不同,最终其生物力学表现特征各有不同。修复材料生物力学性能研究对于移植后膝关节生物塑形、愈合过程以及功能恢复都有很重要的作用。           

Relaxin Receptor RXFP1 and RXFP2 Expression in Ligament,Tendon, and Shoulder Joint Capsule of Rats

Numerous musculoskeletal disorders are caused by thickened ligament, tendon stiffness, or fibrosis of joint capsule. Relaxin, a peptide hormone, can exert collagenolytic effect on ligamentous and fibrotic tissues. We hypothesized that local injection of relaxin could be used to treat entrapment neuropathy and adhesive capsulitis. Because hormonal effect depends on the receptor of the hormone on the target cell, it is important to confirm the presence of such hormonal receptor at the target tissue before the hormone therapy is initiated. The aim of this study was to determine whether there were relaxin receptors in the ligament, tendon, and joint capsular tissues of rats and to identify the distribution of relaxin receptors in these tissues. Transverse carpal ligaments (TCLs), inguinal ligaments, anterior cruciate ligaments (ACLs), Achilles tendons, and shoulder joint capsules were obtained from male Wistar rats. Western blot analysis was used to identify relaxin receptor isoforms RXFP1 and RXFP2. The distribution of relaxin receptors was determined by immunohistochemical staining. The RXFP1 isoform was found in all tissues examined. The RXFP2 isoform was present in all tissues but the TCLs. Its expression in ACLs tissues was relatively weak compared to that in other tissues. Our results revealed that RXFP1 and RXFP2 were distributed in distinctly different patterns according to the type of tissue (vascular endothelial cells, fibroblast-like cells) they were identified.     

Mechanism of muscle–tendon–bone complex development in the head

The musculoskeletal system comprises muscles, tendons, ligaments, and bones. The connection site between muscle and tendon is termed “myotendinous junction,” while the junction between tendon/ligament and bone is termed “enthesis.” These two regions are the center of physical function, but how this functional complex is formed during development is unclear. In this review, we discussed recent findings about the development of tissues constituting the musculoskeletal system and the interactions among these tissues during development. The musculoskeletal system of the head develops in the mid-embryonic stage. In addition, head mesoderm-derived cells (muscle anlagen) and cranial neural crest cells (tendon and bone anlagen) interact with each other. Myogenesis initiates in the head without difficulty, even in the absence of cranial neural crest cells; however, muscle tissue does not grow under these conditions and remains small. Tendons, which differentiate from cranial neural crest cells, form myotendinous junctions at the stage at which desmin accumulates in the tendon-side muscle stump, leading to morphological maturation. Therefore, individual tissues (i.e., muscles, tendons, ligaments, and bones) constituting the musculoskeletal system form a functionally important complex, while mutually influencing one another.