Decreasing inflammatory response of injured patellar tendons results in increased collagen fibril diameters

Tissue inflammation is essential in the healing process, but its effect on the quality of the healing tissue is not clear. This study determines the effect of decreasing early inflammation during wound healing in genetic deficient mice on collagen fibril diameter. Two strains of mice were used: three C3H/HeJ mice and three C3H/HeN mice for each of two time points (7 and 14 days postinjury). C3H/HeJ mice have a genetic deficiency in the production of tumor necrosis factor by macrophages and other cytokines in response to endotoxin, and C3H/HeN mice have no genetic deficiency. The right patellar tendon of both mouse strains was transversely transected, whereas the left patellar tendon was left intact for control. After 7 and 14 days, both right and left patellar tendons were harvested, and tendon samples were examined with transmission electron microscopy. We found that at 7 days, transected tendons of C3H/HeJ mice exhibited on average 1.6 times larger collagen fibril diameters than transected C3H/HeN tendons, whereas at 14 days, collagen fibril diameters of the C3H/HeJ mice were 1.3 times that of C3H/HeN mice. Also, at both 7 days and 14 days, collagen fibrils in C3H/HeJ mice appeared more organized than C3H/HeN mice. In addition, control tendons in both mouse strains showed no significant differences in collagen fibril diameter and organization. Therefore, these results suggest that decreasing the inflammatory response in the early stages of tendon wound healing enhances the quality of the healing tendon through increased collagen fiber diameter and better organization.     

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 determinants of murine achilles tendon strength during healing

The mechanisms by which tendon strength is established during growth and development and restored following injury are not completely understood and are likely to be complex, multifactorial processes. Several studies examining the relationship between mechanical behavior and ultrastructural characteristics of tendons and ligaments during growth and maturation suggest that collagen fibril diameter is strongly correlated with tendon strength. Because of the similarities between development and repair processes of musculoskeletal tissues, increases in tendon strength during healing may be related to increases in fibril ultrastructural parameters such as fibril size, numerical density, and area fraction. In this study, we compared murine Achilles tendons at various time points after tenotomy with sham-operated controls in tensile tests to failure and examined tendons using electron microscopy to assess collagen fibril ultrastructure. We found that in the 6-week period following Achilles tenotomy, fibril mean diameter remained significantly smaller than sham-side diameter by a factor of 2-3. Despite the persistently small fibril size, increasing numerical density resulted in a gradual increase in fibril area fraction. Biomechanical strength did not reach that of intact tendons until some time between 5 and 7 weeks, approximately the same time period when fibril area fraction began to approach sham values. These data suggest that parameters other than collagen fibril size are most responsible for increased tendon strength during healing.     

Ultrastructural Determinants of Murine Achilles Tendon Strength During Healing

The mechanisms by which tendon strength is established during growth and development and restored following injury are not completely understood and are likely to be complex, multifactorial processes. Several studies examining the relationship between mechanical behavior and ultrastructural characteristics of tendons and ligaments during growth and maturation suggest that collagen fibril diameter is strongly correlated with tendon strength. Because of the similarities between development and repair processes of musculoskeletal tissues, increases in tendon strength during healing may be related to increases in fibril ultrastructural parameters such as fibril size, numerical density, and area fraction. In this study, we compared murine Achilles tendons at various time points after tenotomy with sham-operated controls in tensile tests to failure and examined tendons using electron microscopy to assess collagen fibril ultrastructure. We found that in the 6-week period following Achilles tenotomy, fibril mean diameter remained significantly smaller than sham-side diameter by a factor of 2–3. Despite the persistently small fibril size, increasing numerical density resulted in a gradual increase in fibril area fraction. Biomechanical strength did not reach that of intact tendons until some time between 5 and 7 weeks, approximately the same time period when fibril area fraction began to approach sham values. These data suggest that parameters other than collagen fibril size are most responsible for increased tendon strength during healing.     

The adaptability of tendon to loading differs in men and women

The reason why women sustain more soft tissue injury than men during physical activity is unknown. Connective tissue properties and extracellular matrix adaptability in human tendon were investigated in models that addressed biochemical, physiological and biomechanical aspects of tendon connective tissue in response to mechanical loading. Habitual training resulted in a larger patellar tendon in men but not in women. Following an acute bout of exercise, men had an elevated tendon collagen synthesis rate and this effect was less pronounced or absent in women. Moreover, levels of circulating oestrogen affected the acute exercise-related increase in collagen synthesis. Finally, the mechanical strength of isolated tendon collagen fascicles in men surpassed that of women. Thus, compared to men, women have (i) an attenuated tendon hypertrophy response to habitual training; (ii) a lower tendon collagen synthesis rate following acute exercise; (iii) a rate of tendon collagen synthesis which is further attenuated with elevated estradiol levels; and (iv) a lower mechanical strength of their tendons. These data indicate that tendons in women have a lower rate of new connective tissue formation, respond less to mechanical loading, and have a lower mechanical strength, which may leave the tissue more susceptible to injury.     

The effect of mechanical stimulation on the maturation of TDSCs-poly(L-lactide-co-e-caprolactone)/collagen scaffold constructs for tendon tissue engineering

Mechanical stimulation plays an important role in the development and remodeling of tendons. Tendon-derived stem cells (TDSCs) are an attractive cell source for tendon injury and tendon tissue engineering. However, these cells have not yet been fully explored for tendon tissue engineering application, and there is also lack of understanding to the effect of mechanical stimulation on the maturation of TDSCs-scaffold construct for tendon tissue engineering. In this study, we assessed the efficacy of TDSCs in a poly(L-lactide-co-ε-caprolactone)/collagen (P(LLA-CL)/Col) scaffold under mechanical stimulation for tendon tissue engineering both in vitro and in vivo, and evaluated the utility of the transplanted TDSCs-scaffold construct to promote rabbit patellar tendon defect regeneration. TDSCs displayed good proliferation and positive expressed tendon-related extracellular matrix (ECM) genes and proteins under mechanical stimulation in vitro. After implanting into the nude mice, the fluorescence imaging indicated that TDSCs had long-term survival, and the macroscopic evaluation, histology and immunohistochemistry examinations showed high-quality neo-tendon formation under mechanical stimulation in vivo. Furthermore, the histology, immunohistochemistry, collagen content assay and biomechanical testing data indicated that dynamically cultured TDSCs-scaffold construct could significantly contributed to tendon regeneration in a rabbit patellar tendon window defect model. TDSCs have significant potential to be used as seeded cells in the development of tissue-engineered tendons, which can be successfully fabricated through seeding of TDSCs in a P(LLA-CL)/Col scaffold followed by mechanical stimulation.     

Diabetes Alters Mechanical Properties and Collagen Fiber Re-Alignment in Multiple Mouse Tendons

Tendons function to transfer load from muscle to bone through their complex composition and hierarchical structure, consisting mainly of type I collagen. Recent evidence suggests that type II diabetes may cause alterations in collagen structure, such as irregular fibril morphology and density, which could play a role in the mechanical function of tendons. Using the db/db mouse model of type II diabetes, the diabetic skin was found to have impaired biomechanical properties when compared to the non-diabetic group. The purpose of this study was to assess the effect of diabetes on biomechanics, collagen fiber re-alignment, and biochemistry in three functionally different tendons (Achilles, supraspinatus, patellar) using the db/db mouse model. Results showed that cross-sectional area and stiffness, but not modulus, were significantly reduced in all three tendons. However, the tendon response to load (transition strain, collagen fiber re-alignment) occurred earlier in the mechanical test, contrary to expectations. In addition, the patellar tendon had an altered response to diabetes when compared to the other two tendons, with no changes in fiber re-alignment and decreased collagen content at the midsubstance of the tendon. Overall, type II diabetes alters tendon mechanical properties and the dynamic response to load.     

Autologous fibrin matrices: a potential source of biological mediators that modulate tendon cell activities

The use of autologous fibrin matrices has been proposed as a therapeutic strategy for the local and physiological delivery of growth factors in the treatment of several clinical conditions requiring tendon healing or tendon graft remodelling. In the present work, we investigated the proliferation, synthesis of type-I collagen and angiogenic factors by tendon cells seeded on platelet-rich (PR) and platelet-poor (PP) matrices. Furthermore, in vivo cellular and vascular effects of each treatment were examined after infiltration in Achilles tendon in sheep. Results showed that the presence of platelets within the fibrin matrices increased significantly the proliferation of tendon cells. Additionally, cultured tendon cells synthesised type I collagen and angiogenic factors such as VEGF and HGF. The synthesis of VEGF, but not of HGF, was significantly higher when platelets were present within the matrix. In the sheep model, the injection of pre-clotted plasma within tendons increased cellular density and promoted neovascularization. These results indicate that administration of fibrin matrices is a safe and easy strategy that may open new avenues for enhancing tissue healing and remodelling and influences the process of regeneration in clinical situations characterised by a poor healing outcome.     

Response of a collagenase-induced tendon injury to treatment with a polysulphated glycosaminoglycan (Adequan)

This study explored the hypothesis that local administration of a polysulphated glycosaminoglycan (PSGAG) in the early phase of healing of a standard collagenase-induced tendon injury in the superficial digital flexor tendon of the rabbit would reduce the degenerative effects of inflammatory mediators and proteases and preserve normal tendon morphology, composition, and biomechanical properties. Histological and ultrastructural changes together with the mechanical properties, dry weight, collagen content, and amount of DNA in healing tissue at the site of the lesion were assessed in treated and untreated animals. In treated lesions 28 days after injury, the normal orientation of tenoblasts and collagen fibrils was well preserved compared with the disorganized scar formation seen in untreated animals. The degree of cellularity was significantly higher in the untreated lesions. At the ultrastructural level the collagen in the healing tissue of the treated animals consisted of a mixture of small diameter, new regenerated fibrils intermingled with well-preserved large diameter, old fibrils, aligned to the long axis of the tendon; in untreated animals small, randomly arranged new fibrils predominated. The diameters of treated tendons had returned to normal, but in untreated animals the injured tendons remained significantly thicker than their controls. The percentage dry weight and collagen contents of treated injured tendons approximated those of control normal tendons, whereas those of untreated tendons were significantly less than those of the control values. The DNA content of injured treated tendons was not significantly different from that of normal contralateral controls, while in the untreated tendons it was significantly higher. There were no significant differences between the normal and the contralateral treated injured tendons in ultimate strength, fatigue strength, stiffness, and maximum absorbed energy. However in the untreated animals, although the tendon diameter was significantly greater, the ultimate strength, fatigue strength, stiffness, and maximum absorbed energy were significantly lower than the contralateral control. These data suggest that polysulphated glycosaminoglycans are effective in restoring the morphological, biochemical, and biomechanical properties of injured soft connective tissues and may be of clinical value in the treatment of acute tendon injury.     

Response of a Collagenase-Induced Tendon Injury to Treatment with a Polysulphated Glycosaminoglycan (Adequan)

This study explored the hypothesis that local administration of a polysulphated glycosaminoglycan (PSGAG) in the early phase of healing of a standard collagenase-induced tendon injury in the superficial digital flexor tendon of the rabbit would reduce the degenerative effects of inflammatory mediators and proteases and preserve normal tendon morphology, composition, and biomechanical properties. Histological and ultrastructural changes together with the mechanical properties, dry weight, collagen content, and amount of DNA in healing tissue at the site of the lesion were assessed in treated and untreated animals. In treated lesions 28 days after injury, the normal orientation of tenoblasts and collagen fibrils was well preserved compared with the disorganized scar formation seen in untreated animals. The degree of cellularity was significantly higher in the untreated lesions. At the ultrastructural level the collagen in the healing tissue of the treated animals consisted of a mixture of small diameter, new regenerated fibrils intermingled with well-preserved large diameter, old fibrils, aligned to the long axis of the tendon; in untreated animals small, randomly arranged new fibrils predominated. The diameters of treated tendons had returned to normal, but in untreated animals the injured tendons remained significantly thicker than their controls. The percentage dry weight and collagen contents of treated injured tendons approximated those of control normal tendons, whereas those of untreated tendons were significantly less than those of the control values. The DNA content of injured treated tendons was not significantly different from that of normal contralateral controls, while in the untreated tendons it was significantly higher. There were no significant differences between the normal and the contralateral treated injured tendons in ultimate strength, fatigue strength, stiffness, and maximum absorbed energy. However in the untreated animals, although the tendon diameter was significantly greater, the ultimate strength, fatigue strength, stiffness, and maximum absorbed energy were significantly lower than the contralateral control. These data suggest that polysulphated glycosaminoglycans are effective in restoring the morphological, biochemical, and biomechanical properties of injured soft connective tissues and may be of clinical value in the treatment of acute tendon injury.     

Collagen Fibrillogenesis and mRNA Levels in the Maturing Rabbit Medial Collateral Ligament and Patellar Tendon

This study compared collagen fibril diameter and mRNA changes in a subset of molecules involved in collagen fibrillogenesis during postnatal development and at maturity of rabbit medial collateral ligament (MCL) and patellar tendon (PT). Tissue was analyzed by RT-PCR for mRNA levels and collagen fibril diameters were measured using transmission electron microscopy. Collagen fibril diameters increased from 3 to 14 weeks with mean fibril diameters of PT significantly greater than MCL at 9, 12, and 14 weeks and maturity. RT-PCR analysis showed decorin and lumican mRNA levels were significantly higher in PT than MCL at all ages. Type I collagen, MMP-11, and procollagen C proteinase enhancer mRNA levels also were higher in the PT than the MCL between 3 and 14 weeks but not at maturity. Further understanding of collagen fibrillogenesis by studying protein synthesis and matrix turnover during maturation may provide insight into the mechanism(s) by which fibrils accrete in maturing connective tissues and how they are altered during healing following injury.     

Collagen fibrillogenesis and mRNA levels in the maturing rabbit medial collateral ligament and patellar tendon

This study compared collagen fibril diameter and mRNA changes in a subset of molecules involved in collagen fibrillogenesis during postnatal development and at maturity of rabbit medial collateral ligament (MCL) and patellar tendon (PT). Tissue was analyzed by RT-PCR for mRNA levels and collagen fibril diameters were measured using transmission electron microscopy. Collagen fibril diameters increased from 3 to 14 weeks with mean fibril diameters of PT significantly greater than MCL at 9, 12, and 14 weeks and maturity. RT-PCR analysis showed decorin and lumican mRNA levels were significantly higher in PT than MCL at all ages. Type I collagen, MMP-11, and procollagen C proteinase enhancer mRNA levels also were higher in the PT than the MCL between 3 and 14 weeks but not at maturity. Further understanding of collagen fibrillogenesis by studying protein synthesis and matrix turnover during maturation may provide insight into the mechanism(s) by which fibrils accrete in maturing connective tissues and how they are altered during healing following injury.     

碳化二亚胺交联改性脱细胞异种猪肌腱修复跟腱缺损

背景：异体肌腱移植是目前修复肌腱缺损的理想方法,但移植后的排斥反应使其使用受到限制。 目的：观察碳化二亚胺交联改性脱细胞处理的版纳近交系微型猪肌腱移植修复兔跟腱缺损的效果。 方法：横向切除40只日本大白兔双侧跟腱,随机分组：实验组以碳化二亚胺交联改性脱细胞版纳近交系微型猪肌腱移植修复,对照组以自体肌腱移植修复。 结果与结论：①组织学观察：两组新生组织内细胞主要都是单核的成纤维细胞和纤维细胞,术后1-4周主要是呈椭圆形或圆形的成纤维细胞,细胞聚集区的细胞周围有新生胶原形成,这些胶原的走向较紊乱;局灶性条索状成熟胶原呈岛状分布,形成所谓“胶原岛”;术后12周时细胞越来越拉长,成为梭形和长条形的纤维细胞。②实验室检测：两组白细胞、C-反应蛋白水平、羟脯氨酸含量及抗拉强度差异均无显著性意义。说明碳化二亚胺交联改性的脱细胞版纳近交系微型猪肌腱能成功修复兔跟腱缺损,且具有组织相容性好、移植排斥反应轻、生物力学性能强的优点。     

Pericellular proteins of the developing mouse tendon: a proteomic analysis

Tendon fibroblasts synthesize and assemble collagen fibrils, the basic structural unit of tendons. Regulation of fibrillogenesis is essential for tendon development and function. Fibril assembly begins within extracellular micro-domains associated with the fibroblast surface. We hypothesize that molecules crucial to the regulation of fibril assembly are membrane associated and/or within the pericellular micro-environment. This report defines proteins in the surfaceome, that is, plasma membrane and pericellular matrix, from mouse flexor digitorum longus tendons. Proteomic analysis identified a set of surfaceome molecules including collagens, fibronectin, integrins, proteoglycans, and receptors in extracts from mouse tendons at postnatal day 1, a developmental stage when collagen protofibril nucleation and initial steps in fibril assembly predominate. The proteomic results were validated for molecules identified with a small number of unique peptides and/or low sequence coverage. For these analyses, proteins were selected based on their potential roles in fibril nucleation, that is, collagen V; organization of fibrillogenesis, that is, integrins and fibronectin; and known localization to the plasma membrane with potential to impact matrix assembly, that is, CD44, syndecan-1, epidermal growth factor receptor, and matrix metalloproteinase 25. These molecules were all detected in extracts of the developing tendon, demonstrating that the surfaceome included molecules hypothesized to regulate fibrillogenesis as well as many with no known function in this capacity. This report, therefore, generates an unbiased set of cell surface-associated molecules, providing a resource to identify novel or unexpected regulatory molecules involved in collagen fibril and matrix assembly.     

bFGF复合膜对同种异体肌腱移植的作用的实验研究

目的　用bFGF复合膜 ,包裹在腱移植处 ,探讨腱内部再生是否快于腱周结缔组织增生 ,从而达到防止或减轻腱粘连的目的。方法　用 - 80℃冰箱冷存 10天的同种异体跟腱缝接 30只兔左肢跟腱缺损 2 .0cm处 ,分两组 :A组在腱移植处包裹bFGF复合非降解膜 (药膜 )为实验组 ,B组在腱移植处包裹无药的非解降解膜 (无药膜 )为对照组 ,术后不同时间 ,切取腱移植部位 ,常规制成光、电镜标本 ,再行镜下观察 ,图像分析仪测定和腱周粘连定量测定。结果　包药膜腱移植段内部的成纤维细胞和胶原纤维明显较无药膜的多 (<0 0 1) ,包无药膜腱周结缔组织的成纤维细胞和胶原纤维明显较包膜的多 (<0 0 1) ,包药膜腱移植段内部的成纤维细胞和胶原纤维明显较腱周结缔组织的多 (<0 1) ,无药膜的明显较腱周的少 (<0 0 1)。结论　bFGF复合膜有增强腱内部再生和减轻或防止腱粘连的作用     

Biomechanical properties and dry weight content of the developing superficial digital flexor tendon in rabbit

Composition and structural organization of tendon changes during aging and these alterations affect the mechanical behaviors of this structure. Therefore, this experiment was designed to study the biomechanical properties together with changes in dry weight content of normal superficial digital flexor tendon of rabbits from pre-natal stage to 112 days post-natally. Forty-two White New Zealand rabbits were assigned to seven different age groups (from 5–7 days before birth to 112 days after birth), each consisting of six animals. The right superficial digital flexor tendons were used for biomechanical studies and the left ones for percentage dry weight investigation. Ultimate tensile strength, stiffness, maximum energy, and percentage dry weight values significantly increased in each higher age group compared to those of the younger group and the yield strain and maximum strain decreased comparatively as a function of age. This improvement in the mechanical behavior of tendons during aging could be correlated with increase in collagen content, alteration in the collagen fibril differentiation and distribution from small-sized unimodal fibrils to trimodally distributed collagen fibrils, improvement in quantity and quality of the cross-linking, fibril continuity, type of collagen, development and maturation of crimp pattern, tissue alignment and organization. Therefore, characterization of mechanical behavior and tissue dry weight, as an index of collagen content, from fetal stage to skeletally mature animals is essential in better understanding the tissue structural development and hierarchical organization coincidental with the material properties of this organ.     

Expression, content, and localization of insulin-like growth factor I in human achilles tendon

In animals insulin-like growth factor I (IGF-I) stimulates collagen production by fibroblasts and is expressed in tendons together with its binding protein 4 (IGFBP-4). However, the presence of IGF-I and IGFBP-4 in human tendon tissue is not described. Tissue IGF-I content was examined by immunoflourometric assay, real-time PCR, and immunohistochemistry used to localize and determine expression of IGF-I and IGFBP-4 in 6 postmortem human Achilles tendons. Tendon tissue concentrations of IGF-I were found to be 0.53 +/- 0.10 ng/g. Furthermore, we demonstrated that IGF-I and IGFBP-4 are localized around the tendon fibroblasts and that mRNA for IGF-I and IGFBP-4 can be determined in human tendon tissue. The present study adds support for the roles of IGF-I and IGFBP-4 in the regulation of tendon adaptive responses to mechanical loading.