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.     

Effects of sodium-hyaluronate and glucosamine-chondroitin sulfate on remodeling stage of tenotomized superficial digital flexor tendon in rabbits: a clinical, histopathological, ultrastructural, and biomechanical study

This study was designed to evaluate the effects of sodium-hyaluronate (NaH) combined with glucosamine HCl-chondroitin sulfate (GlcN-CS) on the post-surgical repair of tendon rupture on day 84 post injury. Twenty white New Zealand female rabbits were divided randomly into two equal groups of injured treated and injured untreated. After tenotomy and surgical repair, using the modified Kessler technique and running pattern, the injured legs were casted for 14 days. NaH was injected subcutaneously over the lesion on days 3, 7, and 10 and was followed by daily oral administration of GlcN-CS on days 3 to 23 post injury. The control animals received normal saline injection and oral placebo similarly. The weight of the animals, tendon diameter, clinical manifestations, and radiographic and ultrasonographic evaluations were conducted for 12 weeks. The rabbits were euthanized 84 days post injury and the tendons were evaluated at macroscopic, histopathologic, and ultrastructural level and were assessed for biomechanical and percentage dry-weight parameters. Treatment significantly reduced the tendon diameter and ultimate and yield strain, and increased the echogenicity, dry-weight content, ultimate and yield strength, and stress and stiffness of the injured tendons compared to those of the untreated ones. Treatment also significantly enhanced the maturation rate of the tenoblasts, fibrillogenesis, the diameters of the collagen fibrils, and fibrillar density. These findings suggest that a combined treatment of NaH and GlcN-CS could be effective in restoring the morphological and biomechanical properties of injured superficial digital flexor tendon of rabbits and might be helpful for future clinical trial studies in tendon ruptures.     

Effects of sodium-hyaluronate and glucosamine-chondroitin sulfate on remodeling stage of tenotomized superficial digital flexor tendon in rabbits: a clinical,histopathological, ultrastructural,and biomechanical study

This study was designed to evaluate the effects of sodium-hyaluronate (NaH) combined with glucosamine HCl-chondroitin sulfate (GlcN-CS) on the post-surgical repair of tendon rupture on day 84 post injury. Twenty white New Zealand female rabbits were divided randomly into two equal groups of injured treated and injured untreated. After tenotomy and surgical repair, using the modified Kessler technique and running pattern, the injured legs were casted for 14 days. NaH was injected subcutaneously over the lesion on days 3, 7, and 10 and was followed by daily oral administration of GlcN-CS on days 3 to 23 post injury. The control animals received normal saline injection and oral placebo similarly. The weight of the animals, tendon diameter, clinical manifestations, and radiographic and ultrasonographic evaluations were conducted for 12 weeks. The rabbits were euthanized 84 days post injury and the tendons were evaluated at macroscopic, histopathologic, and ultrastructural level and were assessed for biomechanical and percentage dry-weight parameters. Treatment significantly reduced the tendon diameter and ultimate and yield strain, and increased the echogenicity, dry-weight content, ultimate and yield strength, and stress and stiffness of the injured tendons compared to those of the untreated ones. Treatment also significantly enhanced the maturation rate of the tenoblasts, fibrillogenesis, the diameters of the collagen fibrils, and fibrillar density. These findings suggest that a combined treatment of NaH and GlcN-CS could be effective in restoring the morphological and biomechanical properties of injured superficial digital flexor tendon of rabbits and might be helpful for future clinical trial studies in tendon ruptures.     

Bone marrow stromal cells in a liquid fibrin matrix improve the healing process of patellar tendon window defects

Following injury, ligaments and tendons do not regain their normal biological and biomechanical status. This study analyzed whether an injection of human bone marrow stromal cells (BMSC) or human fibroblast in a liquid fibrin matrix influences the histological results, ultrastructural morphology, mRNA expression of essential extracellular matrix proteins, and material properties of the healing tissue. Standardized full-thickness, full-length defects of the central portion of patellar tendons were created in 96 immunodeficient rats, and filled with human BMSC in a fibrin matrix (BMSC group), human fibroblasts in a fibrin matrix (fibroblast group), or fibrin matrix only (matrix group), or left untreated (defect group). Histological sections revealed more mature tissue formation with more regular patterns of cell distribution in the BMSC group, without signs of ectopic tissue formation into bone or cartilage. Mean collagen fibril diameter and relative area covered by collagen fibrils were significantly higher at 10 and 20 days postoperatively in the BMSC group compared to the defect and matrix groups, and comparable to normal tendon tissue. Further, collagen I mRNA expression, collagen I/collagen III mRNA ratio, and Young's modulus were significantly increased at 20 days postoperatively in comparison to the defect and matrix groups. In the fibroblast group, only mean collagen fibril diameter was significantly higher compared to the defect group, whereas the other biological and biomechanical parameters were not significantly improved. This study reveals that an injection of BMSC in a liquid fibrin matrix stimulates histological, ultrastructural, molecular biologic, and biomechanical parameters of patellar tendon healing, whereas injection of fibroblasts in fibrin matrix had only minor effects on the stimulation of tendon healing.     

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.     

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 role of three-dimensional pure bovine gelatin scaffolds in tendon healing,modeling, and remodeling: an in vivo investigation with potential clinical value

Aim of the study: Large tendon defects involving extensive tissue loss present complex clinical problems. Surgical reconstruction of such injuries is normally performed by transplanting autogenous and allogenous soft tissues that are expected to remodel to mimic a normal tendon. However, the use of grafts has always been associated with significant limitations. Tissue engineering employing artificial scaffolds may provide acceptable alternatives. Gelatin is a hydrolyzed form of collagen that is bioactive, biodegradable, and biocompatible. The present study has investigated the suitability of gelatin scaffold for promoting healing of a large tendon-defect model in rabbits. Materials and methods: An experimental model of a large tendon defect was produced by partial excision of the Achilles tendon of the left hind leg in adult rabbits. To standardize and stabilize the length of the tendon defect a modified Kessler core suture was anchored in the sectioned tendon ends. The defects were either left untreated or filled with three-dimensional gelatin scaffold. Before euthanasia 60 days after injury, the progress of healing was evaluated clinically. Samples of healing tendon were harvested at autopsy and evaluated by gross, histopathologic, scanning, and transmission electron microscopy, and by biomechanical testing. Results: The treated animals showed superior weight-bearing and physical activity compared with those untreated, while frequency of peritendinous adhesions around the healing site was reduced. The gelatin scaffold itself was totally degraded and replaced by neo-tendon that morphologically had significantly greater numbers, diameters, density, and maturation of collagen fibrils, fibers, and fiber bundles than untreated tendon scar tissue. It also had mechanically higher ultimate load, yield load, stiffness, maximum stress and elastic modulus, when compared to the untreated tendons. Conclusion: Gelatin scaffold may be a valuable option in surgical reconstruction of large tendon defects.     

A Novel Application of Biosynthetic Tissue-Engineered Tridimensional Implant on Large Tendon Defects: A Comprehensive,Detailed, In Vivo Investigation with Significant Clinical Value

This study was designed to investigate the effect of a biosynthetic implant on tendon healing in vivo. Fifty white New Zealand male rabbits were randomly divided into two groups, namely treated (n = 25) and control (n = 25) groups. A large gap was created in the Achilles tendon and was maintained by Kessler pattern. In the treated group, the implant was inserted in the injured area. No implant was used in the control group. Contrast radiography, hematology, and clinical examination were conducted during the course of the experiment. The animals were euthanized at 60 days post injury (DPI) and their Achilles tendons were subjected to the gross, histopathologic, and biomechanical analyses and the hydroxyproline content of these tendons was also evaluated. Another five treated animals, as a pilot group, were used to define the inflammatory reaction at 10 DPI. Severe inflammatory reaction was initiated by the partially degraded implant, at 10 DPI. However, at 60 DPI, the inflammation subsided, the implant was mostly removed but a few small remnants were still present in the injured area. The newly formed tendon, properly aligned along the longitudinal axis of the Achilles tendon replaced the collagen implant. In the control tendons, a loose areolar connective tissue which tightly adhered to the peri-tendinous tissue was the only regenerated structure in the injured area. At this stage, the treated tendons showed significantly higher ultimate strength (p = 0.001), yield strength (p = 0.001), and stiffness (p = 0.001) compared with the control ones. Application of the biosynthetic implant was a safe and effective option in managing the large tendon defects and could be considered as a substitute for autografts in clinical practice.     

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.     

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.     

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.     

The Influence of Inflammation-Mediated Osteopenia (IMO) on the Structure of Rabbit Bone and Skin Collagen Fibrils

The influence of Inflammation Mediated Osteopenia (IMO) on rabbit skin and bone (trabecular and cortical) collagen fibrils was studied by electron microscopy. Severe abnormalities in collagen fibril structure were detected, at the ultrastructural level, in skin and bone specimens from IMO rabbits. In treated animals the arrangement of fibrils is anarchic. The overall collagen fibril architecture is disturbed compared to normal. IMO collagen fibrils' mean diameter values were significantly larger than those from controls, in all examined tissues. However, the banding patterns of fibrils were normal in all cases. Computer analysis shows no differences in charged amino acid composition between IMO and untreated samples. Our results show a correlation between the effects induced by osteopenia on skin and bone collagen.     

GDF-5 deficiency in mice leads to disruption of tail tendon form and function

Although the biological factors which regulate tendon homeostasis are poorly understood, recent evidence suggests that Growth and Differentiation Factor-5 (GDF-5) may play a role in this important process. The purpose of this study was to investigate the effect of GDF-5 deficiency on mouse tail tendon using the brachypodism mouse model. We hypothesized that GDF-5 deficient tail tendon would exhibit altered composition, ultrastructure, and biomechanical behavior when compared to heterozygous control littermates. Mutant tail tendons did not display any compositional differences in sulfated glycosaminoglycans (GAG/DNA), collagen (hydroxyproline/DNA), or levels of fibromodulin, decorin, or lumican. However, GDF-5 deficiency did result in a 17% increase in the proportion of medium diameter (100-225 nm) collagen fibrils in tail tendon (at the expense of larger fibrils) when compared to controls (p < 0.05). Also, mutants exhibited a trend toward an increase in irregularly-shaped polymorphic fibrils (33% more, p > 0.05). While GDF-5 deficient tendon fascicles did not demonstrate any significant differences in quasistatic biomechanical properties, mutant fascicles relaxed 11% more slowly than control tendons during time-dependent stress-relaxation tests (p < 0.05). We hypothesize that this subtle alteration in time-dependent mechanical behavior is most-likely due to the increased prevalence of irregularly shaped type I collagen fibrils in the mutant tail tendons. These findings provide additional evidence to support the conclusion that GDF-5 may play a role in tendon homeostasis in mice.     

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。     

Curcumin improves tendon healing in rats: a histological,biochemical, and functional evaluation

Curcumin, a compound extracted from the roots of Zingiberaceae, has been proposed as a treatment for tissue injury but studies are yet to be done on its effect on tendon healing. Therefore, we performed a series of experiments to test our hypothesis that curcumin has positive effects on tendon repair. Patellar tendon window defect was created in Sprague–Dawley rats and these were divided into two groups: (i) control and (ii) curcumin-treated. Curcumin (100 mg/kg body weight) was applied by oral gavage. Its potential for promoting tendon healing was assessed by histological evaluation, mRNA expression of tenocyte-related genes, malondialdehyde (MDA) levels, manganese-dependent superoxide dismutase (MnSOD) activity, quantification of hydroxyproline (HOPro), and biomechanical testing. In this tendon injury model, curcumin significantly improved the healing properties as evidenced by extensive deposition of well-organized collagen fibers, decreased MDA levels, and increase in the biomechanical properties and MnSOD activity of the regenerated tendon tissues. The current study showed that curcumin can improve the quality of tendon rupture healing, and thus represents a promising strategy in the management of injured tendon tissue.     

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.     

Ultrastructural changes of the patellar tendon as a cruciate ligament substitute (one year and two year results)

In four black-faced sheep, the posterior cruciate ligament was replaced with a free autogenous patellar tendon transplant. Tissue samples from the transplants were investigated by light and electron microscopy 1 year and 2 years after surgery. The normal contralateral posterior cruciate ligament and the normal contralateral patellar tendon were used as controls. The structural differences concerned cells, collagen fibrils, elastic tissue and proteoglycans. Most of the cells of the contralateral patellar tendon were spindle-shaped, whereas those of the transplant were frequently chondroid. In the central region of the transplant as well as in the area far from the bone, cell degenerations, and occasionally hypo- or even acellular zones were found. Measurements of the diameter of collagen fibrils in both contralateral patellar tendon and posterior cruciate ligament showed a more or less pronounced bimodal distribution. A unimodal distribution with mainly thin fibrils (20-60 nm) was demonstrated in the transplant tissue which also revealed some morphological alterations of the collagen fibrils. Thin elastic fibers (microfibrils and amorphous material) were randomly scattered among the collagen fibrils of the control samples, bundles of microfibrils (without amorphous material) characterized the transplant. Staining with Alcian blue in the presence of 0.3 M MgCl2 demonstrated a close relationship between proteoglycans and collagen fibrils as well as elastic components in patellar tendon. This arrangement was lost in the transplant where abundant proteoglycans were revealed which, however, composed a tight irregular network between the collagen fibrils. The results serve as a baseline for understanding the impaired biochemical properties of a free autogenous patellar tendon transplant.