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.     

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.     

体内移植羊膜对家兔肌腱损伤模型肌腱愈合的影响

研究的目的在于观察羊膜体内移植对肌腱损伤修复的影响。60 只跟腱损伤家兔动物模型采用同体对照动物实验方法,大体观察伤口愈合、肌腱粘连及肌腱肥大情况,并分别于术后第 2、4、6 周等3个时间点取跟腱,HE染色观察损伤区域炎细胞浸润和成纤维细胞数量,免疫组化染色观察胶原纤维排列塑形状况,拉伸试验测定肌腱弹性模量。另取 4 只家兔不做肌腱切断术作为正常对照,在第 0周 测定肌腱弹性模量。全部实验动物皮肤切口在术后 1 周 内基本愈合,未发生伤口感染情况。羊膜实验组跟腱粘连动物数量和肌腱断裂区域肥大程度低于阴性对照组(P<0.05)。术后第 2 周,两组动物炎症反应均达到最高值,但羊膜实验组炎细胞（包括中性粒细胞和单核细胞）浸润数量低于阴性对照组(P<0.05)。两组实验动物样本中成纤维细胞数量和胶原纤维积累数量无明显差异（P >0.05）,但羊膜实验组成纤维细胞和胶原纤维排列更具有序性,尤其是在术后第 6 周,明显好于阴性对照组。术后第 2 周和第4 周,羊膜实验组肌腱弹性模量均优于阴性对照组(P <0.05)。羊膜具有促进肌腱早期愈合和抗炎、抗粘连作用。     

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.     

Knitted poly-lactide-co-glycolide scaffold loaded with bone marrow stromal cells in repair and regeneration of rabbit Achilles tendon

The objectives of this study were to evaluate the morphology and biomechanical function of Achilles tendons regenerated using knitted poly-lactide-co-glycolide (PLGA) loaded with bone marrow stromal cells (bMSCs). The animal model used was that of an adult female New Zealand White rabbit with a 10-mm gap defect of the Achilles tendon. In group I, 19 hind legs with the created defects were treated with allogeneic bMSCs seeded on knitted PLGA scaffold. In group II, the Achilles tendon defects in 19 hind legs were repaired using the knitted PLGA scaffold alone, and in group III, 6 hind legs were used as normal control. The tendon-implant constructs of groups I and II were evaluated postoperatively at 2, 4, 8, and 12 weeks using macroscopic, histological, and immunohistochemical techniques. In addition, specimens from group I (n = 7), group II (n = 7), and group III (n = 6) were harvested for biomechanical test 12 weeks after surgery. Postoperatively, at 2 and 4 weeks, the histology of group I specimens exhibited a higher rate of tissue formation and remodeling as compared with group II, whereas at 8 and 12 weeks postoperation, the histology of both group I and group II was similar to that of native tendon tissue. The wound sites of group I healed well and there was no apparent lymphocyte infiltration. Immunohistochemical analysis showed that the regenerated tendons were composed of collagen types I and type III fibers. The tensile stiffness and modulus of group I were 87 and 62.6% of normal tendon, respectively, whereas those of group II were about 56.4 and 52.9% of normal tendon, respectively. These results suggest that the knitted PLGA biodegradable scaffold loaded with allogeneic bone marrow stromal cells has the potential to regenerate and repair gap defect of Achilles tendon and to effectively restore structure and function.     

Adenovirus-mediated gene transfer of growth and differentiation factor-5 into tenocytes and the healing rat Achilles tendon

Growth and differentiation factor-5 (GDF-5) is known to induce tendon tissue and stimulate tendon healing. The hypothesis was that adenoviral GDF-5 transfer leads to transitory transgene expression and improves Achilles tendon healing. In vitro experiments were first performed with rat tenocytes. Transgene expression was evaluated by RT-PCR, Western blotting and GDF-5-ELISA. In vivo virus dosage and transgene expression were examined by a marker gene transfer (LacZ and luciferase). In the main experiment in 131 rats, adenovirus particles (3 x 10(10)) were injected into transected Achilles tendons. The time course of GDF-5 mRNA expression was assessed by real-time RT-PCR. Histology and biomechanical testing were used to evaluate tendon healing and tensile strength. In vitro GDF-5 was secreted with a maximum after 2 weeks (330 ng GDF-5/10(6) cells per 24 hr). In vivo GDF-5 transgene expression showed a maximum at 4 weeks. At 8 weeks, GDF-5 specimens were thicker (p<0.05) with a trend to higher strength (p=0,064). Histology showed greater cartilage formation in type II collagen stains than in controls. Injection of adenovirus particles successfully can deliver the GDF-5 gene in healing tendons and leads to thicker tendon regenerates after 8 weeks. This technique might become a new approach for nonsurgical treatment of tendon injuries.     

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.     

Spatial and temporal expression of molecular markers and cell signals during normal development of the mouse patellar tendon

Tendon injuries are common clinical problems and are difficult to treat. In particular, the tendon-to-bone insertion site, once damaged, does not regenerate its complex zonal arrangement. A potential treatment for tendon injuries is to replace injured tendons with bioengineered tendons. However, the bioengineering of tendon will require a detailed understanding of the normal development of tendon, which is currently lacking. Here, we use the mouse patellar tendon as a model to describe the spatial and temporal pattern of expression of molecular markers for tendon differentiation from late fetal life to 2 weeks after birth. We found that collagen I, fibromodulin, and tenomodulin were expressed throughout the tendon, whereas tenascin-C, biglycan, and cartilage oligomeric protein were concentrated in the insertion site during this period. We also identified signaling pathways that are activated both throughout the developing tendon, for example, transforming growth factor beta and bone morphogenetic protein, and specifically in the insertion site, for example, hedgehog pathway. Using a mouse line expressing green fluorescent protein in all tenocytes, we also found that tenocyte cell proliferation occurs at highest levels during late fetal life, and declines to very low levels by 2 weeks after birth. These data will allow both the functional analysis of specific signaling pathways in tenocyte development and their application to tissue-engineering studies in vitro.     

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

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

Regenerative Versus Reparative Healing in Tendon: A Study of Biomechanical and Histological Properties in Fetal Sheep

Previous studies have shown fetal tissues heal in a regenerative fashion without scar formation. The objective of this study is to compare the healing properties of adult and fetal tendons. Time-mated pregnant ewes at 80–85 days of gestation were utilized. A partial, midsubstance tenotomy was performed in the lateral extensor fetal tendons, and analogous tenotomies were created in the maternal limbs. One week after injury, the fetal and adult animals were sacrificed, and tendons were histologically and mechanically evaluated. Immunohistochemical staining for transforming growth factor beta isoform 1 (TGF-1) was performed. Histologically, a gap with granulation tissue and inflammatory cells was visible in the site of wounding in the adult tendons. In the fetal tendons, no abnormalities were noted in the wound, with reconstitution of collagen architecture. TGF-1 expression was low in fetal but upregulated in the adult wounds. No significant differences were found in the biomechanical properties between groups. We identified regenerative healing properties in injured fetal tendon, while adult tendon tissue healed reparatively with scar formation. Fetal tendons demonstrated a limited recovery of mechanical properties after injury that was no better than that of the adult tendons at seven days. A better understanding of the mechanisms of fetal healing may lead to novel therapeutic strategies in the clinical setting. © 2003 Biomedical Engineering Society. PAC2003: 8719Rr, 8718La     

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 Tendon Injury Response is Influenced by Decorin and Biglycan

Defining the constituent regulatory molecules in tendon is critical to understanding the process of tendon repair and instructive to the development of novel treatment modalities. The purpose of this study is to define the structural, expressional, and mechanical changes in the tendon injury response, and elucidate the roles of two class I small leucine-rich proteoglycans (SLRPs). We utilized biglycan-null, decorin-null and wild type mice with an established patellar tendon injury model. Mechanical testing demonstrated functional changes associated with injury and the incomplete recapitulation of mechanical properties after 6 weeks. In addition, SLRP deficiency influenced the mechanical properties with a marked lack of improvement between 3 and 6 weeks in decorin-null tendons. Morphological analyses of the injury response and role of SLRPs demonstrated alterations in cell density and shape as well as collagen alignment and fibril structure resulting from injury. SLRP gene expression was studied using RT-qPCR with alterations in expression associated with the injured tendons. Our results show that in the absence of biglycan initial healing may be impaired while in the absence of decorin later healing is clearly diminished. This suggests that biglycan and decorin may have sequential roles in the tendon response to injury.     

Reconstruction and restoration of neglected ruptured patellar tendon using semitendinosus and gracilis tendons with preserved distal insertions: two case reports

Neglected rupture of the patellar tendon is rare but becomes more difficult to repair the longer it is left untreated. The most common rupture sites are the inferior pole of the patella and distal insertion. Proximal retraction of the patella and extensor mechanism adhesions makes the treatment more difficult than acute tendon rupture. We report two patients with neglected patellar tendon rupture treated by reconstruction and restoration using semitendinosus-gracilis (STG) tendons with preserved distal insertions. Preserved distal insertion provided sufficient blood supply to accelerate healing, while combined fixation with tension-reducing wire, offered the initial stability of the closed-loop sutured tendon. Both patients reacquired near normal strength and stability of the patellar tendon and restoration of function after operation and rehabilitation.     

The cell specific temporal expression of nitric oxide synthase isoforms during Achilles tendon healing

OBJECTIVE AND DESIGN: We have previously shown that nitric oxide synthase (NOS) activity is upregulated following tendon injury, and that this activity is important to Achilles tendon healing. The aim of this study was to identify the cellular distribution of nitric oxide synthase isoforms during tendon healing. MATERIAL OR SUBJECTS: Surgical division of the right Achilles tendon was performed in eighty-five male Sprague-Dawley rats. Healing Achilles tendons were harvested at 4, 7, 14 and 21 days following the surgery. The un-injured left Achilles tendons were used as controls. Using RNase protection assays, in situ hybridization and immunohistochemistry, mRNA and protein of NOS isoforms were evaluated. RESULTS: Minimal NOS expression was found in un-injured tendon. A cell specific temporal pattern for the mRNA and protein for all three NOS isoforms was found following injury to the Achilles tendon. iNOS was maximal on day 4 in macrophages and fibroblasts. eNOS was maximal on day 4 in endothelial cells and fibroblasts. bNOS expression gradually increased up to day 21 and was found only in fibroblasts. CONCLUSIONS: These results suggest that all three nitric oxide synthase isoforms are expressed by fibroblasts in a coordinated temporal sequence during tendon healing. The sequential pattern of NOS expression in healing fibroblasts suggests that each NOS isoform may play a different role in the healing process and provides opportunities to modify tendon healing in the clinical setting.     

保留止点自体肌腱移植治疗陈旧性髌韧带断裂

背景：髌韧带在膝关节的运动中起着非常重要的的作用,因其断裂比较少见,临床上误诊率较高。目前重建髌韧带的方法较多,疗效报道不一,治疗不当容易出现髌骨位置不良,会严重影响膝关节的功能。 目的：探讨保留止点自体肌腱移植治疗陈旧性髌韧带断裂的临床疗效。 方法：对8例单侧陈旧性髌韧带断裂的患者行保留止点的半肌腱、股薄肌重建髌韧带,髌韧带重建后行半环形石膏后托固定屈膝15°1周,髌韧带重建后2周拆除切口缝线,3周去除石膏行股四头肌等长等张练习,6周时佩戴可调节膝关节护具进行膝关节活动度锻炼,3次/d,每周增加10°。髌韧带重建3个月后允许完全负重行走。 结果与结论：所有患者髌韧带重建后无并发症,X射线示髌骨恢复正常高度。膝关节功能恢复良好。所有患者髌韧带重建后24个月行走2 km以上膝关节无不适。与髌韧带重建前相比,髌韧带重建后24个月患者的Lysholm评分明显增加(P < 0.05),说明保留止点的半腱肌、股薄肌重建髌韧带可重建治疗陈旧性髌韧带断裂。     

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.     

Reconstruction of the canine Achilles and patellar tendons using dacron mesh silicone prosthesis. I. Clinical and biocompatibility evaluation.

Surgical replacement of the Achilles and patellar tendons using Dacron mesh silicone prostheses was performed in 15 mature beagle dogs. This part of the study includes clinical evaluation, gross inspection at autopsy of regrown tendons, and histological determination of biocompatibility of prosthetic implants. Functional continuity and integrity of prosthetic patellar tendons have been established by evaluating the biologic-prosthetic interface and the mechanical properties of regrown tendon tissue around and through the Dacron silicone replacements. Results of prosthetic Achilles tendons were less satisfactory because of difficulties in suturing to a muscle and its fascia. Although the prosthetic tendon did not regrow through the tube, it provided a structure for regrowth around it. The regrown tendons became nearly ten times the normal cross-sectional area after three to four months and decreased to two to three times after 13 months. Histological studies indicate that in the absence of infection, the Dacron mesh silicone tendon was well tolerated for periods up to 13 months. Overall results are encouraging and warrant further investigation although the regenerative capacity of human patellar and Achilles tendons is unknown.