Differential expression of transforming growth factor-beta receptors in a rabbit zone II flexor tendon wound healing model

Flexor tendon repair in zone II is complicated by adhesions that impair normal postoperative gliding. Transforming growth factor-beta (TGF-beta) is a family of growth factors that has been implicated in scar formation. The TGF-beta family of proteins binds to three distinct classes of membrane receptors, termed RI, RII, and RIII. In this study, we analyzed the temporal and spatial distribution of TGF-beta receptor isoforms (RI, RII, and RIII) in a rabbit zone II flexor tendon wound healing model.Twenty-eight adult New Zealand White rabbit forepaws underwent isolation of the middle digit flexor digitorum profundus tendon in zone II. The tendons underwent transection in zone II and immediate repair. The tendons were harvested at increasing time points: 1, 3, 7, 14, 28, and 56 days postoperatively (n = 4 at each time point). The control flexor tendons were harvested without transection and repair (n = 4). Immunohistochemical analysis was used to detect the expression patterns for TGF-beta receptors RI, RII, and RIII.Immunohistochemical staining of the transected and repaired tendons demonstrated up-regulation of TGF-beta RI, RII, and RIII protein levels. TGF-beta receptor production in the experimental group (transection and repair) was concentrated in the epitenon and along the repair site. Furthermore, the TGF-beta receptor expression levels peaked at day 14 and decreased by day 56 postoperatively. In contrast, minimal receptor expression was observed in the untransected and unrepaired control tendons.These data provide evidence that (1) TGF-beta receptors are up-regulated after injury and repair; (2) peak levels of TGF-beta receptor expression occurred at day 14 and decreased by day 56 after wounding and repair; and (3) both the tendon sheath and epitenon have the highest receptor expression, and both may play critical roles in flexor tendon wound healing. Understanding the up-regulation of TGF-beta isoforms and the up-regulation of their corresponding receptors during flexor tendon wound healing provides new targets for biomolecular modulation of postoperative scar formation.     

屏障材料预防肌腱粘连的研究进展

肌腱与腱周组织粘连是肌腱损伤修复术后最主要的并发症,使用屏障材料预防肌腱粘连是一种最直接有效的方法。传统使用的不可吸收材料常常会导致严重免疫反应或阻隔肌腱营养而导致肌腱愈合不良,且常需二次手术取出,已基本淘汰。可吸收膜和水凝胶等具有良好通透性及组织相容性的可吸收大分子材料是目前研究的重点。使用自体或异体组织材料重建腱鞘也可预防肌腱粘连,但自体组织重建腱鞘损伤较大且手术复杂,异体组织常常受到安全性、伦理学等诸多限制,而组织工程化腱鞘可能是未来预防肌腱粘连的一个重要研究方向。     

Tendon tissue engineering: progress, challenges, and translation to the clinic

The tissue engineering field has made great strides in understanding how different aspects of tissue engineered constructs (TECs) and the culture process affect final tendon repair. However, there remain significant challenges in developing strategies that will lead to a clinically effective and commercially successful product. In an effort to increase repair quality, a better understanding of normal development, and how it differs from adult tendon healing, may provide strategies to improve tissue engineering. As tendon tissue engineering continues to improve, the field needs to employ more clinically relevant models of tendon injury such as degenerative tendons. We need to translate successes to larger animal models to begin exploring the clinical implications of our treatments. By advancing the models used to validate our TECs, we can help convince our toughest customer, the surgeon, that our products will be clinically efficacious. As we address these challenges in musculoskeletal tissue engineering, the field still needs to address the commercialization of products developed in the laboratory. TEC commercialization faces numerous challenges because each injury and patient is unique. This review aims to provide tissue engineers with a summary of important issues related to engineering tendon repairs and potential strategies for producing clinically successful products.     

The cell biology of suturing tendons

Trauma by suturing tendon form areas devoid of cells termed “acellular zones” in the matrix. This study aimed to characterise the cellular insult of suturing and acellular zone formation in mouse tendon. Acellular zone formation was evaluated using single grasping sutures placed using flexor tendons with time lapse cell viability imaging for a period of 12 h. Both tension and injury were required to induce cell death and cell movement in the formation of the acellular zone. DNA fragmentation studies and transmission electron microscopy indicated that cells necrosed.Parallel in vivo studies showed that cell-to-cell contacts were disrupted following grasping by the suture in tensioned tendon. Without tension, cell death was lessened and cell-to-cell contacts remained intact. Quantitative immunohistochemistry and 3D cellular profile mapping of wound healing markers over a one year time course showed that acellular zones arise rapidly and showed no evidence of healing whilst the wound healing response occurred in the surrounding tissues. The acellular zones were also evident in a standard modified “Kessler” clinical repair. In conclusion, the suture repair of injured tendons produces acellular zones, which may potentially cause early tendon failure.     

屏障材料预防肌腱粘连的研究进展

肌腱与腱周组织粘连是肌腱损伤修复术后最主要的并发症,使用屏障材料预防肌腱粘连是一种最直接有效的方法。传统使用的不可吸收材料常常会导致严重免疫反应或阻隔肌腱营养而导致肌腱愈合不良,且常需二次手术取出,已基本淘汰。可吸收膜和水凝胶等具有良好通透性及组织相容性的可吸收大分子材料是目前研究的重点。使用自体或异体组织材料重建腱鞘也可预防肌腱粘连,但自体组织重建腱鞘损伤较大且手术复杂,异体组织常常受到安全性、伦理学等诸多限制,而组织工程化腱鞘可能是未来预防肌腱粘连的一个重要研究方向。     

Mechanical property changes during neonatal development and healing using a multiple regression model

During neonatal development, tendons undergo a well orchestrated process whereby extensive structural and compositional changes occur in synchrony to produce a normal tissue. Conversely, during the repair response to injury, structural and compositional changes occur, but a mechanically inferior tendon is produced. As a result, developmental processes have been postulated as a potential paradigm for elucidation of mechanistic insight required to develop treatment modalities to improve adult tissue healing. The objective of this study was to compare and contrast normal development with injury during early and late developmental healing. Using backwards multiple linear regressions, quantitative and objective information was obtained into the structure-function relationships in tendon. Specifically, proteoglycans were shown to be significant predictors of modulus during early developmental healing but not during late developmental healing or normal development. Multiple independent parameters predicted percent relaxation during normal development, however, only biglycan and fibril diameter parameters predicted percent relaxation during early developmental healing. Lastly, multiple differential predictors were observed between early development and early developmental healing; however, no differential predictors were observed between late development and late developmental healing. This study presents a model through which objective analysis of how compositional and structural parameters that affect the development of mechanical parameters can be quantitatively measured. In addition, information from this study can be used to develop new treatment and therapies through which improved adult tendon healing can be obtained.     

The interface of mechanical loading and biological variables as they pertain to the development of tendinosis

Different tendons are designed to withstand different mechanical loads in their individual environments. Variable physiologic loading ranges and correspondingly different injury thresholds lead to tendon heterogeneity. Also, tendon heterogeneity is evident when examining how different tendons regulate their response to changes in mechanical loading (over- and under-loading). The response of tendons to changes in mechanical loading plays an important role in the induction and progression of tendinosis which is tendon degeneration without inflammation. Tendon overuse injury is likely related to abnormal mechanical loading that deviates from normal mechanical loading in magnitude, frequency, duration and/or direction. Mechanical loading that results in tendon overuse injury can initiate a repair process but, after failed initial repair, non-resolving chronic attempted repair appears to lead to a "smoldering" fibrogenesis. Contributions of regulatory components, including minor components in the "nerve-mast cell-myofibroblast axis", are key features in the development and progression of tendinosis. Hormonal and genetic factors may also influence risk for tendinosis. Further understanding of how tendinosis induction is related to mechanical use/overuse, how tendinosis progression is related to abnormal regulation of attempted repair, and how induction and/or progression are modulated by other risk factors may lead to interventions that mitigate risk and enhance functional repair.     

The role of repair tension on tendon to bone healing in an animal model of chronic rotator cuff tears

Rotator cuff tendon tears are one of the most common shoulder injuries. Although surgical repair is typically beneficial, re-tearing of the tendons frequently occurs. It is generally accepted that healing is worse for chronic tears than acute tears, but the reasons for this are unknown. One potential cause may be the large tensions that are sometimes required to repair chronically torn tendons back to bone (i.e., repair tension). Therefore, the objective of this study was to utilize an animal model of chronic rotator cuff repairs to investigate the role of increased repair tension on tendon to bone healing. We hypothesized that an increase in repair tension would be related to detrimental changes to the healing insertion site. To test this hypothesis, the supraspinatus tendon of rats was surgically detached and then repaired immediately or after a delay of 2, 4, or 16 weeks. The repair tension was measured using a tensiometer and the mechanical properties, collagen organization, and protein expression of the healing insertion site were evaluated 4 and/or 16 weeks following repair. We found that the repair tension increased with time following detachment, and was related to a decrease in the failure properties and viscoelastic peak stress and an increase in cross-sectional area and stiffness of the insertion site. Therefore, repair tension should be minimized in the clinical setting. Future studies will include additional animal model studies involving the relationship between tension and muscle properties and a clinical study investigating the role of repair tension on repair failure.     

Murine patellar tendon biomechanical properties and regional strain patterns during natural tendon-to-bone healing after acute injury

Tendon-to-bone healing following acute injury is generally poor and often fails to restore normal tendon biomechanical properties. In recent years, the murine patellar tendon (PT) has become an important model system for studying tendon healing and repair due to its genetic tractability and accessible location within the knee. However, the mechanical properties of native murine PT, specifically the regional differences in tissue strains during loading, and the biomechanical outcomes of natural PT-to-bone healing have not been well characterized. Thus, in this study, we analyzed the global biomechanical properties and regional strain patterns of both normal and naturally healing murine PT at three time points (2, 5, and 8 weeks) following acute surgical rupture of the tibial enthesis. Normal murine PT exhibited distinct regional variations in tissue strain, with the insertion region experiencing approximately 2.5 times greater strain than the midsubstance at failure (10.80±2.52% vs. 4.11±1.40%; mean±SEM). Injured tendons showed reduced structural (ultimate load and linear stiffness) and material (ultimate stress and linear modulus) properties compared to both normal and contralateral sham-operated tendons at all healing time points. Injured tendons also displayed increased local strain in the insertion region compared to contralateral shams at both physiologic and failure load levels. 93.3% of injured tendons failed at the tibial insertion, compared to only 60% and 66.7% of normal and sham tendons, respectively. These results indicate that 8 weeks of natural tendon-to-bone healing does not restore normal biomechanical function to the murine PT following injury.     

Development and evaluation of multiple tendon injury models in the mouse

The mouse has proven to be an advantageous animal model system in basic science research focused on aiding in development and evaluation of potential treatments; however, the small size of mouse tendons makes consistent and reproducible injury models and subsequent biomechanical evaluation challenging for studying tendon healing. In this study, we investigated the feasibility and reproducibility of multiple mouse tendon injury models. Our hypothesis was that incisional (using a blade) and excisional (using a biopsy punch) injuries would result in consistent differences in tendon material properties. At 16 weeks of age, 17 C57BL/6 mice underwent surgery to create defects in the flexor digitorum longus, Achilles, or patellar tendon. Each animal received 1-2 full-thickness, central-width incisional or excisional injuries per limb; at least one tendon per limb remained uninjured. The injuries were distributed such that each tendon type had comparable numbers of uninjured, incisionally injured, and excisionally injured specimens. Three weeks after injury, all animals were euthanized and tendons were harvested for mechanical testing. As hypothesized, differences were detected for all three different tendon types at three weeks post-injury. While all models created injuries that produced predictable outcomes, the patellar tendon model was the most consistent in terms of number and size of significant differences in injured tendons compared to native properties, as well as in the overall variance in the data. This finding provides support for its use in fundamental tendon healing studies; however, future work may use any of these models, based on their appropriateness for the specific question under study.     

Development of collagen fibril organization and collagen crimp patterns during tendon healing

Normal tendon comprises coaxially aligned bundles of crimped collagen fibres each of which possesses a fibrillar substructure. In acute traumatic injury this level of organization is disrupted and the mechanical function of the tendon impaired. During repair, a degree of recovery of the fibrillar structure takes place. In this tudy we have assessed the re-establishment of tendon organization after injury on the basis of the collagen fibril diameter distribution and the collagen crimp parameters. Crimp became undetectable following injury but one month later was present throughout the tissue. At this time the periodicity was greatly reduced by comparison with that of the normal tendon and normal values were not re-established within 14 months following injury. Collagen fibril diameters remained abnormally small over this same period of time. In particular, fibrils of diameters in excess of 100 nm, commonly found in normal and contralateral tendons, were totally absent from the observed distributions in the healing tendons. Such large diameter fibrils often account for as much as 50% of the total mass of collagen present in the uninjured tissue. Thus the mechanical properties of the healing tendon may remain significantly different from those of normal tendon for a minimum time of 14 months after injury.     

Insulin-dependent diabetes impairs the inflammatory response and delays angiogenesis following Achilles tendon injury

Although impaired wound healing associated with type 1 diabetes mellitus has been well studied in skin tissue, the influence of this metabolic disorder on tendon healing and recovery has not been extensively investigated. Because tendons are known to have limited repair potential, we studied the tendon-healing process by using a diabetic rat tendonitis model. We tested the hypothesis that diabetes influences the inflammatory response, cell proliferation, and angiogenesis in injured Achilles tendons. Diabetes was induced by injecting streptozotocin at 45 mg/kg body wt. Non-diabetic rats as well as diabetic and insulin-treated diabetic animals were then injected with collagenase. The accumulation of inflammatory cells was quantified in transversal sections of Achilles tendon by using immunohistochemical staining at days 0, 1, 3, 7, 14, and 28 posttrauma. The number of proliferative cells and the extent of neovascularization was also quantified in the paratenon and the core of the tendon at days 0, 3, 7, 14, and 28 posttrauma. Relative to nondiabetic and insulin-treated diabetic animals, the numbers of accumulated neutrophils and ED1(+) and ED2(+) macrophages in diabetic rats decreased by 46, 43, and 52%, respectively, in the first 3 days after injury compared with levels in nondiabetic and insulin-treated diabetic animals. The density of newly formed blood vessels decreased by 35 and 29% in the paratenon and the core of tendon, respectively, at days 3 and 7 after injury. Lastly, the concentration of proliferative cells decreased by 34% in the paratenon at day 7 posttrauma in injured tendons from diabetic rats relative to nondiabetic rats. These results indicate that alterations in inflammatory, angiogenic, and proliferative processes occurred in the diabetic state that might eventually perturb tendon healing and remodeling.     

Effect of vascular endothelial growth factor on rat Achilles tendon healing

This study evaluated the effect of exogenous vascular endothelial growth factor (VEGF) on tendon healing and regulation of other growth factors in a rat Achilles tendon model. Fifty Sprague-Dawley rats were used. In the experimental group, the left Achilles tendon was transected and repaired with the modified Kessler suture technique, and the right Achilles tendon was transected and repaired with resection of plantaris tendon. VEGF, 100 mul (50 mug/ml), was injected into each tendon at the repair site. The same surgical procedures were performed in the control group, with the same volume of saline injected into the repair sites. At intervals of 1, 2, and 4 weeks, the animals were killed and the tendons were harvested and evaluated for tensile strength (1, 2, and 4 weeks) and gene expression (postoperative day 4). At 1 week postoperatively, when plantaris tendon was preserved, the tensile strength of the repaired tendons with VEGF treatment (3.63 +/- 0.62 MPa) was significantly higher than the tensile strength of the repaired tendons with saline treatment (2.20 +/- 0.36 MPa). There was no difference in tensile strength between the two groups without the plantaris tendon support. At 2 weeks postoperatively, the tensile strength was 11.34 +/- 3.89 MPa in the group with VEGF treatment and plantaris tendon preservation, which was significantly higher than the tensile strength in the other groups. There was no significant difference in tensile strength among the groups at 4 weeks postoperatively. The gene expression showed that transforming growth factor-beta in the VEGF-treated tendon was up-regulated in the early stage of tendon healing, whereas expression of platelet-derived growth factor, basic fibroblast growth factor, and insulin-like growth factor-1 was not significantly different among the groups. In conclusion, administration of exogenous VEGF can significantly improve tensile strength early in the course of the rat Achilles tendon healing and was associated with increased expression of transforming growth factor-beta.     

The rigidity of repaired flexor tendons increases following ex vivo cyclic loading

Transected flexor tendons are typically treated by suture repair followed by rehabilitation that generates repetitive tendon loading. Recent results in an in vivo canine model indicate that during the first 10 days after injury and repair, there is an increase in the rigidity of the tendon repair site. Our objective was to determine whether or not ex vivo cyclic loading of repaired flexor tendons causes a similar increase in repair-site rigidity. We simulated 10 days of rehabilitation by applying 6000 loading cycles to repaired canine flexor tendons ex vivo at force levels generated during passive motion rehabilitation; we then evaluated their tensile mechanical properties. High-force (peak force, 17 N) cyclic loading increased repair-site rigidity by 100% and decreased repair-site strain by 50%, whereas low-force (5 N) loading did not change the properties of the repair site. This mechanical conditioning effect may explain, in part, the changes in tensile properties observed after only 10 days of healing in vivo. Mechanical conditioning of repaired flexor tendons by repetitive forces applied during rehabilitation may lead to increases in repair-site rigidity and decreases in strain, thereby altering the mechanical loading environment of tissues and cells at the repair site.     

Effect of supraspinatus tendon repair technique on the infraspinatus tendon

Supraspinatus tendon tears are common and often propagate into larger tears that include the infraspinatus tendon, resulting in loss of function and increased pain. Previously, we showed that the supraspinatus and infraspinatus tendons mechanically interact through a range of rotation angles, potentially shielding the torn supraspinatus tendon from further injury while subjecting the infraspinatus tendon to increased risk of injury. Surgical repair of torn supraspinatus tendons is common, yet the effect of the repair on the infraspinatus tendon is unknown. Since we have established a relationship between strain in the supraspinatus and infraspinatus tendons the success of a supraspinatus tendon repair depends on its effect on the loading environment in the infraspinatus tendon. More specifically, the effect of transosseous supraspinatus tendon repair in comparison to one that utilizes suture anchors, as is commonly done with arthroscopic repairs, on this interaction through these joint positions will be evaluated. We hypothesize that at all joint positions evaluated, both repairs will restore the interaction between the two tendons. For both repairs, (1) increasing supraspinatus tendon load will increase infraspinatus tendon strain and (2) altering the rotation angle from internal to external will increase strain in the infraspinatus tendon. Strains were measured in the infraspinatus tendon insertion through a range of joint rotation angles and supraspinatus tendon loads, for the intact, transosseous, and suture anchor repaired supraspinatus tendons. Images corresponding to specific supraspinatus tendon loads were isolated for the infraspinatus tendon insertion for analysis. The effect of supraspinatus tendon repair on infraspinatus tendon strain differed with joint position. Altering the joint rotation did not change strain in the infraspinatus tendon for any supraspinatus tendon condition. Finally, increasing supraspinatus tendon load resulted in an increase in average maximum and decrease in average minimum principal strain in the infraspinatus tendon. There is a significant difference in infraspinatus tendon strain between the intact and arthroscopically (but not transosseous) repaired supraspinatus tendons that increases with greater loads. Results suggest that at low loads neither supraspinatus tendon repair technique subjects the infraspinatus tendon to potentially detrimental loads; however, at high loads, transosseous repairs may be more advantageous over arthroscopic repairs for the health of the infraspinatus tendon. Results emphasize the importance of limiting loading of the repaired supraspinatus tendon and that at low loads, both repair techniques restore the interaction to the intact supraspinatus tendon case.     

REPAIR OF TENDONS IN THE HAND

The physiologic processes by which tendons of the hand heal after injury differ from one part of the hand to another.Although definitive operation immediately after injury is advisable in many cases to avoid infection, factors other than infection may be more important and dictate delay.While early exercise to mobilize the tendon soon after repair would seem logical, actually the process of healing is such that during the third week the tendon is spontaneously freed from adherence to surrounding tissue. Motion earlier than that causes irritation at the point of suture of the two ends of the tendon and increases scar. After completion of the healing process, motion serves to increase the strength of the new tendon fibrils.     

Stem cell therapy in the management of shoulder rotator cuff disorders

Rotator cuff tears are frequent shoulder problems that are usually dealt with surgical repair. Despite improved surgical techniques, the tendon-to-bone healing rate is unsatisfactory due to difficulties in restoring the delicate transitional tissue between bone and tendon. It is essential to understand the molecular mechanisms that determine this failure. The study of the molecular environment during embryogenesis and during normal healing after injury is key in devising strategies to get a successful repair. Mesenchymal stem cells (MSC) can differentiate into different mesodermal tissues and have a strong paracrine, anti-inflammatory, immunoregulatory and angiogenic potential. Stem cell therapy is thus a potentially effective therapy to enhance rotator cuff healing. Promising results have been reported with the use of autologous MSC of different origins in animal studies: they have shown to have better healing properties, increasing the amount of fibrocartilage formation and improving the orientation of fibrocartilage fibers with less immunologic response and reduced lymphocyte infiltration. All these changes lead to an increase in biomechanical strength. However, animal research is still inconclusive and more experimental studies are needed before human application. Future directions include expanded stem cell therapy in combination with growth factors or different scaffolds as well as new stem cell types and gene therapy.     

Early response to tendon fatigue damage accumulation in a novel in vivo model

This study describes the development and application of a novel rat patellar tendon model of mechanical fatigue for investigating the early in vivo response to tendon subfailure injury. Patellar tendons of adult female Sprague-Dawley rats were fatigue loaded between 1–35 N using a custom-designed loading apparatus. Patellar tendons were subjected to Low-, Moderate- or High-level fatigue damage, defined by grip-to-grip strain measurement. Molecular response was compared with that of a laceration-repair injury. Histological analyses showed that progression of tendon fatigue involves formation of localized kinked fiber deformations at Low damage, which increased in density with presence of fiber delaminations at Moderate damage, and fiber angulation and discontinuities at High damage levels. RT-PCR analysis performed at 1- and 3-day post-fatigue showed variable changes in type I, III and V collagen mRNA expression at Low and Moderate damage levels, consistent with clinical findings of tendon pathology and were modest compared with those observed at High damage levels, in which expression of all collagens evaluated were increased markedly. In contrast, only type I collagen expression was elevated at the same time points post-laceration. Findings suggest that cumulative fatigue in tendon invokes a different molecular response than laceration. Further, structural repair may not be initiated until reaching end-stage fatigue life, where the repair response may unable to restore the damaged tendon to its pre-fatigue architecture.     

Repair of tendons in the hand

The physiologic processes by which tendons of the hand heal after injury differ from one part of the hand to another. Although definitive operation immediately after injury is advisable in many cases to avoid infection, factors other than infection may be more important and dictate delay.While early exercise to mobilize the tendon soon after repair would seem logical, actually the process of healing is such that during the third week the tendon is spontaneously freed from adherence to surrounding tissue. Motion earlier than that causes irritation at the point of suture of the two ends of the tendon and increases scar. After completion of the healing process, motion serves to increase the strength of the new tendon fibrils.