Effects of Pulsed Electromagnetic Field Therapy on Rat Achilles Tendon Healing

The Achilles tendon is frequently injured. Data to support specific treatment strategies for complete and partial tears is inconclusive. Regardless of treatment, patients risk re-rupture and typically have long-term functional deficits. We previously showed that pulsed electromagnetic field (PEMF) therapy improved tendon-to-bone healing in a rat rotator cuff model. This study investigated the effects of PEMF on rat ankle function and Achilles tendon properties after (i) complete Achilles tendon tear and repair with immobilization, (ii) partial Achilles tendon tear without repair and with immobilization, and (iii) partial Achilles tendon tear without repair and without immobilization. We hypothesized that PEMF would improve tendon properties, increase collagen organization, and improve joint function, regardless of injury type. After surgical injury, animals were assigned to a treatment group: (i) no treatment control, (ii) 1 h of PEMF per day, or (iii) 3 h of PEMF per day. Animals were euthanized at 1, 3, and 6 weeks post-injury. Joint mechanics and gait analysis were assessed over time, and fatigue testing and histology were performed at each time point. Results indicate no clear differences in Achilles healing with PEMF treatment. Some decreases in tendon mechanical properties and ankle function suggest PEMF may be detrimental after complete tear. Some early improvements were seen with PEMF after partial tear with immobilization; however, immobilization was found to be a confounding factor. This body of work emphasizes the distinct effects of PEMF on tendon-to-bone healing and supports trialing potential treatment strategies pre-clinically across tendons before applying them clinically. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:70–81, 2020     

Unfavorable effect of knee immobilization on Achilles tendon healing in rabbits

This study was undertaken to assess the effect of knee immobilization on the treatment of Achilles tendon rupture. After their Achilles tendons were severed, rabbits were divided into 2 groups. In Group A, only the ankle joint was immobilized. In Group B, both the knee and ankle joints were immobilized. At 4 weeks after surgery, both the ultimate tensile force and stiffness of the severed tendons were significantly greater in Group A than in Group B. In Group A, dense collagen fibers were seen in the repaired tendons, and the bundles of collagen fibers were parallel to one another along the axis of the tendons. In contrast, in Group B, dilated veins and capillaries were seen in the repaired tendons, and the proliferation of connective tissue containing collagen fibers was severely reduced around these veins and capillaries and was in general irregular and uneven. These results suggest that knee immobilization retards the healing of a ruptured Achilles tendon without suture, due to congestion and tension deprivation produced by keeping the tendon static.     

Unfavorable effect of knee immobilization on Achilles tendon healing in rabbits

This study was undertaken to assess the effect of knee immobilization on the treatment of Achilles tendon rupture. After their Achilles tendons were severed, rabbits were divided into 2 groups. In Group A, only the ankle joint was immobilized. In Group B, both the knee and ankle joints were immobilized. At 4 weeks after surgery, both the ultimate tensile force and stiffness of the severed tendons were significantly greater in Group A than in Group B. In Group A, dense collagen fibers were seen in the repaired tendons, and the bundles of collagen fibers were parallel to one another along the axis of the tendons. In contrast, in Group B, dilated veins and capillaries were seen in the repaired tendons, and the proliferation of connective tissue containing collagen fibers was severely reduced around these veins and capillaries and was in general irregular and uneven. These results suggest that knee immobilization retards the healing of a ruptured Achilles tendon without suture, due to congestion and tension deprivation produced by keeping the tendon static.     

Pulsed ultrasound treatment accelerates the repair of Achilles tendon rupture in rats.

A rat tenotomy model was used to investigate the effect of combined conservative management and pulsed ultrasound (PUS) on the repair of tenotomized Achilles tendon. Hemitenotomy of right medial Achilles tendon was performed in 48 rats without suture, and patella tenotomy was performed to mimic immobilization and limb disuse of an injured limb. PUS and sham PUS were applied to the healing wound for the treatment group and control group for 5 min, 3 times per week for 2 or 4 weeks, respectively. Tensile tests showed that the ultimate tensile strength (UTS) and stiffness of the repaired tendon in the treatment group at 2 weeks reached 48.92+/-8.39% and 62.48+/-32.46% of the contralateral normal tendon strength, which were significantly higher than those of the control group (UTS, 30.36+/-15.46%; stiffness, 33.90+/-17.59; p<0.05). At 4 weeks, UTS increased to 77.09+/-15.31% and stiffness to 92.48+/-31.12% in the treatment group, significantly higher than those in the control group (UTS, 54.33+/-18.40%, p<0.01; stiffness, 65.02+/-25.48%, p<0.05). Light microscopy revealed more regular, denser, and better aligned collagen fibers in the healing scar of the PUS-treated healing tendons. The findings suggested that PUS were able to accelerate the healing of the ruptured tendons.     

Unfavorable effect of knee immobilization on Achilles tendon healing in rabbits

This study was undertaken to assess the effect of knee immobilization on the treatment of Achilles tendon rupture. After their Achilles tendons were severed, rabbits were divided into 2 groups. In Group A, only the ankle joint was immobilized. In Group B, both the knee and ankle joints were immobilized. At 4 weeks after surgery, both the ultimate tensile force and stiffness of the severed tendons were significantly greater in Group A than in Group B. In Group A, dense collagen fibers were seen in the repaired tendons, and the bundles of collagen fibers were parallel to one another along the axis of the tendons. In contrast, in Group B, dilated veins and capillaries were seen in the repaired tendons, and the proliferation of connective tissue containing collagen fibers was severely reduced around these veins and capillaries and was in general irregular and uneven. These results suggest that knee immobilization retards the healing of a ruptured Achilles tendon without suture, due to congestion and tension deprivation produced by keeping the tendon static.     

Biomechanical comparison of new Achilles tendon rupture repair technique the “Giftbox” versus the Krackow technique in New Zealand white rabbits: An experimental animal study

IntroductionTo compare the strength between the Achilles tendons repaired with the “Giftbox” and the Krackow techniques in New Zealand white rabbits post six weeks of tendon healing.Materials and MethodsEight rabbits were randomized into Giftbox and Krackow groups. Tenotomy was performed on the Achilles tendon of one side of the lower limb and repaired with the respective techniques. The contralateral limb served as control. Subjects were euthanized six weeks post-operative, and both repaired and control Achilles tendons were harvested for biomechanical tensile test.ResultsThe means of maximum load to rupture and tenacity in the Giftbox group (156.89 ± 38.49 N and 159.98 ± 39.25 gf/tex) were significantly different than Krackow's (103.55 ± 27.48 N and 104.91 ± 26.96 gf/tex, both p = 0.043).ConclusionThe tendons repaired with Giftbox technique were biomechanically stronger than those repaired with Krackow technique after six weeks of tendon healing.     

Effects of transforming growth factor-beta 1 on the early stages of healing of the Achilles tendon in a rat model.

We studied the effects of transforming growth factor-beta 1 (TGF-beta 1) on the genetic expression of procollagen type I and III and its effects on structural properties in the early stages of healing in rat Achilles tendon. The Achilles tendons in 90 rats were transsected and repaired immediately. TGF-beta 1 dissolved in phosphate-buffered saline was injected locally at the repair site using two different doses, and outcomes in both groups were compared to that in the control group given phosphate-buffered saline only. Five animals in each group were killed at one, two, and four weeks postoperatively, and the healing tendon was evaluated. A dose-dependent increase in the expression of procollagen type I and III mRNA was found one week postoperatively. The failure load and stiffness of the healing tendon were increased by TGF-beta 1 at two and four weeks.     

An in-vivo experimental evaluation of He–Ne laser photostimulation in healing Achilles tendons

There is no method of treatment that has been proven to accelerate the rate of tendon healing or to improve the quality of the regenerating tendon. Low level laser photostimulation has gained a considerable attention for enhancing tissue repair in a wide spectrum of applications. However, there is controversy regarding the effectiveness of laser photostimulation for improvement of the healing process of surgically repaired tendons. Accordingly, the present study was conducted to evaluate the role of helium–neon (He–Ne) laser photostimulation on the process of healing of surgically repaired Achilles tendons. Thirty unilateral Achilles tendons of 30 Raex rabbits were transected and immediately repaired. Operated Achilles tendons were randomly divided into two equal groups. Tendons at group A were subjected to He–Ne laser (632.8 nm) photostimulation, while tendons at group B served as a control group. Two weeks later, the repaired Achilles tendons were histopathologically and biomechanically evaluated. The histopathological findings suggest the favorable qualitative pattern of the newly synthesized collagen of the regenerating tendons after He–Ne laser photostimulation. The biomechanical results support the same favorable findings from the functional point of view as denoted by the better biomechanical properties of the regenerating tendons after He–Ne laser photostimulation with statistical significance (p ≤ 0.01) at most of the biomechanical parameters. He–Ne laser photostimulation reported a great value after surgical repair of ruptured and injured tendons for a better functional outcome. It could be applied safely and effectively in humans, especially with respect to the proposed long-term clinical outcome.     

Immune modulation with primed mesenchymal stem cells delivered via biodegradable scaffold to repair an Achilles tendon segmental defect

Tendon healing is a complex coordinated series of events resulting in protracted recovery, limited regeneration, and scar formation. Mesenchymal stem cell (MSC) therapy has shown promise as a new technology to enhance soft tissue and bone healing. A challenge with MSC therapy involves the ability to consistently control the inflammatory response and subsequent healing. Previous studies suggest that preconditioning MSCs with inflammatory cytokines, such as IFN‐γ, TNF‐α, and IL‐1β may accelerate cutaneous wound closure. The objective of this study was to therefore elucidate these effects in tendon. That is, the in vivo healing effects of TNF‐α primed MSCs were studied using a rat Achilles segmental defect model. Rat Achilles tendons were subjected to a unilateral 3 mm segmental defect and repaired with either a PLG scaffold alone, MSC‐seeded PLG scaffold, or TNF‐α‐primed MSC‐seeded PLG scaffold. Achilles tendons were analyzed at 2 and 4 weeks post‐injury. In vivo, MSCs, regardless of priming, increased IL‐10 production and reduced the inflammatory factor, IL‐1α. Primed MSCs reduced IL‐12 production and the number of M1 macrophages, as well as increased the percent of M2 macrophages, and synthesis of the anti‐inflammatory factor IL‐4. Primed MSC treatment also increased the concentration of type I procollagen in the healing tissue and increased failure stress of the tendon 4 weeks post‐injury. Taken together delivery of TNF‐α primed MSCs via 3D PLG scaffold modulated macrophage polarization and cytokine production to further accentuate the more regenerative MSC‐induced healing response. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:269–280, 2017.

     

Structural and biomechanical characteristics after early mobilization in an achilles tendon rupture model: operative versus nonoperative treatment

Acute Achilles tendon ruptures are common sports injuries; however, treatment remains a clinical challenge. Studies show a superior effect of early mobilization and full weight bearing on tendon healing and clinical outcome; however, few data exist on structural and biomechanical characteristics in the early healing phase. This study investigated the histological and biomechanical characteristics of early mobilization and full weight bearing in an Achilles tendon rupture model. Eighty rats underwent dissection of a hindpaw Achilles tendon; 40 rats were treated conservatively and 40 underwent open repair of the transected Achilles tendon by suturing. Early mobilization and full weight bearing were allowed in both groups. At 1, 2, 4, and 8 weeks after tenotomy, tensile strength, stiffness, thickness, tissue characteristics (histological analysis), and length were determined. Dissected Achilles tendons healed in all animals during full weight-bearing early mobilization. One and 2 weeks after tenotomy, rats in the operative group showed increased tensile strength and stiffness compared with the nonoperative group. Repair-site diameters were increased at 1, 2, and 8 weeks after tenotomy. Tendon length was decreased in the operative group throughout observation, whereas the nonoperative group showed increased structural characteristics on the cellular level and a more homogeneous collagen distribution. Surgical treatment of dissected rat Achilles tendons showed superior biomechanical characteristics within the first 2 weeks. Conservative treatment resulted in superior histological findings but significant lengthening of the tendon in the early healing phase (weeks 1-8).     

The effects of growth and differentiation factor 5 on bone marrow stromal cell transplants in an in vitro tendon healing model

The effects of growth differentiation factor-5 (GDF-5) and bone marrow stromal cells (BMSCs) on tendon healing were investigated under in?vitro tissue culture conditions. BMSCs and GDF-5 placed in a collagen gel were interpositioned between the cut ends of dog flexor digitorum profundus tendons. The tendons were randomly assigned into four groups: 1) repaired tendon without gel; 2) repaired tendon with BMSC-seeded gel; 3) repaired tendon with GDF-5 gel without cells; and 4) repaired tendon with GDF-5 treated BMSC-seeded gel. At 2 and 4 weeks, the maximal strength of repaired tendons with GDF-5 treated BMSCs-seeded gel was significantly higher than in tendons without gel interposition. However, neither BMSCs nor GDF-5 alone significantly increased the maximal strength of healing tendons at 2 or 4 weeks. These results suggest that the combination of BMSCs and GDF-5 accelerates tendon healing, but either BMSCs or GDF-5 alone are not effective in this model.     

Matrix remodeling in healing rabbit Achilles tendon

Biochemical, biomechanical and ultrastructural properties of the connective tissue matrix were investigated during the early remodeling phase of tissue repair in experimentally tenotomized and repaired rabbit Achilles tendons. Sterile surgical tenotomy was performed on the right Achilles tendons of 14 rabbits and allowed to heal for 15 days. The animals were euthanized and the Achilles tendons excised from both limbs. The left contralateral Achilles tendon of each rabbit was used as a control in the experiments. Prior to biochemical analysis, both intact and healing tendons were tested for their biomechanical integrity. The results revealed that the healing tendons had regained some of their physicochemical characteristics, but differed significantly from the intact left tendons. The healing tendons regained 48% tensile strength, 30% energy absorption, 20% tensile stress, and 14% Young's modulus of elasticity of intact tendons. In contrast, biochemical analysis showed that the healing tendons had 80% of the collagen and 60% of the collagen crosslinks (hydroxypyridinium) of normal tendons. Sequential extraction of collagen from the tissues yielded more soluble collagen in the healing tendons than intact tendons, suggesting either an increase in collagen synthesis and/or enhanced resorption of mature collagen in healing tendons compared to intact tendons. Electron microscopic studies revealed remarkable differences in the ultrastructure between intact and healing tendons. These observations could explain, in part, the connective tissue response to healing during the early phases of tissue remodeling.     

Biomechanical and biochemical studies of tendon healing after conservative and surgical treatment

Based on clinical studies, it has been proposed that conservative treatment of Achilles tendon ruptures is feasible. However, no biomechanical or biochemical confirmation of this proposal has been presented. In the present investigation the biomechanical and biochemical properties of tendons treated surgically and conservatively after transverse tenotomy were studied. Sixty-eight New Zealand White rabbits were used. A transverse tenotomy of the plantaris longus tendon of the left hind leg was performed. In half of the animals the tenotomy was repaired with a criss-cross silk suture and the limb was immobilized; the other half were treated with immobilization alone. The immobilization was maintained for 4 weeks with a long-leg plastic splint in both groups. After 4, 8, and 16 weeks the tendons were examined regarding biomechanical and biochemical parameters. There were no detectable differences in biomechanical parameters or in tendon elongation between the groups, nor was there any difference in the total hydroxyproline content, the amount of mature and of immature collagen, or the water content. The present findings are compatible with previous favorable reports on conservative treatment of Achilles tendon ruptures and thus suggest interesting clinical possibilities.     

Exercise following a short immobilization period is detrimental to tendon properties and joint mechanics in a rat rotator cuff injury model

Rotator cuff tears are a common clinical problem that can result in pain and disability. Previous studies in a rat model showed enhanced tendon to bone healing with postoperative immobilization. The objective of this study was to determine the effect of postimmobilization activity level on insertion site properties and joint mechanics in a rat model. Our hypothesis was that exercise following a short period of immobilization will cause detrimental changes in insertion site properties compared to cage activity following the same period of immobilization, but that passive shoulder mechanics will not be affected. We detached and repaired the supraspinatus tendon of 22 Sprague‐Dawley rats, and the injured shoulder was immobilized postoperatively for 2 weeks. Following immobilization, rats were prescribed cage activity or exercise for 12 weeks. Passive shoulder mechanics were determined, and following euthansia, tendon cross‐sectional area and mechanical properties were measured. Exercise following immobilization resulted in significant decreases compared to cage activity in range of motion, tendon stiffness, modulus, percent relaxation, and several parameters from both a structurally based elastic model and a quasi‐linear viscoelastic model. Therefore, we conclude that after a short period of immobilization, increased activity is detrimental to both tendon mechanical properties and shoulder joint mechanics, presumably due to increased scar production. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:841–845, 2010     

富血小板血浆对大鼠跟腱断裂早期愈合的影响

目的富血小板血浆(platelet-rich plasma,PRP)具有促进损伤组织修复作用。通过观察PRP局部注射对大鼠跟腱断裂早期愈合的影响,为临床应用提供实验依据。方法 SPF级SD大鼠46只,雌雄不限,体重190～240 g。取10只大鼠心脏动脉血制备PRP及贫血小板血浆(platelet-poor plasma,PPP);其余36只随机分为3组(n=12),分别为空白对照组、PPP组及PRP组。大鼠制备双侧跟腱断裂模型后,PPP组和PRP组跟腱周围局部对应注射PPP及PRP,每侧100μL,每周1次至处死;空白对照组不作处理。术后观察大鼠一般情况,于1、2、3、4周取双侧跟腱,行大体、组织学及免疫组织化学染色观察,测量新生跟腱Ⅰ型胶原纤维含量;并于4周行生物力学测试。结果大鼠均存活至实验完成。随着时间延长,各组大鼠跟腱水肿逐渐减退,滑动性逐渐改善;术后3周内各组跟腱粘连逐渐加重,4周时减轻,1、4周时各组跟腱粘连程度分级差异均无统计学意义(P>0.05)。术后1周PRP组炎性细胞浸润、毛细血管及胶原纤维增殖较空白对照组、PPP组明显,之后炎性反应及毛细血管生成逐渐减少。各时间点各组均可见Ⅰ型胶原纤维阳性表达,术后1、2、3周PRP组Ⅰ型胶原纤维阳性密度值多于空白对照组和PPP组(P<0.05),4周时3组差异无统计学意义(P>0.05)。生物力学测试:术后4周3组跟腱最大滑动距离比较,差异均无统计学意义(P>0.05);PRP组跟腱弹性模量及最大抗拉力明显高于空白对照组及PPP组(P<0.05)。结论大鼠跟腱断裂早期于断端周围注射PRP能促进跟腱愈合。     

Synergy of tendon stem cells and platelet-rich plasma in tendon healing

Injured rat Achilles tendons were treated with botulism toxin to create a mechanically unloaded condition (unloaded) or left untreated (loaded), and then treated with phosphate‐buffered saline (PBS), platelet‐rich plasma (PRP), tendon stem cells (TSCs), or a combination (TSCs + PRP). mRNA and protein expression of collagen I, collagen III, tenascin C, and Smad 8 were determined by real time PCR and immunostaining, respectively. Loaded tendons treated with PBS, PRP, or TSCs for 3 or 14 days had higher collagen I mRNA expression than unloaded tendons. Loaded tendons treated with PBS for 3 or 14 days or with PRP for 3 days had higher collagen I protein levels than unloaded tendons. Loaded tendons treated for 3 days with PBS, for 14 days with PRP or TSCs or TSCs + PRP for 3 or 14 days had higher collagen III protein levels than unloaded tendons. Collagen I mRNA levels were higher in TSCs + PRP‐treated loaded tendons compared to PBS‐treated loaded tendons on day 3 of treatment. Based on changes in the expression of tendon‐healing genes, our data suggest that the combination of TSCs and PRP has synergistic effects on tendon healing under both loaded and unloaded conditions, and loaded conditions improve tendon healing. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:991–997, 2012     

Genetic expression for type I procollagen in the early stages of flexor tendon healing.

To determine the precise mechanism by which contact tendon healing occurs at the cellular level, the production of pro alpha (I) collagen messenger RNA (mRNA) produced by fibroblasts of healing intrasynovial flexor tendons was determined by an in situ hybridization technique. The repair site and the proximal and distal tendon stumps of repaired tendons treated with early controlled passive mobilization were fixed and buffered in formalin, 3, 7, 10, and 17 days after repair. A complimentary DNA (cDNA) probe corresponding to alpha (I) procollagen mRNA was labeled with [32P]d-CTP. After hybridization, autoradiography, and staining of the sections, the level of procollagen mRNA was assessed by microscopic examination. Rising levels of procollagen mRNA, indicating progressively increasing levels of synthetic collagen activity, were detected in the healing tendons through 10 days. A moderate decrease in procollagen mRNA was seen at 17 days. Genetic expression for procollagen mRNA was localized specifically to the epitenon cells on the tendon surface overlying the repair site and to cells in the gap between the tendon stumps. No detectable expression was noted in endotenon fibroblasts. The finding of high levels of expression for procollagen type I mRNA in the surface layer of healing tendons demonstrates that cells intrinsic to tendon epitenon contribute the greatest quantity of native tendon collagen to the repair site during these important early intervals after tendon suture.     

Ten weeks of treadmill running decreases stiffness and increases collagen turnover in tendons of old mice

Increased tendon stiffness in response to mechanical loading is well established in young animals. Given that tendons stiffen with aging, we aimed to determine the effect of increased loading on tendons of old animals. We subjected 28‐month‐old mice to 10 weeks of uphill treadmill running; sedentary 8‐ and 28‐month‐old mice served as controls. Following training, plantaris tendon stiffness and modulus were reduced by approximately half, such that the values were not different from those of tendons from adult sedentary animals. The decrease in plantaris tendon stiffness was accompanied by a similar reduction in the levels of advanced glycation end‐product protein adducts in tibialis anterior tendons of trained compared with sedentary old mice. In Achilles tendons, elevated mRNA levels for collagen type 1, matrix‐metalloproteinase‐8, and lysyl oxidase following training suggest that collagen turnover was likely also increased. The dramatic mechanical and structural changes induced by training occurred independent of changes in cell density or tendon morphology. Finally, Achilles tendon calcification was significantly reduced following exercise. These results demonstrate that, in response to exercise, tendons from old animals are capable of replacing damaged and dysfunctional components of extracellular matrix with tissue that is mechanically and structurally comparable to adult tissue. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:346–353, 2016.