Immunomodulatory Behavior of Tendon Magnetic Cell Sheets can be Modulated in Hypoxic Environments under Magnetic Stimulus.

Tissue environments play a crucial role in orchestrating cell behavior, guided by a complex interplay of various factors. Long lasting inflammatory signals compromise tendon homeostasis and promote tissue degeneration, while tissue oxygen levels affect local cells' responses with hypoxic environments influencing apoptosis, inflammatory mediators, and matrix production. Recent works have unveiled the therapeutic potential of pulsed electromagnetic field (PEMF) in modulating inflammatory signals expressed by human tendon cells (hTDCs), and in mitigating the hypoxia-induced effects on the regulation of inflammatory cytokines. Thus, we sought to investigate the role of hypoxic environments, namely, 1 and 2% oxygen tension, in the inflammatory profiles of magnetic cell sheets (magCSs) formed by magnetic nanoparticles internalized in contiguous hTDCs with intact cell-cell junctions and deposited matrix. We also aimed to explore the impact of PEMF over hypoxia-treated magCSs, including IL-1ß-primed-magCSs, with the objective of harnessing magnetic stimulation to guide abnormal inflammatory cell responses toward efficient treatments supporting tendon regenerative potential. Our findings revealed that low oxygen tensions amplified the expression of hypoxia-associated genes and of inflammatory markers in IL-1ß-primed-magCSs with an involvement of the NF-κB signaling pathway. Encouragingly, when PEMF was applied to IL-1ß-primed-magCSs under hypoxic conditions, it successfully modulated inflammatory cues by favoring IL-10 and IL-4, via the NF-κB pathway. These results signify the remarkable potential of PEMF in driving proregenerative strategies and opens up new approaches in tendon therapies, highlighting the transformative impact of immunomodulatory magnetic cell sheets.

A2A adenosine receptors are involved in the reparative response of tendon cells to pulsed electromagnetic fields.

Tendinopathy is a degenerative disease in which inflammatory mediators have been found to be sometimes present. The interaction between inflammation and matrix remodeling in human tendon cells (TCs) is supported by the secretion of cytokines such as IL-1ß, IL-6 and IL-33. In this context, it has been demonstrated that pulsed electromagnetic fields (PEMFs) were able to reduce inflammation and promote tendon marker synthesis. The aim of this study was to evaluate the anabolic and anti-inflammatory PEMF-mediated response on TCs in an in vitro model of inflammation. Moreover, since PEMFs enhance the anti-inflammatory efficacy of adenosine through the adenosine receptors (ARs), the study also focused on the role of A2AARs. Human TCs were exposed to PEMFs for 48 hours. After stimulation, A2AAR saturation binding experiments were performed. Along with 48 hours PEMF stimulation, TCs were treated with IL-1ß and A2AAR agonist CGS-21680. IL-1Ra, IL-6, IL-8, IL-10, IL-33, VEGF, TGF-ß1, PGE2 release and SCX, COL1A1, COL3A1, ADORA2A expression were quantified. PEMFs exerted A2AAR modulation on TCs and promoted COL3A1 upregulation and IL-33 secretion. In presence of IL-1ß, TCs showed an upregulation of ADORA2A, SCX and COL3A1 expression and an increase of IL-6, IL-8, PGE2 and VEGF secretion. After PEMF and IL-1ß exposure, IL-33 was upregulated, whereas IL-6, PGE2 and ADORA2A were downregulated. These findings demonstrated that A2AARs have a role in the promotion of the TC anabolic/reparative response to PEMFs and to IL-1ß.

Magnetic Stimulation Drives Macrophage Polarization in Cell to-Cell Communication with IL-1ß Primed Tendon Cells.

Inflammation is part of the natural healing response, but it has been simultaneously associated with tendon disorders, as persistent inflammatory events contribute to physiological changes that compromise tendon functions. The cellular interactions within a niche are extremely important for healing. While human tendon cells (hTDCs) are responsible for the maintenance of tendon matrix and turnover, macrophages regulate healing switching their functional phenotype to environmental stimuli. Thus, insights on the hTDCs and macrophages interactions can provide fundamental contributions on tendon repair mechanisms and on the inflammatory inputs in tendon disorders. We explored the crosstalk between macrophages and hTDCs using co-culture approaches in which hTDCs were previously stimulated with IL-1ß. The potential modulatory effect of the pulsed electromagnetic field (PEMF) in macrophage-hTDCs communication was also investigated using the magnetic parameters identified in a previous work. The PEMF influences a macrophage pro-regenerative phenotype and favors the synthesis of anti-inflammatory mediators. These outcomes observed in cell contact co-cultures may be mediated by FAK signaling. The impact of the PEMF overcomes the effect of IL-1ß-treated-hTDCs, supporting PEMF immunomodulatory actions on macrophages. This work highlights the relevance of intercellular communication in tendon healing and the beneficial role of the PEMF in guiding inflammatory responses toward regenerative strategies.

Effects of SCCO2, Gamma Irradiation, and Sodium Dodecyl Sulfate Treatments on the Initial Properties of Tendon Allografts.

Sterile and decellularized allograft tendons are viable biomaterials used in reconstructive surgeries for dense connective tissue injuries. Established allograft processing techniques including gamma irradiation and sodium dodecyl sulfate (SDS) can affect tissue integrity. Supercritical carbon dioxide (SCCO2) represents a novel alternative that has the potential to decellularize and sterilize tendons with minimized exposure to denaturants, shortened treatment time, lack of toxic residues, and superior tissue penetration, and thus efficacy. This study attempted to develop a single-step hybrid decellularization and sterilization protocol for tendons that involved SCCO2 treatment with various chemical additives. The processed tendons were evaluated with mechanical testing, histology, scanning electron microscopy (SEM), and Fourier-transform infrared (FTIR) spectroscopy. Uniaxial mechanical testing showed that tendons treated with SCCO2 and additive NovaKillTM Gen2 and 0.1% SDS had significantly higher (p < 0.05) ultimate tensile stress (UTS) and Young's modulus compared to gamma-irradiated and standard-SDS-treated tendons. This was corroborated by the ultrastructural intactness of SCCO2-treated tendons as examined by SEM and FTIR spectroscopy, which was not preserved in gamma-irradiated and standard SDS-treated tendons. However, complete decellularization was not achieved by the experimented SCCO2-SDS protocols used in this study. The present study therefore serves as a concrete starting point for development of an SCCO2-based combined sterilization and decellularization protocol for allograft tendons, where additive choice is to be optimized.

The use of allograft tendons in primary ACL reconstruction.

PURPOSE: Graft choice in primary anterior cruciate ligament (ACL) reconstruction remains controversial. The use of allograft has risen exponentially in recent years with the attraction of absent donor site morbidity, reduced surgical time and reliable graft size. However, the published evidence examining their clinical effectiveness over autograft tendons has been unclear. The aim of this paper is to provide a current review of the clinical evidence available to help guide surgeons through the decision-making process for the use of allografts in primary ACL reconstruction. METHODS: The literature in relation to allograft healing, storage, sterilisation, differences in surgical technique and rehabilitation have been reviewed in addition to recent comparative studies and all clinical systematic reviews and meta-analyses. RESULTS: Early reviews have indicated a higher risk of failure with allografts due to association with irradiation for sterilisation and where rehabilitation programs and post-operative loading may ignore the slower incorporation of allografts. More recent analysis indicates a similar low failure rate for allograft and autograft methods of reconstruction when using non-irradiated allografts that have not undergone chemically processing and where rehabilitation has been slower. However, inferior outcomes with allografts have been reported in young (< 25 years) highly active patients, and also when irradiated or chemically processed grafts are used. CONCLUSION: When considering use of allografts in primary ACL reconstruction, use of irradiation, chemical processing and rehabilitation programs suited to autograft are important negative factors. Allografts, when used for primary ACL reconstruction, should be fresh frozen and non-irradiated. Quantification of the risk of use of allograft in the young requires further evaluation. LEVELS OF EVIDENCE: III.

Effects of supercritical fluid CO2 and 25 kGy gamma irradiation on the initial mechanical properties and histological appearance of tendon allograft.

Tendon allografts, when autograft options are limited or when obtaining an autograft is not aligned with the patients' best interest, play an important role in tendon and ligament reconstruction. To minimize the risk of infectious disease transmission tissue banks perform screening tests and the allografts cleaned are sterilized. The current study examines and compares the initial mechanical properties and histological appearance of supercritical CO2 (SCCO2)-treated and gamma-irradiated porcine extensor tendons. Thirty intact porcine forelimb extensor tendons randomized equally into three groups: control group, gamma-irradiation group, and SCCO2-treated group. Once treated, histological assessment and histomorphologic measurements were made on the histological sections obtained from each tendon while stiffness and ultimate failure loads were evaluated from tensile testing. Histological evaluation of gamma-irradiated tendons showed significant disruption to the hierarchical morphology of the fascicle bundles, which was not evident in SCCO2-treated specimens. Histomorphologic measurements showed a significant increase for measured dead space (void) between tendon fibrils of the gamma-irradiated group comparing to both control and SCCO2 treated groups (p < 0.01). There was a significant reduction in the ultimate failure load for tendons treated by gamma-irradiation compared to the control group (p < 0.05). No statistically significant difference was detected between control and SCCO2-treated tendons in the ultimate failure load. Stiffness values were not significantly different between three-study groups. This study suggests that while gamma-irradiation has a deleterious effect on mechanical properties of tendon tissue, SCCO2 does not alter the biomechanical properties and the histological structure of porcine extensor tendons.

Magnetotherapy: The quest for tendon regeneration.

Tendons are mechanosensitive tissues that connect and transmit the forces generated by muscles to bones by allowing the conversion of mechanical input into biochemical signals. These physical forces perform the fundamental work of preserving tendon homeostasis assuring body movements. However, overloading causes tissue injuries, which leads us to the field of tendon regeneration. Recently published reviews have broadly shown the use of biomaterials and different strategies to attain tendon regeneration. In this review, our focus is the use of magnetic fields as an alternative therapy, which has demonstrated clinical relevance in tendon medicine because of their ability to modulate cell fate. Yet the underlying cellular and molecular mechanisms still need to be elucidated. While providing a brief outlook about specific signalling pathways and intracellular messengers as framework in play by tendon cells, application of magnetic fields as a subcategory of physical forces is explored, opening up a compelling avenue to enhance tendon regeneration. We outline here useful insights on the effects of magnetic fields both at in vitro and in vivo levels, particularly on the expression of tendon genes and inflammatory cytokines, ultimately involved in tendon regeneration. Subsequently, the potential of using magnetically responsive biomaterials in tendon tissue engineering is highlighted and future directions in magnetotherapy are discussed.

Uncovering the effect of low-frequency static magnetic field on tendon-derived cells: from mechanosensing to tenogenesis.

Magnetotherapy has been receiving increased attention as an attractive strategy for modulating cell physiology directly at the site of injury, thereby providing the medical community with a safe and non-invasive therapy. Yet, how magnetic field influences tendon cells both at the cellular and molecular levels remains unclear. Thus, the influence of a low-frequency static magnetic field (2 Hz, 350 mT) on human tendon-derived cells was studied using different exposure times (4 and 8 h; short-term studies) and different regimens of exposure to an 8h-period of magnetic stimulation (continuous, every 24 h or every 48 h; long-term studies). Herein, 8 h stimulation in short-term studies significantly upregulated the expression of tendon-associated genes SCX, COL1A1, TNC and DCN (p < 0.05) and altered intracellular Ca2+ levels (p < 0.05). Additionally, every 24 h regimen of stimulation significantly upregulated COL1A1, COL3A1 and TNC at day 14 in comparison to control (p < 0.05), whereas continuous exposure differentially regulated the release of the immunomodulatory cytokines IL-1ß and IL-10 (p < 0.001) but only at day 7 in comparison to controls. Altogether, these results provide new insights on how low-frequency static magnetic field fine-tune the behaviour of tendon cells according to the magnetic settings used, which we foresee to represent an interesting candidate to guide tendon regeneration.

The effects of irradiation dose and storage time following treatment on the viscoelastic properties of decellularised porcine super flexor tendon.

Decellularised porcine super flexor tendon (pSFT) offers a promising solution to the replacement of damaged anterior cruciate ligament. It is desirable to package and terminally sterilise the acellular grafts to eliminate any possible harmful pathogens. However, irradiation techniques can damage the collagen structure and consequently reduce the mechanical properties. The aims of this study were to investigate the effects of irradiation sterilisation of varying dosages on the viscoelastic properties of the decellularised pSFT. Decellularised pSFT tendons were subjected to irradiation sterilisation using either 30kGygamma, 55kGygamma, 34kGy E-beam, 15kGygamma, 15kGy E-beam and (15+15) kGy E-beam (fractionated dose). Specimens then underwent stress relaxation testing at 0 and 12months post sterilisation to determine whether any effect on the viscoelastic properties was progressive. Significant differences were found which demonstrated that all irradiation treatments had an effect on the time-independent and time-dependent viscoelastic properties of irradiated tendons compared to peracetic acid only treated controls. No significant differences were found between the irradiated groups and no significant differences were found between groups at 0 and 12months. These results indicate the decellularised pSFT graft has a stable shelf-life.

Does a different dose of gamma irradiation have the same effect on five different types of tendon allografts? - a biomechanical study.

INTRODUCTION: The goals of our study were to evaluate the biomechanical differences between five tendons and the changes in biomechanical properties caused by irradiation. METHODS: Achilles, quadriceps, semitendinosus + gracilis (STG), tibialis anterior (TA) and the peroneus longus (PL) were harvested from 30 donors. Group A contained 50 tendons without gamma irradiation. The groups were irradiated with a dose of 21 kGy (group B 50 tendons) and with a dose of 42 kGy (group C 50 tendons). The grafts were soaked in a radio-protectant solution and frozen at -80 °C. Cyclic loading tests were performed followed by load to failure tests. Young modulus of elasticity, maximum force, strain at tensile strength and strain at rupture were calculated. RESULTS: The Achilles tendons had significantly lower Young modulus than the TA (p = 0.0036) in group A. The Achilles showed significantly lower than PL (p = 0.000042) and TA (p = 0.00142) in group B and C. The quadriceps and the ST (p = 0.0037) provided poorer values than the TA (p = 0.0432) in group C. We found no difference in maximum loads among the tendons in group A. The maximum load of the Achilles and quadriceps showed better results than the PL (p = 0.0016), (p = 0.0018) and the STG (p = 0.0066), (p = 0.0019) in group C. The TA had similar results like the Achilles and quadriceps. DISCUSSION AND CONCLUSIONS: The vulnerability of gamma irradiation of TA was less than Achilles and quadriceps tendons.

Ex vivo penetration of low-level laser light through equine skin and flexor tendons.

OBJECTIVE To measure penetration efficiencies of low-level laser light energy through equine skin and to determine the fraction of laser energy absorbed by equine digital flexor tendons (superficial [SDFT] and deep [DDFT]). SAMPLE Samples of skin, SDFTs, and DDFTs from 1 metacarpal area of each of 19 equine cadavers. PROCEDURES A therapeutic laser with wavelength capabilities of 800 and 970 nm was used. The percentage of energy penetration for each wavelength was determined through skin before and after clipping and then shaving of hair, through shaved skin over SDFTs, and through shaved skin, SDFTs, and DDFTs (positioned in anatomically correct orientation). Influence of hair color; skin preparation, color, and thickness; and wavelength on energy penetration were assessed. RESULTS For haired skin, energy penetration was greatest for light-colored hair and least for dark-colored hair. Clipping or shaving of skin improved energy penetration. Light-colored skin allowed greatest energy penetration, followed by medium-colored skin and dark-colored skin. Greatest penetration of light-colored skin occurred with the 800-nm wavelength, whereas greatest penetration of medium- and dark-colored skin occurred with the 970-nm wavelength. As skin thickness increased, energy penetration of samples decreased. Only 1% to 20% and 0.1% to 4% of energy were absorbed by SDFTs and DDFTs, respectively, depending on skin color, skin thickness, and applied wavelength. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that most laser energy directed through equine skin was absorbed or scattered by the skin. To achieve delivery of energy doses known to positively affect cells in vitro to equine SDFTs and DDFTs, skin preparation, color, and thickness and applied wavelength must be considered.

Maximum load to failure of high dose versus low dose gamma irradiation of anterior cruciate ligament allografts: A meta-analysis.

BACKGROUND: The objective of this study was to systematically evaluate the existing literature to compare the biomechanical effects of low dose and high dose gamma irradiation on commonly used ACL allografts. METHODS: A systematic search was performed in PubMed, Cumulative Index for Nursing and Allied Health Literature (CINAHL), Cochrane Reviews, SCOPUS, and SportDiscus. Nine studies were identified that met the following inclusion criteria: 1) controlled laboratory study, 2) investigation of standard allografts for anterior cruciate ligament reconstruction (ACLR), 3) gamma irradiation (dose reported) and a negative control group, and 4) mechanical loading (results reported). RESULTS: Nine studies met all inclusion and exclusion criteria. There was a dose-dependent relationship between radiation and decreased mechanical tendon integrity. Low dose radiation (<2.5Mrad [Mrad]) showed graft weakening with an average of 4.3% decrease in load to failure (standardized mean difference [SMD], 0.23; 95% CI 0.216, 0.68; p=0.31), whereas high-dose radiation showed a significantly larger (32.4% average) decrease in load to failure (SMD, 1.79; 95% CI 1.194, 2.38; p<0.001). CONCLUSIONS: Gamma irradiation has a negative effect on tendon allograft strength that is dose-dependent, with particularly large effects noted at irradiation doses of ≥2.5Mrad.

In vitro and in vivo assessment of magnetically actuated biomaterials and prospects in tendon healing.

AIM: To expand our understanding on the effect of magnetically actuated biomaterials in stem cells, inflammation and fibrous tissue growth. MATERIALS & METHODS: Magnetic biomaterials were obtained by doping iron oxide particles into starch poly-ϵ-caprolactone (SPCL) to create two formulations, magSPCL-1.8 and 3.6. Stem cell behavior was assessed in vitro and the inflammatory response, subcutaneously in Wistar rats. RESULTS: Metabolic activity and proliferation increased significantly overtime in SPCL and magSPCL-1.8. Electromagnetic fields attenuated the presence of mast cells and macrophages in tissues surrounding SPCL and magSPCL-1.8, between weeks 1 and 9. Macrophage reduction was more pronounced for magSPCL-1.8, which could explain why this material prevented growth of fibrous tissue overtime. CONCLUSION: Magnetically actuated biomaterials have potential to modulate inflammation and the growth of fibrous tissue.

Low-level laser therapy modulates pro-inflammatory cytokines after partial tenotomy.

Tendon injuries give rise to substantial morbidity, and current understanding of the mechanisms involved in tendon injury and repair is limited. This lesion remains a clinical issue because the injury site becomes a region with a high incidence of recurrent rupture and has drawn the attention of researchers. We already demonstrated that low-level laser therapy (LLLT) stimulates the synthesis and organization of collagen I, MMP-9, and MMP-2 and improved the gait recovery of the treated animals. The aim of this study was to evaluate the effects of LLLT in the nitric oxide and cytokines profile during the inflammatory and remodeling phases. Adult male rats were divided into the following groups: G1--intact, G2-- injured, G3--injured + LLLT (4 J/cm(2) continuous), G4--injured + LLLT (4 J/cm(2)-20 Hz--pulsed laser). According to the analysis, the animals were euthanized on different dates (1, 4, 8, or 15 days after injury). ELISA assay of TNF-α, IL-1ß, IL-10, and TGF-ß was performed. Western blotting of isoform of nitric oxide synthase (i-NOS) and nitric oxide dosage experiments was conducted. Our results showed that the pulsed LLLT seems to exert an anti-inflammatory effect over injured tendons, with reduction of the release of proinflammatory cytokines, such as TNF-α and the decrease in the i-NOS activity. Thanks to the pain reduction and the facilitation of movement, there was a stimulation in the TGF-ß and IL-1ß release. In conclusion, we believe that pulsed LLLT worked effectively as a therapy to reestablish the tendon integrity after rupture.

The effect of low-intensity pulsed ultrasound on bone-tendon junction healing: Initiating after inflammation stage.

The purpose of this study was to explore the effect of low-intensity pulsed ultrasound (LIPUS) treatment initiating after inflammation stage on the process of bone-tendon junction (BTJ) healing in a rabbit model. Thirty-six rabbits undergoing partial patellectomy were randomly divided into two groups: control and LIPUS. The period of initial inflammatory stage is 2 weeks. So LIPUS treatment was initiated at postoperative week 2 and continued until the patella-patellar tendon (PPT) complexes were harvested at postoperative weeks 4, 8, and 16. At each time point, the PPT complexes were harvested for qRT-PCR, histology, radiographs, synchroton radiation micro computed tomography (SR-µCT), and biomechanical testing. The qRT-PCR results showed that LIPUS treatment beginning at postoperative week 2 played an anti-inflammatory role in BTJ healing. Histologically, the LIPUS group showed more advanced remodeling of the lamellar bone and marrow cavity than the control group. The area and length of the new bone in the LIPUS group were significantly greater than the control group at postoperative weeks 8 and 16. SR-µCT demonstrated that new bone formation and remodeling in the LIPUS group were more advanced than the control group. Biomechanical test results demonstrated that the failure load, ultimate strength and energy at failure were significantly higher than those of the control group. In conclusion, LIPUS treatment beginning at postoperative week 2 was able to accelerate bone formation during the bone-tendon junction healing process and significantly improved the healing quality of BTJ injury. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1697-1706, 2016.

Focal Adhesion Kinase Signaling Mediated the Enhancement of Osteogenesis of Human Mesenchymal Stem Cells Induced by Extracorporeal Shockwave.

Extracorporeal shockwave (ESW) has been shown of great potential in promoting the osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but it is unknown whether this osteogenic promotion effect can also be achieved in other MSCs (i.e., tendon-derived stem cells (TDSCs) and adipose-derived stem cells (ADSCs)). In the current study, we aimed not only to compare the osteogenic effects of BMSCs induced by ESW to those of TDSCs and ADSCs; but also to investigate the underlying mechanisms. We show here that ESW (0.16 mj/mm(2)) significantly promoted the osteogenic differentiation in all the tested types of MSCs, accompanied with the downregulation of miR-138, but the activation of FAK, ERK1/2, and RUNX2. The enhancement of osteogenesis in these MSCs was consistently abolished when the cells were pretreated with one of the following conditions: overexpression of miR-138, FAK knockdown using specific siRNA, and U0126, implying that all of these elements are indispensable for mediating the effect of ESW. Moreover, our study provides converging genetic and molecular evidence that the miR-138-FAK-ERK1/2-RUNX2 machinery can be generally activated in ESW-preconditioned MSCs, suggesting that ESW may be a promising therapeutic strategy for the enhancement of osteogenesis of MSCs, regardless of their origins.

Extracorporeal Shock Wave Treatment (ESWT) enhances the in vitro-induced differentiation of human tendon-derived stem/progenitor cells (hTSPCs).

Extracorporeal shock wave therapy (ESWT) is a non-invasive and innovative technology for the management of specific tendinopathies. In order to elucidate the ESWT-mediated clinical benefits, human Tendon-derived Stem/Progenitor cells (hTSPCs) explanted from 5 healthy semitendinosus (ST) and 5 ruptured Achilles (AT) tendons were established. While hTSPCs from the two groups showed similar proliferation rates and stem cell surface marker profiles, we found that the clonogenic potential was maintained only in cells derived from healthy donors. Interestingly, ESWT significantly accelerated hTSPCs differentiation, suggesting that the clinical benefits of ESWT may be ascribed to increased efficiency of tendon repair after injury.

Effect of a novel sterilization method on biomechanical properties of soft tissue allografts.

PURPOSE: Evaluate allograft tissue commonly used in soft tissue reconstruction to determine whether stiffness and strength were significantly altered after grafts were treated with different sterilization methods. Unprocessed, irradiated, and grafts treated with supercritical CO2 were compared. METHODS: Thirty-eight anterior or posterior tibialis tendons were obtained from a tissue bank (Allograft Innovations, Gainesville, FL). Group I was unprocessed, group II was sterilized with gamma irradiation (20-28 kGy), and group III was sterilized with supercritical CO2. The grafts were pretensioned to 89 N for 300 s. Specimens were then loaded from 50 to 300 N at 0.5 Hz for 250 cycles before being loaded to failure at 50 mm/min. Dependent variables were compared between sterilization groups with one-way ANOVA (P < 0.05) and equivalence trial. RESULTS: There was no significant difference in load to failure or failure stress among groups I, II, and III. Group III resulted in 27-36 % lower stiffness than group I and II. This difference was significant at 1, 10, 50, 100, and 250 cycles. There was no significant difference in stiffness between group I and group II. CONCLUSION: The two sterilization methods tested in this study do not affect allograft strength. The supercritical CO2 sterilization method resulted in significantly lower stiffness than unprocessed and irradiated allografts. However, the stiffness and strength of all groups tested were greater than that of published values of the native intact anterior cruciate ligament (ACL). This study provides previously unpublished mechanical test data on a new sterilization technique that will assist surgeons to decide which allograft to use in ACL reconstruction surgery. LEVEL OF EVIDENCE: III.

Effect of phototherapy with light-emitting diodes (890 nm) on tendon repair: an experimental model in sheep.

The effect of phototherapy with 890-nm light-emitting diodes (LEDs) on the healing of experimentally induced tendinitis in sheep was evaluated in this study. Partial tenotomies measuring 0.2 cm wide × 0.5 cm long were performed on the second third of the superficial digital flexor tendons of 10 healthy sheep. The animals were divided into two groups: "treated" (TG), treated with LEDs at the aforementioned wavelength, and "control" (CG), a control group treated with a placebo. Kinesiotherapy, which consisted of 5-min walks on grassy ground, was performed on both groups. B-mode and power Doppler ultrasonographies (US) were performed to evaluate the tendon healing process during the first 14 days after surgery and on the 21st and 28th postoperative days. Biopsies were performed on day 28 for the histopathological assessment of neovascularisation and the pattern of the tendon fibres. The absence of lameness and a significant improvement (p < 0.05) in the sensitivity to pain during palpation were observed in the treated group. Furthermore, a significant reduction in oedema and an increased number of vessels (p < 0.05) were observed in this group with the B-mode and power Doppler US, respectively. No significant difference in the evolution of the lesion was found. There was a histological difference (p < 0.05) in neovascularisation in the treated group. Phototherapy with 890-nm light-emitting diodes decreases the inflammatory process.

Effects of gamma irradiation on collagen damage and remodeling.

PURPOSE: To evaluate the dose-time dependences of structural changes occurring in collagen within 24 hours to three months after gamma-irradiation at doses from 2-40 Gy in vivo. MATERIALS AND METHODS: Rat's tail tendon was chosen as in vivo model, with its highly ordered collagen structure allowing the changes to be interpreted unambiguously. Macromolecular level (I) was investigated by differential scanning calorimetry (DSC); fibers and bundles level (II) by laser scanning microscopy (LSM), and bulk tissue microstructural level (III) by cross-polarization optical coherence tomography (CP-OCT). RESULTS: For (I), the formation of molecular cross-links and breaks appeared to be a principal mechanism of collagen remodeling, with the cross-links number dependent on radiation dose. Changes on level (II) involved primary, secondary and tertiary bundles splitting in a day and a week after irradiation. Bulk collagen microstructure (III) demonstrated early widening of the interference fringes on CP-OCT images observed to occur in the tendon as result of this splitting. At all three levels, the observed collagen changes demonstrated complete remodeling within ∼ a month following irradiation. CONCLUSION: The time course and dose dependencies of the observed collagen changes at different levels of its hierarchy further contribute to elucidating the role of connective tissue in the radiotherapy process.