Organization Of Collagen Fibers Research Articles

Molecular and structural insights into skin collagen reveals several factors that influence its architecture.

Although the biomechanical properties of skin and its molecular components have been extensively studied, little research has been devoted to understanding the links between them. Here, a comprehensive analysis of the molecular components of deer and cow skins was undertaken in order to understand the basis of their physical properties. These skins were chosen because they are known to be strong yet supple, exhibiting properties that have been exploited by man for centuries. Firstly, the tensile strength, tear strength and denaturation temperature of deer and cow skins were measured. Secondly, the organisation of the collagen fibrils and presence of glycosaminoglycans in each skin was investigated using polarising microscopy (PM), laser scanning confocal microscopy (LSCM), transmission electron microscopy (TEM), nuclear magnetic resonance (NMR) and small angle X-ray scattering (SAXS). Finally, amino acid, crosslink and glycosaminoglycan analyses were carried out on both skins in the study. The results of the study showed that individual physical properties such as tensile strength of the skin are derived from different combinations of biomolecular components which are reflected in collagen architecture. The "wavy" organisation of collagen fibres in deer skin was associated with a small fibril diameter, uniform glycosaminoglycan distribution and higher proportion of trivalent crosslinks. In contrast, the collagen fibrils in cow skin were large, contained a diverse glycosaminoglycan distribution and a higher proportion of tetravalent crosslinks, resulting in straight fibres. This study showed for the first time that the relationship between the structure of collagen in skin and its biomechanical functions is complex, arising from different architectural and molecular features including organisation of collagen fibres, diameters of collagen fibrils, distribution and amount of glycosaminoglycans and types and concentrations of crosslinks.

Wrapping of tendon tissues with diclofenac-immobilized polycaprolactone fibrous sheet improves tendon healing in a rabbit model of collagenase-induced Achilles tendinitis

We fabricated diclofenac-immobilized polycaprolactone (DFN/PCL) fibrous sheets to improve tendon healing of rabbits with collagenase-induced Achilles tendinitis. DFN/PCL have anti-inflammatory effects related to suppression of mRNA levels of pro-inflammatory factors including COX-2, IL-6, TNF-α, and MMP-3 in inflamed tenocytes and tendon tissues of rabbits with Achilles tendinitis as well as those of anti-inflammatory cytokines including IL-4, IL-10, and IL-13. In vivo studies, wrapping of tendon tissues with DFN/PCL fibrous sheets may afford superior tendon healing, with decreased inflammatory cells, increased collagen content, restored collagen fiber organization, and improved mechanical strength. This approach will be a promising treatment from Achilles tendinitis.

Transected Tendon Treated with a New Fibrin Sealant Alone or Associated with Adipose-Derived Stem Cells.

Tissue engineering and cell-based therapy combine techniques that create biocompatible materials for cell survival, which can improve tendon repair. This study seeks to use a new fibrin sealant (FS) derived from the venom of Crotalus durissus terrificus, a biodegradable three-dimensional scaffolding produced from animal components only, associated with adipose-derived stem cells (ASC) for application in tendons injuries, considered a common and serious orthopedic problem. Lewis rats had tendons distributed in five groups: normal (N), transected (T), transected and FS (FS) or ASC (ASC) or with FS and ASC (FS + ASC). The in vivo imaging showed higher quantification of transplanted PKH26-labeled ASC in tendons of FS + ASC compared to ASC on the 14th day after transection. A small number of Iba1 labeled macrophages carrying PKH26 signal, probably due to phagocytosis of dead ASC, were observed in tendons of transected groups. ASC up-regulated the Tenomodulin gene expression in the transection region when compared to N, T and FS groups and the expression of TIMP-2 and Scleraxis genes in relation to the N group. FS group presented a greater organization of collagen fibers, followed by FS + ASC and ASC in comparison to N. Tendons from ASC group presented higher hydroxyproline concentration in relation to N and the transected tendons of T, FS and FS + ASC had a higher amount of collagen I and tenomodulin in comparison to N group. Although no marked differences were observed in the other biomechanical parameters, T group had higher value of maximum load compared to the groups ASC and FS + ASC. In conclusion, the FS kept constant the number of transplanted ASC in the transected region until the 14th day after injury. Our data suggest this FS to be a good scaffold for treatment during tendon repair because it was the most effective one regarding tendon organization recovering, followed by the FS treatment associated with ASC and finally by the transplanted ASC on the 21st day. Further investigations in long-term time points of the tendon repair are needed to analyze if the higher tissue organization found with the FS scaffold will improve the biomechanics of the tendons.

PEDF-derived peptide promotes tendon regeneration through its mitogenic effect on tendon stem/progenitor cells

BackgroundTendon stem/progenitor cells (TSPC) exhibit a low proliferative response to heal tendon injury, leading to limited regeneration outcomes. Exogenous growth factors that activate TSPC proliferation have emerged as a promising approach for treatment. Here, we evaluated the pigment epithelial-derived factor (PEDF)-derived short peptide (PSP; 29-mer) for treating acute tendon injury and to determine the timing and anatomical features of CD146- and necleostemin-positive TSPC in the tendon healing process.MethodsTendon cells were isolated from rabbit Achilles tendons, stimulated by the 29-mer and analyzed for colony-forming capacity. The expression of the TSPC markers CD146, Oct4, and nestin, induced by the 29-mer, was examined by immunostaining and western blotting. Tendo-Achilles injury was induced in rats by full-thickness insertion of an 18-G needle and immediately treated topically with an alginate gel, loaded with 29-mer. The distribution of TSPC in the injured tendon and their proliferation were monitored using immunohistochemistry with antibodies to CD146 and nucleostemin and by BrdU labeling.ResultsTSPC markers were enriched among the primary tendon cells when stimulated by the 29-mer. The 29-mer also induced the clonogenicity of CD146+ TSPC, implying TSPC stemness was retained during TSPC expansion in culture. Correspondingly, the expanded TSPC differentiated readily into tenocyte-like cells after removal of the 29-mer from culture. 29-mer/alginate gel treatment caused extensive expansion of CD146+ TSPC in their niche on postoperative day 2, followed by infiltration of CD146+/BrdU− TSPC into the injured tendon on day 7. The nucleostemin+ TSPC were located predominantly in the healing region of the injured tendon in the later phase (day 7) and exhibited proliferative capacity. By 3 weeks, 29-mer-treated tendons showed more organized collagen fiber regeneration and higher tensile strength than control tendons. In culture, the mitogenic effect of the 29-mer was found to be mediated by the phosphorylation of ERK2 and STAT3 in nucleostemin+ TSPC.ConclusionsThe anatomical analysis of TSPC populations in the wound healing process supports the hypothesis that substantial expansion of resident TSPC by exogenous growth factor is beneficial for tendon healing. The study suggests that synthetic 29-mer peptide may be an innovative therapy for acute tendon rupture.

Effect of the magnetotherapy in the regeneration after tenotomy in animals

Background: The Achilles tendon is the largest and most resistant tendon in the human body, being one of the most common areas of overload injury among athletes. The rupture occurs more frequently in football athletes, running and jumping, because these activities cause a great stress on the tendon during eccentric muscle contraction of the sural triceps. Among the different physiotherapy resources used to repair tendon injuries, there is magnetotherapy, which is a feature that uses a pulsatile electromagnetic field, a suggested inducer of the acceleration of regeneration. Objective: To investigate the effects of magnetotherapy on the organization of collagen and elastic fibers and inflammatory process induced in different days and phases of the healing process of Achilles tendon injury in rats. Method: This study is a randomized controlled trial. Thirty female Wistar rats were randomly divided into 5 groups (each with n = 6), submitted to tenotomy by transverse shear in the middle third of the right Achilles tendon, except for group 1, which was control group without injury, sacrificed on day 14. Group 2, control with injury, sacrificed on day 7; group 3 was treated and received 5 pulsed electromagnetic field (PEMF) applications from 24h post injury and sacrificed on day 7; group 4, control with injury and sacrificed on day 14; and group 5 received 12 PEMF applications from 24h post injury and sacrificed on day 14. The parameters used were 20 mT, 50Hz and 30 minutes of application in each session, using the Magnetherp 330 digital device (Meditea). At the end of treatment, a histological study was performed to evaluate the amount of collagen and elastic cells and fibers and to compare the degree of organization of collagen fibers. Results: G - 3, compared to its control G - 2 showed lower acute inflammatory response; G - 5 did not show acute and chronic inflammation and possessed an accelerated process of repair without fibrosis, unlike its control. Conclusion: The magnetotherapy decreased the signs of inflammation in the acute phase, accelerated the repair process with the onset of fibrosis in 7 days; and at 14 days the tendon tissue was healed, with organizing fibrosis, without acute or chronic inflammation.

Evaluation of biological behavior of odontogenic keratocyst and orthokeratinized odontogenic cyst using picrosirius red stain: A clinicopathological retrospective study

Background: Odontogenic keratocyst (OKC) and orthokeratinized odontogenic cyst (OOC) show considerable difference in biological behavior, and the study of the organization of collagen fibers may be helpful in understanding the nature of these variants. Aims: The purpose of this study is to determine the clinicopathological features and to analyze the birefringence of collagen fibers in OKC and OOC using picrosirius red stain to delineate its exact role in biological behavior. Materials and Methods: Clinical data were obtained from the case records of 71 patients, and for the analysis of birefringence, paraffin-embedded blocks of 10 cases each of OKC and OOC were retrieved from the archives of our department during the period of 2010–2018. Tissue sections were obtained and stained with picrosirius red stain to determine the birefringence of collagen fibers in both the variants. Statistical analysis was done by unpaired t-test. Results: Out of 71 cases, 61 were OKC and 10 were OOC. The clinicopathological data revealed that the most common affected age group was third decade with male predilection and the most common site noted was posterior mandible, and considerable difference in birefringence was noted in the collagen fibers of OKC and OOC. Conclusion: The study results analyzing the clinicopathological parameters of OKC and OOC could act as a viable record for the distribution of odontogenic lesions in the sample population. Significant differences in birefringence were demonstrated in OKC and OOC, suggesting that different collagen fibers may exist in the two lesions.

Effect of varying the crosslinking degree of a pericardial implant with oligourethane on the repair of a rat full-thickness abdominal wall defect.

The stability and bioactivity of biologic implants rely mainly on the control of the crosslinking process of collagen. However, the most common methods have no control on the crosslinking degree producing it excessively. This study outlines the role of crosslinking of collagen-based implants with oligourethane on the host response following reconstruction of a rat full-thickness abdominal wall defect. We decellularized and crosslinked bovine pericardial tissue to achieve two crosslinking degrees. For the decellularized implants, named as non-crosslinked (N-CL), the collagen-amines were 0.42 ± 0.02 mmol/mg. Crosslinking by the oligourethane reduced the primary amine concentration to 0.28 ± 0.01 and 0.19 ± 0.01 mmol/mg; these values were classified as low (∼30%, L-CL) and medium crosslinking (∼50%, M-CL), respectively. By imaging the implants using second harmonic generation microscopy, we observed undulated bundles of collagen fibers organized in multi-directed layers localized in N-CL and L-CL samples. Post-implantation, a negligible change in the organization of collagen fibers in the crosslinked implants was observed, suggesting that the in vivo biodegradation was delayed. An enlargement of the implant area was also observed, without rupture, in all three (N-CL, L-CL, M-CL) materials, whereas adhesion to the omentum, but not to the bowel, was observed. The number of blood vessels after 90-day implantation in N-CL and L-CL was 13 ± 1 and 12 ± 1 per field, respectively, while the number significantly decreased to 2 ± 1 in M-CL. The results suggest that the controlled degree of crosslinking in oligourethane-modified biologic implants can be used as a strategy to balance biodegradation and remodeling in surgical repair of soft tissues.

Engineered tendon-fibrocartilage-bone composite and bone marrow-derived mesenchymal stem cell sheet augmentation promotes rotator cuff healing in a non-weight-bearing canine model

Reducing rotator cuff failure after repair remains a challenge due to suboptimal tendon-to-bone healing. In this study we report a novel biomaterial with engineered tendon-fibrocartilage-bone composite (TFBC) and bone marrow-derived mesenchymal stem cell sheet (BMSCS); this construct was tested for augmentation of rotator cuff repair using a canine non-weight-bearing (NWB) model. A total of 42 mixed-breed dogs were randomly allocated to 3 groups (n = 14 each). Unilateral infraspinatus tendon underwent suture repair only (control); augmentation with engineered TFBC alone (TFBC), or augmentation with engineered TFBC and BMSCS (TFBC + BMSCS). Histomorphometric analysis and biomechanical testing were performed at 6 weeks after surgery. The TFBC + BMSCS augmented repairs demonstrated superior histological scores, greater new fibrocartilage formation and collagen fiber organization at the tendon-bone interface compared with the controls. The ultimate failure load and ultimate stress were 286.80 ± 45.02 N and 4.50 ± 1.11 MPa for TFBC + BMSCS group, 163.20 ± 61.21 N and 2.60 ± 0.97 MPa for control group (TFBC + BMSCS vs control, P = 1.12E-04 and 0.003, respectively), 206.10 ± 60.99 N and 3.20 ± 1.31 MPa for TFBC group (TFBC + BMSCS vs TFBC, P = 0.009 and 0.045, respectively). In conclusion, application of an engineered TFBC and BMSCS can enhance rotator cuff healing in terms of anatomic structure, collagen organization and biomechanical strength in a canine NWB model. Combined TFBC and BMSCS augmentation is a promising strategy for rotator cuff tears and has a high potential impact on clinical practice.

P3.03-19 The Lysyl Oxidase like-1 Promotes NSCLC Tumorigenecity Through Increased Matrix Reorganization and Stiffness

The importance of the stroma in tumorigenicity has been increasingly recognized in the past few years and targeting tumor stroma has become a new strategy for the development of new therapies. The lysyl oxidase like-1 (LOXL1) is a matrix cross-linking enzyme, essentially secreted by cancer-associated fibroblasts (CAFs) in the stroma. We previously showed that LOXL1 expression strongly correlated with integrin α11 expression, a collagen receptor that promotes NSCLC tumorigenecity, suggesting that LOXL1 may also contribute to lung tumor growth and metastasis by modifying the stromal collagen matrix. Primary CAFs (CAF094) isolated from a NSCLC resection specimen and immortalized with hTERT were infected with full length LOXL1 (LOXL1 overexpression), LOXL1 shRNA (LOXL1 knockdown) or with their respective controls, using a lentiviral system. To investigate the impact of LOXL1 on collagen matrix reorganization, we allowed CAFs expressing different levels of LOXL1 to contract collagen matrices. Contracted collagen matrices have then been submitted to second harmonic generation to analyze collagen fibers. We established the role of LOXL1 in lung carcinoma invasion and growth using an in vitro organotypic model and an in vivo xenograft model, respectively. LOXL1-overexpressing CAFs embedded in collagen lattices displayed greater ability to reorganize the collagen matrix than those with lower LOXL1 expression. Furthermore, analysis of the collagen matrix demonstrated that LOXL1 contributed to more linear and dense collagen fibers organization. As a consequence of collagen matrix reorganization, invasion of the lung carcinoma H661 cell line significantly increased in an organotypic model in presence of LOXL1-overexpressing CAFs. Moreover, we showed that loss of Loxl1 in mice inhibited subcutaneous growth of the lung adenocarcinoma H4006 cell line compared to the mouse wild-type counterparts. We demonstrated that overexpression of LOXL1 in NSCLC tumor stroma results in an increased of tumor growth and invasion, due to a change in the matrix reorganization. Thus, LOXL1 appears as an interesting target to improve the outcome of lung cancer.

Toxicity of the biocide polycarbamate, used for aquaculture nets, to some marine fish species

We investigated toxic effects of the antifouling biocide polycarbamate (PC) on marine fish by conducting acute, early-life stage toxicity (ELS), and embryo toxicity tests. Mummichog (Fundulus heteroclitus) 96-h LC50 values for hatched larvae (body weight about 2.0 mg) and juveniles (660 ± 36 mg) were about 12 and 630 μg/L, respectively. The ELS test using mummichog embryos yielded a lowest-observed-effect concentration of 3.9 μg/L and a no-observed-effect concentration of 2.1 μg/L with growth as the most sensitive endpoint. The embryo toxicity test for spotted halibut (Verasper variegatus) revealed a 10-d EC50 of 8.1 μg/L with abnormality as an endpoint. During the ELS and embryo toxicity tests, morphological abnormalities (notochord undulation) were induced in the embryos. Biochemical and gene-expression analysis suggest that PC-induced morphological abnormalities involve disruption of lysyl oxidase-mediated collagen fiber organization, essential for notochord formation, and inhibition of gene expression related to notochord formation.

Human Tendon-Derived Hydrogel Improves Healing of Chronic Tendon–Bone Interface Injuries

Grant support received from: 2016 AFSH Basic Science Grant There is no financial information to disclose. Previously our laboratory reported the positive impact of thermoresponsive, type 1 collagen–rich, decellularized human tendon-derived hydrogel (tHG) on tendon healing using a rat Achilles tendon acute injury model. We hypothesized tHG would facilitate healing of chronic injuries at tendon–bone interface (TBI) in a rotator cuff model. Rotator cuff tears are important causes of pain and disability of upper extremity and involve injury at TBI. In this study a rat chronic rotator cuff injury model was used to evaluate TBI healing after repair. Twenty Sprague–Dawley rats underwent bilateral detachment of supraspinatus (SSp) tendons. Eight weeks later, both SSp tendons were anatomically reattached to humerus. One shoulder underwent a suture to bone repair using bone tunnels (control group, n = 20) and the other underwent the same repair plus tHG injection at repair site (tHG group, n = 20). Eight weeks after repair, SSp tendon–muscle units with humeral bone were harvested after animal death. Biomechanical tests were performed on 15 samples in each group. Five additional samples in each group were evaluated via micro CT, followed by histological examinations. Paired t tests were performed for statistical analyses. Biomechanical testing revealed significantly larger load to failure (27.5 ± 11.8N vs 19.7 ± 5.4N, P = .03) and higher stiffness (15.7 ± 6.3 N/mm vs 11.5 ± 3.5 N/mm, P = .02) in the tHG group versus control. Safranin O staining showed the significantly larger area of new cartilage formation in the tHG group (0.48 ± 0.1 mm2 vs 0.32 ± 0.06 mm2, P = .03). Based on birefringence under polarized microscopy in Picosirius red–stained slides collagen fiber organization was greater in tHG group compared with the control (40.4 ± 9.8 gray scale vs 31.1 ± 4.2 gray scale, P = .07), although it did not significantly differ. Micro CT demonstrated higher bone mineral density (296.1 ± 21.9 mg/cm3 vs 274.6 ± 17.2 mg/cm3, P = .05) and larger trabecular number (1.7 ± 0.1/mm vs 1.5 ± 0.2, P = .07) at the supraspinatus insertion area of the humerus in tHG group than those in the control, although the difference did not reach statistical significance. •Injection of collagen-rich tHG at the rotator cuff repair site statistically significantly enhanced biomechanical properties and increased fibrocartilage formation at the TBI in a chronic injury model.•An improvement collagen fiber organization, higher bone mineral density, and larger trabecular number were also seen in the tHG group versus control.•This human-derived collagen gel is a promising scaffold to augment healing of chronic TBI injuries during surgical intervention. This research was supported by a Basic Science Grant from the American Foundation for Surgery of the Hand.

Injured Achilles Tendons Treated with Adipose-Derived Stem Cells Transplantation and GDF-5.

Tendon injuries represent a clinical challenge in regenerative medicine because their natural repair process is complex and inefficient. The high incidence of tendon injuries is frequently associated with sports practice, aging, tendinopathies, hypertension, diabetes mellitus, and the use of corticosteroids. The growing interest of scientists in using adipose-derived mesenchymal stem cells (ADMSC) in repair processes seems to be mostly due to their paracrine and immunomodulatory effects in stimulating specific cellular events. ADMSC activity can be influenced by GDF-5, which has been successfully used to drive tenogenic differentiation of ADMSC in vitro. Thus, we hypothesized that the application of ADMSC in isolation or in association with GDF-5 could improve Achilles tendon repair through the regulation of important remodeling genes expression. Lewis rats had tendons distributed in four groups: Transected (T), transected and treated with ADMSC (ASC) or GDF-5 (GDF5), or with both (ASC+GDF5). In the characterization of cells before application, ADMSC expressed the positive surface markers, CD90 (90%) and CD105 (95%), and the negative marker, CD45 (7%). ADMSC were also differentiated in chondrocytes, osteoblast, and adipocytes. On the 14th day after the tendon injury, GFP-ADMSC were observed in the transected region of tendons in the ASC and ASC+GDF5 groups, and exhibited and/or stimulated a similar genes expression profile when compared to the in vitro assay. ADMSC up-regulated Lox, Dcn, and Tgfb1 genes expression in comparison to T and ASC+GDF5 groups, which contributed to a lower proteoglycans arrangement, and to a higher collagen fiber organization and tendon biomechanics in the ASC group. The application of ADMSC in association with GDF-5 down-regulated Dcn, Gdf5, Lox, Tgfb1, Mmp2, and Timp2 genes expression, which contributed to a lower hydroxyproline concentration, lower collagen fiber organization, and to an improvement of the rats’ gait 24 h after the injury. In conclusion, although the literature describes the benefic effect of GDF-5 for the tendon healing process, our results show that its application, isolated or associated with ADMSC, cannot improve the repair process of partial transected tendons, indicating the higher effectiveness of the application of ADMSC in injured Achilles tendons. Our results show that the application of ADMSC in injured Achilles tendons was more effective in relation to its association with GDF-5.

Cell regulation of collagen fibril macrostructure during corneal morphogenesis

While tissue form and function is highly dependent upon tissue-specific collagen composition and organization, little is known of the mechanisms controlling the bundling of collagen fibrils into fibers and larger structural designs that lead to the formation of bones, tendons and other tissues. Using the cornea as a model system, our previous 3 dimensional mapping of collagen fiber organization has demonstrated that macrostructural organization of collagen fibers involving interweaving, branching and anastomosing plays a critical role in controlling mechanical stiffness, corneal shape and refractive power. In this work, the cellular and mechanical mechanisms regulating critical events in the assembly of collagen macrostructure are analysed in the developing chicken cornea. We elucidated the temporal events leading to adult corneal structure and determined the effects of intraocular pressure (IOP) on the organization of the collagen macrostructure. Our findings indicate that the complex adult collagen organization begins to appear on embryonic day 10 (E10) after deposition of the primary stroma and full invasion of keratocytes. Importantly, organizational changes in keratocytes appearing at E9 preceded and predicted later changes in collagen organization. Corneal collagen organization remained unaffected when the development of IOP was blocked at E4. These findings support a primary role for keratocytes in controlling stromal organization, mechanical stiffness and corneal shape that are not regulated by the IOP. Our findings also suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. Statement of significanceThis work by using an ex ovo model system, begins to investigate the potential mechanisms controlling collagen fibril macrostructure. In particular, this work highlights a convergent role for the corneal keratocytes in organizing the complex collagen macrostructure, necessary to support high visual acuity. Our data supports that the intraocular pressure does not influence collagen fibril macrostructure and suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. Clearly understanding the cellular and molecular mechanisms that underlie collagen fibril macrostructure will be highly beneficial for future tissue engineering and regenerative medicine applications.

Effect of 3 Preservation Methods (Freezing, Cryopreservation, and Freezing + Irradiation) on Human Menisci Ultrastructure: An Ex Vivo Comparative Study With Fresh Tissue as a Gold Standard

Background: Three main meniscus preservation methods have been advocated: freezing (–80°C), freezing with gamma irradiation (–80°C + 25 kGy), and cryopreservation (–140°C). Hypothesis: All preservation methods will result in structural and architectural properties similar to those of fresh meniscus, defined as the gold standard. Study Design: Controlled laboratory study. Methods: Five human intact menisci were collected from 5 patients undergoing total knee arthroplasty. The inclusion criteria were patients <70 years old with primary unilateral (medial) femorotibial knee osteoarthritis and without surgical or traumatic history on the operated knee. Four cubes (9 mm3) were cut inside of the white, or avascular, area of each specimen’s middle horn and divided into 4 groups: “fresh” control, frozen (–80°C), cryopreserved (–140°C), and frozen + irradiated (–80°C + 25 kGy). Specimens of the control group were evaluated at day 1, and specimens from the frozen, cryopreserved, and frozen + irradiated groups were evaluated after 1 month of storage. Evaluation was performed with electron microscopy according a validated protocol to analyze (1) mean diameters of the collagen fibers in longitudinal and transverse sections in 5 points per section and (2) validated architectural scores. Results: No significant difference was found between the control and cryopreserved groups regarding mean transverse and longitudinal diameters (transverse: 95.39 ± 15.87 nm vs 99.62 ± 19.23 nm, P = .1; longitudinal: 96.31 ± 13.96 nm vs 94.57 ± 16.42 nm, P = .1). Significant differences were found between the control and frozen groups (transverse: 95.39 ± 15.87 nm vs 70.20 ± 13.94 nm, P < .001; longitudinal: 96.31 ± 13.96 nm vs 71.28 ± 10.64 nm, P < .001) and the control and frozen + irradiated groups (transverse: 95.39 ± 15.87 nm vs 63.1 ± 15.57 nm, P < .001; longitudinal: 96.31 ± 13.96 nm vs 60.9 ± 14.8 nm, P < .001). Regarding architectural score calculation, there were significant differences between the control and frozen groups (4.5 ± 1.3 vs 2.3 ± 1.4, P = .02) and the control and frozen + irradiated groups (4.5 ± 1.3 vs 1.4 ± 0.9, P = .02). Conclusion: Cryopreservation is the only method that preserves fresh meniscus architectural specificities. Freezing and freezing + irradiation methods modify histologic properties of meniscal allograft. Irradiation deeply alters diameters and the organization of collagen fibers, and this method should be used with caution to preserve and sterilize meniscus tissue. Clinical Relevance: The results of our study exhibited detrimental effects of simple freezing and freezing + irradiation on the collagen network of sample meniscus. If those effects occur in menisci prepared for allograft procedures, important differences could appear on the basis of the preservation procedure in terms of the graft’s mechanical properties and, thus, the patient’s outcomes.

Anisotropy of thePassive and Active Rat Vagina Under Biaxial Loading.

Pelvic organ prolapse, the descent of the pelvic organs from their normal anatomical position, is a common condition among women that is associated with mechanical alterations of the vaginal wall. In order to characterize the complex mechanical behavior of the vagina, we performed planar biaxial tests of vaginal specimens in both the passive (relaxed) and active (contracted) states. Specimens were isolated from virgin, female Long-Evans rats (n = 16) and simultaneously stretched along the longitudinal direction (LD) and circumferential direction (CD) of the vagina. Tissue contraction was induced by electric field stimulation (EFS) at incrementally increasing values of stretch and, subsequently, by KCl. On average, the vagina was stiffer in the CD than in the LD (p < 0.001). The mean maximum EFS-induced active stress was significantly higher in the CD than in the LD (p < 0.01). On the contrary, the mean KCl-induced active stress was lower in the CD than in the LD (p < 0.01). When comparing the mean maximum EFS-induced active stress to the mean KCl-induced active stress, no differences were found in the CD (p = 0.366) but, in the LD, the mean active stress was much higher in response to the KCl stimulation (p < 0.001). Collectively, these results suggest that the anisotropic behavior of the vaginal tissue is determined not only by collagen and smooth muscle fiber organization but also by the innervation.

Severe Burn-Induced Inflammation and Remodeling of Achilles Tendon in a Rat Model.

ABSTRACTSevere burn causes systemic inflammation and hypercatabolism, resulting in damage to multiple organs distant to the burn site, including the musculoskeletal system. Bone mass and muscle loss have been reported. However, tendon that connects bone and muscle has not been studied in comparable detail. Here we aimed to characterize the molecular and functional changes in Achilles tendon triggered by severe burn. Forty male Sprague–Dawley rats received 40% total body surface area scald burn. Achilles tendons were collected up to 14 days postburn. Sham-treated animals served as a control group. We analyzed tendons for changes in expression of IL-6, IL-1β, TNF, MMP9, MMP13, TGFβ1, Collagens I and III, and for morphological and biomechanical changes. Gene expression of IL-6 and IL-1β as well as MMP9 and MMP13 increased in rat tendon 3 days after burn. Col3a1 increased at day 3 and col1a1 at day 7. At day 14, TGFβ1 increased, whereas the protein ratio for collagens I/III decreased, indicating tendon remodeling. Histological analysis with H&E and Picrosirius red staining further revealed a decrease in organized collagen fibers 14 days after burn. Biomechanical analysis showed a decrease in stiffness and ultimate force of tendons in burn rats.We conclude that tendinopathy was observed in Achilles tendon 14 days after severe burn, via the induction of inflammation and remodeling. The present study provides a model of tendinopathy that may be used for the development of therapeutic approaches after burn.

Biomechanical Characterization of Intracranial Aneurysm Wall: A Multiscale Study

Aneurysm wall biomechanics are not yet an integral part of aneurysm rupture risk evaluation. We aimed to develop a new technique describing the biomechanical properties of aneurysm wall and correlating them to rupture status. Aneurysm wall samples collected during surgery were submitted before and after freezing to tensile tests or as fresh samples to indentation tests. The lateral stiffness or the Young's modulus of the different samples was determined as a function of the mechanical test used. The impact of freezing on biomechanical properties was evaluated. The correlation of clinical and radiologic data with the biomechanical profile of the aneurysm samples was investigated. Two-photon microscopy was used to study collagen fiber organization. Sixteen aneurysm samples (11 unruptured and 5 ruptured) were included. Freezing decreased tissue stiffness. No significant difference was found between ruptured and unruptured aneurysm wall samples regarding demographic characteristics, ethnicity, smoking status, arterial hypertension, site, size and shape of the aneurysm, PHASES score, mechanical profile, or overall Young's modulus. Indentation tests found that the rupture occurred in a restricted area of increased elastic capacity and unruptured areas had increased stiffness. Two-photon microscopy found disruption of the collagen fiber network in rupture zones. The indentation test of fresh aneurysm wall samples described the heterogeneity of biomechanical properties of the tissue and found increased elastic capacity in the rupture zone and increased stiffness in the remainder of the aneurysm. This study could be a basis for further research aimed at building a biomechanical-based model of aneurysm rupture risk.

Mitochondrial dynamics (fission and fusion) and collagen production in a rat model of diabetic wound healing treated by photobiomodulation: comparison of 904 nm laser and 850 nm light-emitting diode (LED)

ObjectiveMitochondrial dysfunction has been associated with the development of diabetes mellitus which is characterized by disorders of collagen production and impaired wound healing. This study analyzed the effects of photobiomodulation (PBM) mediated by laser and light-emitting diode (LED) on the production and organization of collagen fibers in an excisional wound in an animal model of diabetes, and the correlation with inflammation and mitochondrial dynamics. MethodsTwenty Wistar rats were randomized into 4 groups of 5 animals. Groups: (SHAM) a control non-diabetic wounded group with no treatment; (DC) a diabetic wounded group with no treatment; (DLASER) a diabetic wounded group irradiated by 904 nm pulsed laser (40 mW, 9500 Hz, 1 min, 2.4 J); (DLED) a diabetic wounded group irradiated by continuous wave LED 850 nm (48 mW, 22 s, 1.0 J). Diabetes was induced by injection with streptozotocin (70 mg/kg). PBM was carried out daily for 5 days followed by sacrifice and tissue removal. ResultsCollagen fibers in diabetic wounded skin were increased by DLASER but not by DLED. Both groups showed increased blood vessels by atomic force microscopy. Vascular endothelial growth factor (VEGF) was higher and cyclooxygenase (COX2) was lower in the DLED group. Mitochondrial fusion was higher and mitochondrial fusion was lower in DLED compared to DLASER. ConclusionDifferences observed between DLASER and DLED may be due to the pulsed laser and CW LED, and to the higher dose of laser. Regulation of mitochondrial homeostasis may be an important mechanism for PBM effects in diabetes.

Kartogenin Enhances Collagen Organization and Mechanical Strength of the Repaired Enthesis in a Murine Model of Rotator Cuff Repair

To investigate the use of kartogenin (KGN) in augmenting healing of the repaired enthesis after rotator cuff repair in a murine model. Seventy-two C57BL/6 wild-type mice underwent unilateral detachment and transosseous repair of the supraspinatus tendon augmented with either fibrin sealant (control group; n= 36) or fibrin sealant containing 100μmol/L of KGN (experimental group; n= 36) applied at the repair site. Postoperatively, mice were allowed free cage activity without immobilization. Mice were humanely killed at 2 and 4weeks postoperatively. Repair site integrity was evaluated histologically through fibrocartilage formation and collagen fiber organization and biomechanically through load-to-failure testing of the supraspinatus tendon-bone construct. At 2weeks, no differences were noted in percent area of fibrocartilage, collagen organization, or ultimate strength between groups. At 4weeks, superior collagen fiber organization (based on collagen birefringence [17.3 ± 2.0 vs 7.0 ± 6.5 integrated density/μm2; P<.01]) and higher ultimate failure loads (3.5 ± 0.6N vs 2.3 ± 1.1N; P= .04) were seen in the KGN group. The percent area of fibrocartilage (13.2 ± 8.4% vs 4.4 ± 5.4%; P= .04) was higher in the control group compared with the KGN group. Rotator cuff repair augmentation with KGN improved the collagen fiber organization and biomechanical strength of the tendon-bone interface at 4weeks in a murine model. These findings have implications for improving the structural integrity of the repaired enthesis and potentially reducing the retear rate after rotator cuff repair, which can ultimately lead to improvements in clinical outcomes.

Experimental model of temporomandibular joint arthritis: Evaluation of contralateral joint and masticatory muscles

ObjectiveTo investigate morphological and biochemical changes in an experimental model of temporomandibular joint (TMJ) arthritis, as well as examine contralateral joint involvement and morphometric aspects of masticatory muscles in affected animals. DesignForty Wistar rats were allocated into three groups, as follows: a healthy control group (HG); and a group with arthritis induced in the left TMJ (IG); and a contralateral group with noninflamed right TMJ (CG). Arthritis was induced by intra-articular injection of 50 μL of Complete Freund's Adjuvant (CFA). Morphological analysis was performed using histological sections of TMJs stained with hematoxylin-eosin, picrosirius and toluidine blue. In addition, histomorphometric analyses of the articular cartilage, articular disc and masticatory muscles, were performed. Biochemical analysis of TMJ tissues included dosage of sulfated glycosaminoglycans (GAGs), zymography of the Matrix Metalloproteinases (MMPs) 2 and 9 and ELISA for the cytokines IL-6, TNF-α and IL-1β. The data were analyzed by paired and unpaired t-tests, with a p < 0.05. ResultsIG animals presented morphological and biochemical changes in their TMJ, including synovial membrane hyperplasia; chronic inflammatory infiltrate; collagen fiber disorganization in the disc; reduced proteoglycans in the cartilage; greater disc thickness; lower cartilage thickness; lower feed intake; higher activity of MMP-9 and MMP-2, and higher concentration of IL-6, TNF-α, and IL-1β, as compared to HG. CG animals showed posterior disc thickening; reduced proteoglycans in the cartilage; lower collagen fiber organization of the disc; active MMP-9, and higher concentration of IL-6 and TNF-α compared to HG animals. Articular inflammation reduced the area and diameter of fibers of the temporal (right), medial pterygoid (left), and lateral pterygoid (right and left) muscles as compared to HG animals. ConclusionTMJ arthritis reduced the area and diameter of masticatory muscle fibers and caused morphological and molecular changes in the contralateral joint.