Impact of the Sensory and Sympathetic Nervous System on Fracture Healing in Ovariectomized Mice

The peripheral nervous system modulates bone repair under physiological and pathophysiological conditions. Previously, we reported an essential role for sensory neuropeptide substance P (SP) and sympathetic nerve fibers (SNF) for proper fracture healing and bone structure in a murine tibial fracture model. A similar distortion of bone microarchitecture has been described for mice lacking the sensory neuropeptide alpha-calcitonin gene-related peptide (alpha-CGRP). Here, we hypothesize that loss of SP, alpha-CGRP, and SNF modulates inflammatory and pain-related processes and also affects bone regeneration during fracture healing under postmenopausal conditions. Intramedullary fixed femoral fractures were set to 28 days after bilateral ovariectomy (OVX) in female wild type (WT), SP-, alpha-CGRP-deficient, and sympathectomized (SYX) mice. Locomotion, paw withdrawal threshold, fracture callus maturation and numbers of TRAP-, CD4-, CD8-, F4/80-, iNos-, and Arg1-positive cells within the callus were analyzed. Nightly locomotion was reduced in unfractured SP-deficient and SYX mice after fracture. Resistance to pressure was increased for the fractured leg in SP-deficient mice during the later stages of fracture healing, but was decreased in alpha-CGRP-deficient mice. Hypertrophic cartilage area was increased nine days after fracture in SP-deficient mice. Bony callus maturation was delayed in SYX mice during the later healing stages. In addition, the number of CD 4-positive cells was reduced after five days and the number of CD 8-positive cells was additionally reduced after 21 days in SYX mice. The number of Arg1-positive M2 macrophages was higher in alpha-CGRP-deficient mice five days after fracture. The alkaline phosphatase level was increased in SYX mice 16 days after fracture. Absence of alpha-CGRP appears to promote M2 macrophage polarization and reduces the pain threshold, but has no effect on callus tissue maturation. Absence of SP reduces locomotion, increases the pain-threshold, and accelerates hypertrophic callus tissue remodeling. Destruction of SNF reduces locomotion after fracture and influences bony callus tissue remodeling during the later stages of fracture repair, whereas pain-related processes are not affected

Spectrum of Tendon Pathologies: Triggers, Trails and End-State

The biggest compartment of the musculoskeletal system is the tendons and ligaments. In particular, tendons are dense tissues connecting muscle to bone that are critical for the integrity, function and locomotion of this system. Due to the increasing age of our society and the overall rise in engagement in extreme and overuse sports, there is a growing prevalence of tendinopathies. Despite the recent advances in tendon research and due to difficult early diagnosis, a multitude of risk factors and vague understanding of the underlying biological mechanisms involved in the progression of tendon injuries, the toolbox of treatment strategies remains limited and non-satisfactory. This review is designed to summarize the current knowledge of triggers, trails and end state of tendinopathies

Cyclooxygenases and prostaglandin E(2 )receptors in growth plate chondrocytes in vitro and in situ – prostaglandin E(2 )dependent proliferation of growth plate chondrocytes

Prostaglandin E(2 )(PGE(2)) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE(2 )pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE(2 )receptors) is essential. We therefore examined the production of PGE(2 )in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE(2 )on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE(2 )receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE(2 )synthesis was determined by mass spectrometry, cell proliferation by DNA [(3)H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE(2 )into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE(2)-dependent proliferation. Exogenously added PGE(2 )stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10(-8 )M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE(2 )was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE(2 )release, which stimulates cell proliferation via the EP1 receptor

Standardised Models for Inducing Experimental Peritoneal Adhesions in Female Rats

Animal models for adhesion induction are heterogeneous and often poorly described. We compare and discuss different models to induce peritoneal adhesions in a randomized, experimental in vivo animal study with 72 female Wistar rats. Six different standardized techniques for peritoneal trauma were used: brushing of peritoneal sidewall and uterine horns (group 1), brushing of parietal peritoneum only (group 2), sharp excision of parietal peritoneum closed with interrupted sutures (group 3), ischemic buttons by grasping the parietal peritoneum and ligating the base with Vicryl suture (group 4), bipolar electrocoagulation of the peritoneum (group 5), and traumatisation by electrocoagulation followed by closure of the resulting peritoneal defect using Vicryl sutures (group 6). Upon second look, there were significant differences in the adhesion incidence between the groups (P < 0.01). Analysis of the fraction of adhesions showed that groups 2 (0%) and 5 (4%) were significantly less than the other groups (P < 0.01). Furthermore, group 6 (69%) was significantly higher than group 1 (48%) (P < 0.05) and group 4 (47%) (P < 0.05). There was no difference between group 3 (60%) and group 6 (P = 0.2). From a clinical viewpoint, comparison of different electrocoagulation modes and pharmaceutical adhesion barriers is possible with standardised models

Aged Tendon Stem/Progenitor Cells Are Less Competent to Form 3D Tendon Organoids Due to Cell Autonomous and Matrix Production Deficits

Tendons are dense connective tissues, which are critical for the integrity and function of our musculoskeletal system. During tendon aging and degeneration, tendon stem/progenitor cells (TSPCs) experience profound phenotypic changes with declined cellular functions that can be linked to the known increase in complications during tendon healing process in elderly patients. Tissue engineering is a promising approach for achieving a complete recovery of injured tendons. However, use of autologous cells from aged individuals would require restoring the cellular fitness prior to implantation. In this study, we applied an established cell sheet model for in vitro tenogenesis and compared the sheet formation of TSPC derived from young/healthy (Y-TSPCs) versus aged/degenerative (A-TSPCs) human Achilles tendon biopsies with the purpose to unravel differences in their potential to form self-assembled three-dimensional (3D) tendon organoids. Using our three-step protocol, 4 donors of Y-TSPCs and 9 donors of A-TSPCs were subjected to cell sheet formation and maturation in a period of 5 weeks. The sheets were then cross evaluated by weight and diameter measurements; quantification of cell density, proliferation, senescence and apoptosis; histomorphometry; gene expression of 48 target genes; and collagen type I protein production. The results revealed very obvious and significant phenotype in A-TSPC sheets characterized by being fragile and thin with poor tissue morphology, and significantly lower cell density and proliferation, but significantly higher levels of the senescence-related gene markers and apoptotic cells. Quantitative gene expression analyses at the mRNA and protein levels, also demonstrated abnormal molecular circuits in the A-TSPC sheets. Taken together, we report for the first time that A-TSPCs exhibit profound deficits in forming 3D tendon tissue organoids, thus making the cell sheet model suitable to investigate the molecular mechanisms involved in tendon aging and degeneration, as well as examining novel pharmacologic strategies for rejuvenation of aged cells

The influence of bone substitute materials on the bone volume after maxillary sinus augmentation: a microcomputerized tomography study

Objectives: This study aims to evaluate the effect of adding bone substitute materials (BSM) to particulated autogenous bone (PAB) on the volume fraction (Vf) of newly formed bone after maxillary sinus augmentation. Materials and methods: Thirty healthy patients undergoing maxillary sinus augmentation were included. PAB (N = 10), mixtures of PAB and beta-tricalciumphosphate (PAB/β-TCP) (N = 10), as well as PAB and β-TCP and hydroxyapatite (PAB/HA/β-TCP) (N = 10) were randomly used for sinus augmentation. A sample of the graft material was maintained from each patient at time of maxillary sinus augmentation, and Vfs of the PAB and/or BSM in the samples were determined by means of microcomputerized tomography (μ-CT). Five months later, samples of the grafted areas were harvested during implantation using a trephine bur. μ-CT analysis of these samples was performed, and the Vf of bone and BSM were compared with the data obtained 5months earlier from the original material. Results: The mean Vf of the bone showed a statistically significant increase (p < 0.05) in all groups after a healing period of 5months without statistically significant difference between the groups. Conclusions: With regard to the increase of bone volume, it is not relevant if PAB is used alone or combined with β-TCP or HA/β-TCP. Clinical relevance: The amount of PAB and associated donor site morbidity may be reduced by adding BSM for maxillary sinus augmentatio