Der Einfluss des sensiblen Neurotransmitters SP und des sympathischen Nervensystems auf die enchondrale Ossifikation im Verlauf der Frakturheilung und auf die allgemeine Knochenstruktur in vivo und in vitro

Nervenfasern des peripheren sensiblen und sympathischen Nervensystems innervieren den Knochen, das Knochenmark und das Periost. Via Neurotransmitter, die von den peripheren Nervenfasern abgegeben werden, moduliert das Nervensystem die enchondrale Ossifikation während des Knochenlängenwachstums und während der Kallusdifferenzierung nach einem Frakturtrauma im adulten Organismus. Die Effekte dieser neuronalen Mediatoren auf den Vorgang der enchondralen Ossifikation während der Embryogenese und nach Frakturtrauma im adulten Organismus sind noch weitgehend unbekannt. In dieser Arbeit konnte gezeigt werden, dass der sensible Neurotransmitter SP und das sympathische Nervensystem unter physiologischen Knochenumbauprozessen und pathophysiologischen Bedingungen der Kallusreifung nach Fraktur – verwendet als adultes Modell der enchondralen Ossifikation – entscheidende trophische Effekte ausüben. Durch die Expression des NK1R und die endogene Produktion von SP sind Chondrozyten im Frakturkallus in der Lage autokrin und parakrin zu agieren. Zusätzlich waren TH-immunoreaktive Nervenfasern während des gesamten Heilungsverlaufs im Periost detektierbar. Wir konnten zeigen, dass Zellen im Frakturkallus α1D- und α2B-AR exprimieren und dadurch in der Lage sind auf sympathische Stimuli zu reagieren. 5 Tage nach Fraktur führte das Fehlen von SP zu einer Reduktion der Berührungssensibilität, die Ablation des SNS (Syx) verringerte 8 Tage nach Fraktur die Berührungssensibilität. Die Analysen der Gewebezusammensetzung im Frakturkallus zeigten, dass SyX-Mäuse 5 Tage nach Fraktur einen größeren Anteil an mesenchymalem Kallusgewebe und einen geringeren Anteil an knorpeligem Softkallus aufwiesen, was auf eine Verzögerung des Heilungsprozesses hindeutet. 13 Tage nach Fraktur verzögerte das Fehlen von SP und die SyX die terminale Differenzierung der Chondrozyten, die Fläche mit hypertrophen Chondrozyten im knorpeligen Softkallus von SyX- und Tac1-/- Mäusen war signifikant geringer. Die geringere Anzahl an Osteoblasten und Osteoklasten in den Frakturkalli von Tac1-/- Mäusen lässt vermuten, dass das Fehlen von SP die Differenzierung osteogener Zellen verzögert. Das Fehlen von SNF während der Frakturheilung scheint hingegen die Osteoklastogenese zu aktivieren, worauf die erhöhte Anzahl an Osteoklasten in Frakturkalli sympathektomierter Mäuse hindeutete. Das Fehlen von SP und die Ablation des SNS resultierten 21 Tage nach Fraktur in signifikant verminderten strukturellen Knochenparametern der frakturierten und nicht-frakturierten Femora, wobei die fehlenden sympathischen Effekte eine drastischere Wirkung erzielten. Die biomechanischen Tests bestätigten die beeinträchtigten mechanischen Eigenschaften der frakturierten und nicht-frakturierten Femora von SyX- und Tac1-/- Mäusen. Die in vitro Analysen von isolierten Osteoblasten aus Tac1-/- Mäusen zeigten, dass diese eine geringere ALP-Aktivität aufwiesen, isolierte Osteoklasten zeigten eine höhere Apoptoserate. Isolierte Osteoblasten aus SyX-Mäusen zeigten in vitro eine erhöhte ALP-Aktivität, Osteoklasten aus sympathektomierten Mäusen zeigten eine reduzierte Apoptoserate und einer erhöhte Kathepsin K Aktivität. Das Fehlen von SP und die chemische Sympathektomie beeinflussen die Expression selektiver Gene, die am Matrix-Umbau und an der Entzündungsreaktion beteiligt sind. Unserer Daten und die aus vorhandener Literatur zeigen, dass das Fehlen von SP die Anzahl und die Resorptionsaktivität von Osteoklasten über den NK1R-vermittelten Signalweg reduziert und so zu einer verringerten Knochenresorptionsrate führt. Gleichzeitig reduziert das Fehlen von SP deutlich die Proliferation und die Aktivität von Osteoblasten, was – trotz verringerter Knochenresorption – zu einer Netto-Reduktion der Knochenbildung führt (Abb. A -1). Die chemische Sympathektomie führt zur selektiven Zerstörung katecholaminerger Nervenfasern, was zu einer geringen NA und Adrenalin Konzentration im Gewebe führt. Da NA in geringen Konzentrationen vornehmlich α-AR stimuliert, vermuten wir, dass NA die α-AR auf den Osteoblasten aktiviert und zu vermehrter Produktion des Osteoklastogenese-fördernden Faktors RANKL führt. Die Aktivierung der α-AR auf Osteoklasten-Vorläuferzellen führt zusätzlich zur vermehrten Produktion von Osteoklastogenese-fördernden Genen. Zudem nehmen sensible Nervenfasern den Wachstumsfaktor NGF auf, was die SP Produktion und die Stimulation der sensiblen Nervenfasern erhöht, woraufhin SP vermehrt in die Peripherie abgegeben werden könnte und die Osteoklastogenese durch Bindung an den NK1R auf den Osteoklasten stimuliert. Diese Mechanismen führen zu einer Induktion der Osteoklastendifferenzierung und -aktivierung und einem Netto-Anstieg der Knochenresorption wohingegen die Rate der Knochenbildung unverändert bleibt

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

Substance P modulates bone remodeling properties of murine osteoblasts and osteoclasts

Clinical observations suggest neuronal control of bone remodeling. Sensory nerve fibers innervating bone, bone marrow and periosteum signal via neurotransmitters including substance P (SP). In previous studies we observed impaired biomechanical and structural bone parameters in tachykinin (Tac) 1-deficient mice lacking SP. Here, we aim to specify effects of SP on metabolic parameters of bone marrow macrophage (BMM)/osteoclast cultures and osteoblasts isolated from Tac1-deficient and wildtype (WT) mice. We demonstrated endogenous SP production and secretion in WT bone cells. Absence of SP reduced bone resorption rate, as we found reduced numbers of precursor cells (BMM) and multinucleated osteoclasts and measured reduced cathepsin K activity in Tac1-/- BMM/osteoclast cultures. However, this might partly be compensated by reduced apoptosis rate and increased fusion potential of Tac1-/- precursor cells to enlarged "super" osteoclasts. Contrarily, increased ALP enzyme activity and apoptosis rate during early osteoblast differentiation accelerated osteogenesis and cell death in the absence of SP together with reduced ALP activity of Tac1-/- osteoblasts during late osteogenic differentiation resulting in reduced bone formation at later stages. Therefore, we suggest that absence of SP presumably results in a slight reduction of bone resorption rate but concomitantly in a critical reduction of bone formation and mineralization rate

Physiological and pathophysiological aspects of primary cilia—a literature review with view on functional and structural relationships in cartilage

Cilia are cellular organelles that project from the cell. They occur in nearly all non-hematopoietic tissues and have different functions in different tissues. In mesenchymal tissues primary cilia play a crucial role in the adequate morphogenesis during embryological development. In mature articular cartilage, primary cilia fulfil chemo- and mechanosensitive functions to adapt the cellular mechanisms on extracellular changes and thus, maintain tissue homeostasis and morphometry. Ciliary abnormalities in osteoarthritic cartilage could represent pathophysiological relationships between ciliary dysfunction and tissue deformation. Nevertheless, the molecular and pathophysiological relationships of 'Primary Cilia' (PC) in the context of osteoarthritis is not yet fully understood. The present review focuses on the current knowledge about PC and provide a short but not exhaustive overview of their role in cartilage

Ultrastructural Evidence of Mitochondrial Dysfunction in Osteomyelitis Patients

Osteomyelitis is a difficult-to-treat disease with high chronification rates. First studies suggest increases in mitochondrial fission and mitochondrial dysfunction as possible contributors to the accumulation of intracellular reactive oxygen species and thereby to the cell death of infected bone cells. The aim of the present study is to analyze the ultrastructural impact of bacterial infection on osteocytic and osteoblastic mitochondria. Human infected bone tissue samples were visualized via light microscopy and transmission electron microscopy. Osteoblasts, osteocytes and their mitochondria were analyzed histomorphometrically and compared with the control group of noninfectious human bone tissue samples. The results depicted swollen hydropic mitochondria including depleted cristae and a decrease in matrix density in the infected samples. Furthermore, perinuclear clustering of mitochondria could also be observed regularly. Additionally, increases in relative mitochondrial area and number were found as a correlate for increased mitochondrial fission. In conclusion, mitochondrial morphology is altered during osteomyelitis in a comparable way to mitochondria from hypoxic tissues. This gives new perspectives on the treatment strategies since the manipulation of mitochondrial dynamics may improve bone cell survival as a potential new target for the therapy of osteomyelitis

Excellent histological results in terms of articular cartilage regeneration after spheroid-based autologous chondrocyte implantation (ACI)

Purpose Traumatic lesions of articular cartilage represent a crucial risk factor for osteoarthritis. Even if several strategies exist to treat such damages, the optimal solution has not yet been found. A new strategy represents the scaffold-free spheroid-based autologous chondrocyte transplantation. In this method, spheroids of chondrocytes are synthesized after chondrocyte isolation and expansion, followed by the implantation in a second intervention. Methods Fine Jamshidi-needle biopsies from five patients (one from each patient, Ø 2 mm) treated with a spheroid-based autologous chondrocyte implantation (ACI) after traumatic lesions of the articular cartilage of the knee were analysed histologically and immunohistologically for collagen II, collagen X and aggrecan expression. The indication for a second look arthroscopy was given by arthrofibrosis or meniscus-lesions, respectively. The time between ACI and second-look arthroscopy ranged between 6 and 16 months. Results In all patients, the histological examinations revealed an avascular cartilage tissue with a homogenic extracellular matrix. The subchondral bone neither showed bleeding, necrosis nor hypertrophy. A homogenous alcian blue staining indicated high amounts of mucopolysaccharides and glycosaminoglycans. Collagen II staining was highly positive, whereas collagen X staining was negative in every patient, ruling out hypertrophic chondrocyte differentiation. In addition, intense aggrecan staining indicated a strong expression of this extracellular matrix component. Conclusion The present case series represents the first histological and immunohistological analyses of spheroid-based ACI in humans. Spheroid-based ACI revealed excellent histological results regarding the regeneration of hyaline articular cartilage. These results indicate that spheroid based ACI is a promising strategy for treating traumatic lesions of the articular cartilage of the knee

First detection of primary cilia in injured human anterior cruciate ligament: A pilot study with pathophysiological reflections

The anterior cruciate ligament (ACL) plays a significant role in knee stability, protects the joint under multiple loading conditions and shows complex biomechanics. Beside mechanical stability, the ACL seems to play a crucial role in proprioception, and it is well known, that ACL injuries can cause functional deficits due to decreased proprioception. However, the mechanism of proprioception is not completely understood yet. In this context, primary cilia (PC), which play a significant role in the signaling between the intra- and extracellular space, could be of interest. However, until today, primary cilia are not yet described in human ACL. In total, seven human ACL’s underwent transmission electron microscopical examination. Three cadaveric ACL’s and four freshly injured ACL’s were examined. Single cells of each ACL were examined regarding the presence of axonemes or basal bodies, which represent components of a PC. In total, 276 cells of the cadaveric ACL’s and 180 cells of the injured ACL’s were examined. Basal bodies could be detected in three of the four specimens of the injured ACL’s as well as in one of the three cadaveric ACL’s, resulting in a mean positivity of 2.54% in the cadaveric group and 2.78% in the injured group. In case of PC-presence, only one PC per cell could be detected. No statistically significant difference regarding the frequency could be detected between both groups. In this pilot-study, we present for the first time an ultrastructural study of human ACLs with respect to the occurrence of PC and any structural and morphological features of these complex and dynamic cell organelles. PCs are present in almost all non-hematopoietic tissues of the human body. However, there are different reports on the number, incidence, orientation, and morphology of these cell organelles in the respective tissues. Compared to other tissues and ligaments of other species, we found a significantly lower rate of PC positive cells. This observation might represent a tissue-specific characteristic of ACL tissue. However, our observations need to be explored in more detail in further studies

In Vivo Comparison of Synthetic Macroporous Filamentous and Sponge-like Skin Substitute Matrices Reveals Morphometric Features of the Foreign Body Reaction According to 3D Biomaterial Designs

Synthetic macroporous biomaterials are widely used in the field of skin tissue engineering to mimic membrane functions of the native dermis. Biomaterial designs can be subclassified with respect to their shape in fibrous designs, namely fibers, meshes or fleeces, respectively, and porous designs, such as sponges and foams. However, synthetic matrices often have limitations regarding unfavorable foreign body responses (FBRs). Severe FBRs can result in unfavorable disintegration and rejection of an implant, whereas mild FBRs can lead to an acceptable integration of a biomaterial. In this context, comparative in vivo studies of different three-dimensional (3D) matrix designs are rare. Especially, the differences regarding FBRs between synthetically derived filamentous fleeces and sponge-like constructs are unknown. In the present study, the FBRs on two 3D matrix designs were explored after 25 days of subcutaneous implantation in a porcine model. Cellular reactions were quantified histopathologically to investigate in which way the FBR is influenced by the biomaterial architecture. Our results show that FBR metrics (polymorph-nucleated cells and fibrotic reactions) were significantly affected according to the matrix designs. Our findings contribute to a better understanding of the 3D matrix tissue interactions and can be useful for future developments of synthetically derived skin substitute biomaterial

Macrophages: From Simple Phagocyte to an Integrative Regulatory Cell for Inflammation and Tissue Regeneration—A Review of the Literature

The understanding of macrophages and their pathophysiological role has dramatically changed within the last decades. Macrophages represent a very interesting cell type with regard to biomaterial-based tissue engineering and regeneration. In this context, macrophages play a crucial role in the biocompatibility and degradation of implanted biomaterials. Furthermore, a better understanding of the functionality of macrophages opens perspectives for potential guidance and modulation to turn inflammation into regeneration. Such knowledge may help to improve not only the biocompatibility of scaffold materials but also the integration, maturation, and preservation of scaffold-cell constructs or induce regeneration. Nowadays, macrophages are classified into two subpopulations, the classically activated macrophages (M1 macrophages) with pro-inflammatory properties and the alternatively activated macrophages (M2 macrophages) with anti-inflammatory properties. The present narrative review gives an overview of the different functions of macrophages and summarizes the recent state of knowledge regarding different types of macrophages and their functions, with special emphasis on tissue engineering and tissue regeneration

Structural Analysis of Mitochondrial Dynamics—From Cardiomyocytes to Osteoblasts: A Critical Review

Mitochondria play a crucial role in cell physiology and pathophysiology. In this context, mitochondrial dynamics and, subsequently, mitochondrial ultrastructure have increasingly become hot topics in modern research, with a focus on mitochondrial fission and fusion. Thus, the dynamics of mitochondria in several diseases have been intensively investigated, especially with a view to developing new promising treatment options. However, the majority of recent studies are performed in highly energy-dependent tissues, such as cardiac, hepatic, and neuronal tissues. In contrast, publications on mitochondrial dynamics from the orthopedic or trauma fields are quite rare, even if there are common cellular mechanisms in cardiovascular and bone tissue, especially regarding bone infection. The present report summarizes the spectrum of mitochondrial alterations in the cardiovascular system and compares it to the state of knowledge in the musculoskeletal system. The present paper summarizes recent knowledge regarding mitochondrial dynamics and gives a short, but not exhaustive, overview of its regulation via fission and fusion. Furthermore, the article highlights hypoxia and its accompanying increased mitochondrial fission as a possible link between cardiac ischemia and inflammatory diseases of the bone, such as osteomyelitis. This opens new innovative perspectives not only for the understanding of cellular pathomechanisms in osteomyelitis but also for potential new treatment options