Mitochondrial respiration during normothermic liver machine perfusion predicts clinical outcome.

BACKGROUND: Reliable biomarkers for organ quality assessment during normothermic machine perfusion (NMP) are desired. ATP (adenosine triphosphate) production by oxidative phosphorylation plays a crucial role in the bioenergetic homeostasis of the liver. Thus, detailed analysis of the aerobic mitochondrial performance may serve as predictive tool towards the outcome after liver transplantation. METHODS: In a prospective clinical trial, 50 livers were subjected to NMP (OrganOx Metra) for up to 24.ßh. Biopsy and perfusate samples were collected at the end of cold storage, at 1.ßh, 6.ßh, end of NMP, and 1.ßh after reperfusion. Mitochondrial function and integrity were characterized by high-resolution respirometry (HRR), AMP, ADP, ATP and glutamate dehydrogenase analysis and correlated with the clinical outcome (L-GrAFT score). Real-time confocal microscopy was performed to assess tissue viability. Structural damage was investigated by histology, immunohistochemistry and transmission electron microscopy. FINDINGS: A considerable variability in tissue viability and mitochondrial respiration between individual livers at the end of cold storage was observed. During NMP, mitochondrial respiration with succinate and tissue viability remained stable. In the multivariate analysis of the 35 transplanted livers (15 were discarded), area under the curve (AUC) of LEAK respiration, cytochrome c control efficiency (mitochondrial outer membrane damage), and efficacy of the mitochondrial ATP production during the first 6.ßh of NMP correlated with L-GrAFT. INTERPRETATIONS: Bioenergetic competence during NMP plays a pivotal role in addition to tissue injury markers. The AUC for markers of outer mitochondrial membrane damage, ATP synthesis efficiency and dissipative respiration (LEAK) predict the clinical outcome upon liver transplantation. FUNDING: This study was funded by a Grant from the In Memoriam Dr. Gabriel Salzner Stiftung awarded to SS and the Tiroler Wissenschaftsfond granted to TH.

GCN5 contributes to intracellular lipid accumulation in human primary cardiac stromal cells from patients affected by Arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disease associated with sudden cardiac death and cardiac fibro-fatty replacement. Over the last years, several works have demonstrated that different epigenetic enzymes can affect not only gene expression changes in cardiac diseases but also cellular metabolism. Specifically, the histone acetyltransferase GCN5 is known to facilitate adipogenesis and modulate cardiac metabolism in heart failure. Our group previously demonstrated that human primary cardiac stromal cells (CStCs) contribute to adipogenesis in the ACM pathology. Thus, this study aims to evaluate the role of GCN5 in ACM intracellular lipid accumulation. To do so, CStCs were obtained from right ventricle biopsies of ACM patients and from samples of healthy cadaveric donors (CTR). GCN5 expression was increased both in ex vivo and in vitro ACM samples compared to CTR. When GCN5 expression was silenced or pharmacologically inhibited by the administration of MB-3, we observed a reduction in lipid accumulation and a mitigation of reactive oxygen species (ROS) production in ACM CStCs. In agreement, transcriptome analysis revealed that the presence of MB-3 modified the expression of pathways related to cellular redox balance. Altogether, our findings suggest that GCN5 inhibition reduces fat accumulation in ACM CStCs, partially by modulating intracellular redox balance pathways.

Bone tissue and histological and molecular events during development of the long bones.

The bones are of mesenchymal or ectomesenchymal origin, form the skeleton of most vertebrates, and are essential for locomotion and organ protection. As a living tissue they are highly vascularized and remodelled throughout life to maintain intact. Bones consist of osteocytes entrapped in a mineralized extracellular matrix, and via their elaborated network of cytoplasmic processes they do not only communicate with each other but also with the cells on the bone surface (bone lining cells). Bone tissue develops through a series of fine-tuned processes, and there are two modes of bone formation, referred to either as intramembranous or endochondral ossification. In intramembranous ossification, bones develop directly from condensations of mesenchymal cells, and the flat bones of the skull, the clavicles and the perichondral bone cuff develop via this process. The bones of the axial (ribs and vertebrae) and the appendicular skeleton (e.g. upper and lower limbs) form through endochondral ossification where mesenchyme turns into a cartilaginous intermediate with the shape of the future skeletal element that is gradually replaced by bone. Endochondral ossification occurs in all vertebrate taxa and its onset involves differentiation of the chondrocytes, mineralization of the extracellular cartilage matrix and vascularization of the intermediate, followed by disintegration and resorption of the cartilage, bone formation, and finally - after complete ossification of the cartilage model - the establishment of an avascular articular cartilage. The epiphyseal growth plate regulates the longitudinal growth of the bones, achieved by a balanced proliferation and elimination of chondrocytes, and the question whether the late hypertrophic chondrocytes die or transform into osteogenic cells is still being hotly debated. The complex processes leading to endochondral ossification have been studied for over a century, and this review aims to give an overview of the histological and molecular events, arising from the long bones' (e.g. femur, tibia) development. The fate of the hypertrophic chondrocytes will be discussed in the light of new findings obtained from cell tracking studies.

miR-19a-3p containing exosomes improve function of ischaemic myocardium upon shock wave therapy.

AIMS: As many current approaches for heart regeneration exert unfavourable side effects, the induction of endogenous repair mechanisms in ischaemic heart disease is of particular interest. Recently, exosomes carrying angiogenic miRNAs have been described to improve heart function. However, it remains challenging to stimulate specific release of reparative exosomes in ischaemic myocardium. In the present study, we sought to test the hypothesis that the physical stimulus of shock wave therapy (SWT) causes the release of exosomes. We aimed to substantiate the pro-angiogenic impact of the released factors, to identify the nature of their cargo, and to test their efficacy in vivo supporting regeneration and recovery after myocardial ischaemia. METHODS AND RESULTS: Mechanical stimulation of ischaemic muscle via SWT caused extracellular vesicle (EV) release from endothelial cells both in vitro and in vivo. Characterization of EVs via electron microscopy, nanoparticle tracking analysis and flow cytometry revealed specific exosome morphology and size with the presence of exosome markers CD9, CD81, and CD63. Exosomes exhibited angiogenic properties activating protein kinase b (Akt) and extracellular-signal regulated kinase (ERK) resulting in enhanced endothelial tube formation and proliferation. A miRNA array and transcriptome analysis via next-generation sequencing were performed to specify exosome content. miR-19a-3p was identified as responsible cargo, antimir-19a-3p antagonized angiogenic exosome effects. Exosomes and target miRNA were injected intramyocardially in mice after left anterior descending artery ligation. Exosomes resulted in improved vascularization, decreased myocardial fibrosis, and increased left ventricular ejection fraction as shown by transthoracic echocardiography. CONCLUSION: The mechanical stimulus of SWT causes release of angiogenic exosomes. miR-19a-3p is the vesicular cargo responsible for the observed effects. Released exosomes induce angiogenesis, decrease myocardial fibrosis, and improve left ventricular function after myocardial ischaemia. Exosome release via SWT could develop an innovative approach for the regeneration of ischaemic myocardium.

Subcutaneous administration of a neutralizing IL-1ß antibody prolongs limb allograft survival.

Cytokine-expression profiles revealed IL-1ß highly upregulated in rejecting skin of limb allografts. We investigate the effect of intragraft treatment with a neutralizing IL-1ß antibody in limb transplantation. Following allogenic hind-limb transplantation, Lewis rats were either left untreated or treated with anti-lymphocyte serum + tacrolimus (baseline); baseline immunosuppression + anti-IL-1ß (1 mg/kg once/week, 6-8 subcutaneous injections) into the transplanted or contralateral limb. Endpoint was rejection grade III or day 100. Graft rejection was assessed by histology, immunohistochemistry, flow cytometry phenotyping of immune cells, and monitoring cytokine expression. Anti-IL-1ß injections into the allograft or contralateral limb resulted in a significant delay of rejection onset (controls: 58.60 ± 0.60; group 3: 75.80 ± 10.87, P = .044; group 4: 73.00 ± 6.49, P = .008) and prolongation of graft survival (controls: 64.60 ± 0.87; group 3: 86.60 ± 5.33, P = .002; group 4: 93.20 ± 3.82, P = .002), compared to controls. Although the phenotype of the graft infiltrating immune cells did not differ between groups, significantly decreased skin protein levels of IL-1ß, IL-4, IL-13, IP-10, MCP-1, and MCP-3 in long-term-survivors indicate an overall decrease of chemoattraction and infiltration of immune cells as the immunosuppressive mechanism of anti-IL-1ß. Inhibition of IL-1ß with short-term systemic immunosuppression prolongs limb allograft survival and represents a promising target for immunosuppression in extremity transplantation.

Critical Ischemia Times and the Effect of Novel Preservation Solutions HTK-N and TiProtec on Tissues of a Vascularized Tissue Isograft.

BACKGROUND: We herein investigate critical ischemia times and the effect of novel preservation solutions such as new histidine-tryptophan-ketoglutarate (HTK-N) and TiProtec on the individual tissues of a rat limb isograft. METHODS: Orthotopic hind-limb transplantations were performed in male Lewis rats after 2 hours, 6 hours, or 10 hours of cold ischemia (CI). Limbs were flushed and stored in HTK-N, TiProtec, HTK, or saline solution. Muscle, nerve, vessel, skin, and bone samples were procured on day 10 for histology, immunohistochemistry, confocal and electron microscopy, and quantitative real-time polymerase chain reaction analysis. RESULTS: Histomorphology of the muscle showed a mainly perivascular inflammatory infiltrate, fibrotic degeneration, and neovascularization after 6 hours and 10 hours of CI. However, centrally aligned nuclei observed in muscle fibers suggest for muscle regeneration in these samples. In addition to Wallerian degeneration, nerve injury was significantly aggravated (P = 0.032) after prolonged CI. Proinflammatory and regulatory cytokines were most significantly upregulated after 2-hour CI. Our data suggest no superiority of novel perfusates HTK-N and TiProtec in terms of tissue preservation, compared with HTK and saline. CONCLUSIONS: Limiting CI time for less than 6 hours is the most significant factor to reduce tissue damage in vascularized tissue transplantation. Signs of muscle regeneration give rise that ischemic muscle damage in limb transplantation might be reversible to a certain extent.

L1CAM in the Early Enteric and Urogenital System.

L1 cell adhesion molecule (L1CAM) is a transmembrane molecule belonging to the L1 protein family. It has shown to be a key player in axonal guidance in the course of neuronal development. Furthermore, L1CAM is also crucial for the establishment of the enteric and urogenital organs and is aberrantly expressed in cancer originating in these organs. Carcinogenesis and embryogenesis follow a lot of similar molecular pathways, but unfortunately, comprehensive data on L1CAM expression and localization in human developing organs are lacking so far. In the present study we, therefore, examined the spatiotemporal distribution of L1CAM in the early human fetal period (weeks 8-12 of gestation) by means of immunohistochemistry and in situ hybridization (ISH). In the epithelia of the gastrointestinal organs, L1CAM localization cannot be observed in the examined stages most likely due to their advanced polarization and differentiation. Despite these results, our ISH data indicate weak L1CAM expression, but only in few epithelial cells. The genital tracts, however, are distinctly L1CAM positive throughout the entire fetal period. We, therefore, conclude that in embryogenesis L1CAM is crucial for further differentiation of epithelia.

Neurosensory Differentiation and Innervation Patterning in the Human Fetal Vestibular End Organs between the Gestational Weeks 8-12.

Balance orientation depends on the precise operation of the vestibular end organs and the vestibular ganglion neurons. Previous research on the assemblage of the neuronal network in the developing fetal vestibular organ has been limited to data from animal models. Insights into the molecular expression profiles and signaling moieties involved in embryological development of the human fetal inner ear have been limited. We present an investigation of the cells of the vestibular end organs with specific focus on the hair cell differentiation and innervation pattern using an uninterrupted series of unique specimens from gestational weeks 8-12. Nerve fibers positive for peripherin innervate the entire fetal crista and utricle. While in rodents only the peripheral regions of the cristae and the extra-striolar region of the statolithic organs are stained. At week 9, transcription factors PAX2 and PAX8 were observed in the hair cells whereas PAX6 was observed for the first time among the supporting cells of the cristae and the satellite glial cells of the vestibular ganglia. Glutamine synthetase, a regulator of the neurotransmitter glutamate, is strongly expressed among satellite glia cells, transitional zones of the utricle and supporting cells in the sensory epithelium. At gestational week 11, electron microscopic examination reveals bouton contacts at hair cells and first signs of the formation of a protocalyx at type I hair cells. Our study provides first-hand insight into the fetal development of the vestibular end organs as well as their pattern of innervation by means of immunohistochemical and EM techniques, with the aim of contributing toward our understanding of balance development.

Characterization of DLK1(PREF1)+/CD34+ cells in vascular stroma of human white adipose tissue.

Sorting of native (unpermeabilized) SVF-cells from human subcutaneous (s)WAT for cell surface staining (cs) of DLK1 and CD34 identified three main populations: ~10% stained cs-DLK1+/cs-CD34-, ~20% cs-DLK1+/cs-CD34+dim and ~45% cs-DLK1-/cs-CD34+. FACS analysis after permeabilization showed that all these cells stained positive for intracellular DLK1, while CD34 was undetectable in cs-DLK1+/cs-CD34- cells. Permeabilized cs-DLK1-/cs-CD34+ cells were positive for the pericyte marker α-SMA and the mesenchymal markers CD90 and CD105, albeit CD105 staining was dim (cs-DLK1-/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45-/CD31-). Only these cells showed proliferative and adipogenic capacity. Cs-DLK1+/cs-CD34- and cs-DLK1+/cs-CD34+dim cells were also α-SMA+ but expressed CD31, had a mixed hematopoietic and mesenchymal phenotype, and could neither proliferate nor differentiate into adipocytes. Histological analysis of sWAT detected DLK1+/CD34+ and DLK1+/CD90+ cells mainly in the outer ring of vessel-associated stroma and at capillaries. DLK1+/α-SMA+ cells were localized in the CD34- perivascular ring and in adventitial vascular stroma. All these DLK1+ cells possess a spindle-shaped morphology with extremely long processes. DLK1+/CD34+ cells were also detected in vessel endothelium. Additionally, we show that sWAT contains significantly more DLK1+ cells than visceral (v)WAT. We conclude that sWAT has more DKL1+ cells than vWAT and contains different DLK1/CD34 populations, and only cs-DLK1-/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45-/CD31- cells in the adventitial vascular stroma exhibit proliferative and adipogenic capacity.

Alteration of inflammatory response by shock wave therapy leads to reduced calcification of decellularized aortic xenografts in mice†.

OBJECTIVES: Tissue-engineered xenografts represent a promising treatment option in heart valve disease. However, inflammatory response leading to graft failure and incomplete in vitro repopulation with recipient cells remain challenging. Shock waves (SWs) were shown to modulate inflammation and to enhance re-epithelialization. We therefore aimed to investigate whether SWs could serve as a feasible adjunct to tissue engineering. METHODS: Porcine aortic pieces were decellularized using sodium deoxycholate and sodium dodecylsulphate and implanted subcutaneously into C57BL/6 mice (n = 6 per group). The treatment (shock wave therapy, SWT) group received SWs (0.1 mJ/mm(2), 500 impulses, 5 Hz) for modulation of inflammatory response directly after implantation; control animals remained untreated (CTR). Grafts were harvested 72 h and 3 weeks after implantation and analysed for inflammatory cytokines, macrophage infiltration and polarization, osteoclastic activity and calcification. Transmission electron microscopy (TEM) was performed. Endothelial cells (ECs) were treated with SWs and analysed for macrophage regulatory cytokines. In an ex vivo experimental set-up, decellularized porcine aortic valve conduits were reseeded with ECs with and without SWT (0.1 mJ/mm(2), 300 impulses, 3 Hz), fibroblasts as well as peripheral blood mononuclear cells (all human) and tested in a pulsatile flow perfusion system for cell coverage. RESULTS: Treated ECs showed an increase of macrophage migration inhibitory factor and macrophage inflammatory protein 1ß, whereas CD40 ligand and complement component C5/C5a were decreased. Subcutaneously implanted grafts showed increased mRNA levels of tumour necrosis factor α and interleukin 6 in the treatment group. Enhanced repopulation with recipient cells could be observed after SWT. Augmented macrophage infiltration and increased polarization towards M2 macrophages was observed in treated animals. Enhanced recruitment of osteoclastic cells in proximity to calcified tissue was found after SWT. Consequently, SWT resulted in decreased areas of calcification in treated animals. The reseeding experiment revealed that fibroblasts showed the best coverage compared with other cell types. Moreover, SW-treated ECs exhibited enhanced repopulation compared with untreated controls. CONCLUSIONS: SWs reduce the calcification of subcutaneously implanted decellularized xenografts via the modulation of the acute macrophage-mediated inflammatory response and improves the in vitro repopulation of decellularized grafts. It may therefore serve as a feasible adjunct to heart valve tissue engineering.

Development of the innervation of the human inner ear.

Studies on the formation of neuronal structures of the human cochlea are rare, presumptively, due to the difficult accessibility of specimens, so that most investigations are performed on mouse models. By means of immunohistochemical and transmission electron microscopic techniques, we investigated an uninterrupted series of unique specimens from gestational week 8 to week 12. We were able to demonstrate the presence of nerve fibers in the prosensory domain at gestational week 8, followed by afferent synaptogenesis at week 11. We identified PAX2 as an early marker for hair cell differentiation. Glutamine synthetase-positive peripheral glial cells occurred at the beginning of week 8. Transcription factor MAF B was used to demonstrate maturation of the spiral ganglion neurons. The early expression of tyrosine hydroxylase could be assessed. This study provides insights in the early assembly of the neural circuit and organization in humans.

Histomorphometric evaluation of ischemia-reperfusion injury and the effect of preservation solutions histidine-tryptophan-ketoglutarate and University of Wisconsin in limb transplantation.

BACKGROUND: The effect of cold ischemia (CI) in vascularized composite allotransplantation is unknown. We herein assess tissue-specific damage, acceptable CI time, and the effect of preservation solutions in a syngenic rat hindlimb transplant model. METHODS: Lewis rat limbs were flushed and stored for 2, 10, or 30 hr CI in saline, histidine-tryptophan-ketoglutarate or University of Wisconsin preservation solution before transplantation. Morphologic alterations, inflammation, and damage of the individual tissues were analyzed on day 10 using histomorphology, confocal, light, and transmission-electron microscopy. RESULTS: Two-hour CI led to mild inflammation of tissues on day 10, whereas 10-hr and 30-hr CI resulted in massive inflammation and tissue damage. Although muscle was mainly affected after prolonged CI (≥10 hr), nerve was affected in all CI groups. A perineural cell infiltrate, hypercellular appearance, pronounced vacuolization, and mucoid degeneration, appearing as Wallerian degeneration, were observed. Staining with propidium iodide and Syto 16 revealed a decrease in viable muscle cell nuclei in the anterior tibial muscle on day 10 in all groups, which was most pronounced in 10-hr and 30-hr CI animals. Transmission-electron microscopy indicated that a large number of mitochondria were degenerated in the 10-hr and 30-hr CI groups. Histidine-tryptophan-ketoglutarate preservation solution slightly decreased inflammation and tissue damage compared to University of Wisconsin-treated and saline-treated animals, especially in skin and muscle when CI times did not exceed 10 hr. CONCLUSION: Severe inflammation and tissue damage are observed after prolonged CI in muscle and nerve. Ischemia times in vascularized composite allotransplantation should be kept as short as possible and certainly below 10 hr.

Novel immunohistochemical data indicate that the female foetal urethra is more than an epithelial tube.

The female urethra has often been neglected in previous studies on the development of the human urogenital system. Our aim has been to reach a consensus on the organogenesis of the female urethra and the vagina with respect to interactions between the epithelia with different evolutionary origins. Therefore we tried to clarify open questions on the spatiotemporal distribution of molecular markers raised against mesenchymal and epithelial structures within the developing human female urethra. Furthermore, we draw comparisons regarding gender-specific aspects in urethral development. To this effect, we used molecular markers such as different cytokeratins (CKs), p63, Ki67, uroplakin III, E-cadherin, vimentin, smooth muscle actin (SMA), cleaved caspase 3 and paired box gene 2 (PAX 2) to phenotype developmental changes. Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay was additionally performed to reveal apoptosis. We examined different gestational stages starting from week (W) 8 until W 15. Immunohistochemistry showed a distinct staining pattern for p63 and CK17, both markers for stem cells, ensuing from the urogenital sinus (UGS) proceeding into the Muellerian duct (MD). This was observed throughout development and might be a stimulus for the formation of the vaginal anlagen that derive from the MD. In the attachment area of the MD we detected a conglomeration of cells with different embryonic origins. The epithelium of the UGS became transitional at W 9 after fertilization, and the differentiation advanced in a cranial to caudal direction. The paraurethral glands showed a slightly different staining profile than the urethral epithelium, which may be able to explain why carcinomas of these structures display various histological appearances. In addition, we could show that during the development of the female urogenital system the primary incidence is the formation of the urethra. This is followed by the establishment of the vagina, which clearly depends on the proper differentiation of the UGS/urethra.

Sepsis otopathy: experimental sepsis leads to significant hearing impairment due to apoptosis and glutamate excitotoxicity in murine cochlea.

Hearing loss is frequent in intensive care patients and can be due to several causes. However, sepsis has not been examined as a possible cause. The aim of this study is to assess the influence of experimental sepsis on hearing thresholds and to evaluate pathological changes in the cochlea. The cecal ligation puncture technique was used to induce sepsis in 18 mice. Results were compared with those from 13 sham-operated and 13 untreated control mice. The hearing thresholds of the animals were evaluated with auditory evoked brainstem responses prior to the induction of sepsis and again at the peak of the disease. Immediately after the second measurement, the mice were sacrificed and the inner ears harvested and prepared for further evaluation. The cochleae were examined with light microscopy, electron microscopy and immunohistochemistry for Bax, cleaved caspase-3 and Bcl-2. The mice with sepsis showed a significant hearing loss but not the control groups. Induction of apoptosis could be shown in the supporting cells of the organ of Corti. Furthermore, excitotoxicity could be shown at the basal pole of the inner hair cells. In this murine model, sepsis leads to significant hearing impairment. The physiological alteration could be linked to apoptosis in the supporting cells of the organ of Corti and to a disturbance of the synapses of the inner hair cells.

The male urethra: spatiotemporal distribution of molecular markers during early development.

The organogenesis of the male human urethra is still a subject of controversy. Although many studies have been conducted, the mechanisms of urethral development still need further investigation to clarify questions concerning the sequences in its development. Our aim has been to elucidate the spatiotemporal distribution of relevant immunohistochemical indicators during the development of the male urethral epithelium and its adjacent mesenchyme. Therefore, we analyzed male human embryos and foetuses between the 6th and 15th week after fertilization using histological and immunohistochemical methods. Monoclonal antibodies raised against cytokeratins (CKs) 7, 8, 13 and 17 as well as Ki67, E-Cadherin, p63, uroplakin III, smooth muscle actin (SMA) and vimentin were applied. Our results revealed that epithelial differentiation starts prior to the rupture of the cloacal membrane. At weeks (W) 8-9 the epithelium became transitional over the whole length of the elongating urethra. The urothelial staining pattern of uroplakin III receded continually, and, at the end of W 11, it had receded in proximal direction to the bladder neck comparable to the epithelial appearance in adults. The urogenital sinus epithelium provided the Wolffian duct with p63-positive cells, leading to the suggestion that the development of the male inner genitals requires a cellular stimulus by this very epithelium. CK 17-positive cells, which were described as epithelial stem cells, could be found in the extending urethral plate. This facilitates new insight into the pathogenesis and treatment of hypospadias, which is one of the most common malformations in newborn males.

Role of tartrate-resistant acid phosphatase (TRAP) in long bone development.

Tartrate resistant acid phosphatase (TRAP) was shown to be critical for skeleton development, and TRAP deficiency leads to a reduced resorptive activity during endochondral ossification resulting in an osteopetrotic phenotype and shortened long bones in adult mice. A proper longitudinal growth depends on a timely, well-coordinated vascularization and formation of the secondary ossification center (SOC) of the long bones epiphysis. Our results demonstrate that TRAP is not essential for the formation of the epiphyseal vascular network. Therefore, in wild type (Wt) controls as well as TRAP deficient (TRAP(-/-)) mutants vascularised cartilage canals are present from postnatal day (P) five. However, in the epiphysis of the TRAP(-/-) mice cartilage mineralization, formation of the marrow cavity and the SOC occur prematurely compared with the controls. In the mutant mice the entire growth plate is widened due to an expansion of the hypertrophic zone. This is not seen in younger animals but first detected at week (W) three and during further development. Moreover, an enhanced number of thickened trabeculae, indicative of the osteopetrotic phenotype, are observed in the metaphysis beginning with W three. Epiphyseal excavation was proposed as an important function of TRAP, and we examined whether TRAP deficiency affects this process. We therefore evaluated the marrow cavity volume (MCV) and the epiphyseal volume (EV) and computed the MCV to EV ratio (MCV/EV). We investigated developmental stages until W 12. Our results indicate that both epiphyseal excavation and establishment of the SOC are hardly impaired in the knockouts. Furthermore, no differences in the morphology of the epiphyseal bone trabeculae and remodeling of the articular cartilage layers are noted between Wt and TRAP(-/-) mice. We conclude that in long bones, TRAP is critical for the development of the growth plate and the metaphysis but apparently not for the epiphyseal vascularization, excavation, and establishment of the SOC.

Development of articular cartilage and the metaphyseal growth plate: the localization of TRAP cells, VEGF, and endostatin.

During long bone development the original cartilaginous model in mammals is replaced by bone, but at the long bone endings the avascular articular cartilage remains. Before the articular cartilage attains structural maturity it undergoes reorganization, and molecules such as vascular endothelial growth factor (VEGF) and endostatin could be involved in this process. VEGF attracts blood vessels, whereas endostatin blocks their formation. The present study therefore focused on the spatio-temporal localization of these two molecules during the development of the articular cartilage. Furthermore, we investigated the distribution of the chondro/osteoclasts at the chondro-osseous junction of the articular cartilage with the subchondral bone. Mice served as our animal model, and we examined several postnatal stages of the femur starting with week (W) 4. Our results indicated that during the formation of the articular cartilage, VEGF and endostatin had an overlapping localization. The former molecule was, however, down-regulated, whereas the latter was uniformly intensely localized until W12. At the chondro-osseous junction, the number of tartrate-resistant acid phosphatase (TRAP)-positive chondro/osteoclasts declined with increasing age. Until W3 the articular cartilage was not well organized but at W8 it appeared structurally mature. At that time only a few TRAP cells were present, indicative of a low resorptive activity at the chondro-osseous junction. Subsequently, we examined the metaphyseal growth plate that is closed when skeletal maturity is attained. Within its hypertrophic zone, localization of endostatin and VEGF was observed until W6 and W8, respectively. At the chondro-osseous junction of the growth plate, chondro/osteoclasts remained numerous until W12 to allow for its complete resorption. According to former findings, VEGF is critical for a normal skeleton development, whereas endostatin has almost no effect on this process. In conclusion, our findings suggest that both VEGF and endostatin play a role in the structural reorganization of the articular cartilage and endostatin may be involved in the maintenance of its avascularity. In the growth plate, however, endostatin does not appear to counteract VEGF, allowing vascular invasion of hypertrophic cartilage and bone growth.

VEGF and its role in the early development of the long bone epiphysis.

In long bones of murine species, undisturbed development of the epiphysis depends on the generation of vascularized cartilage canals shortly after birth. Despite its importance, it is still under discussion how this event is exactly regulated. It was suggested previously that, following increased hypoxia in the epiphyseal core, angiogenic factors are expressed and hence stimulate the ingrowth of the vascularized canals. In the present study, we tested this model and examined the spatio-temporal distribution of two angiogenic molecules during early development in mice. In addition, we investigated the onset of cartilage hypertrophy and mineralization. Our results provide evidence that the vascular endothelial growth factor is expressed in the epiphyseal resting cartilage prior to the moment of canal formation and is continuously expressed until the establishment of a large secondary ossification centre. Interestingly, we found no expression of secretoneurin before the establishment of the canals although this factor attracts blood vessels under hypoxic conditions. Epiphyseal development further involves maturation of the resting chondrocytes into hypertrophic ones, associated with the mineralization of the cartilage matrix and eventual death of the latter cells. Our results suggest that vascular endothelial growth factor is the critical molecule for the generation of the epiphyseal vascular network in mice long bones. Secretoneurin, however, does not appear to be a player in this event. Hypertrophic chondrocytes undergo cell death by a mechanism interpreted as chondroptosis.

Lasp1 misexpression influences chondrocyte differentiation in the vertebral column.

The mouse mutant wavy tail Tg(Col1a1-lacZ)304ng was created through transgene insertion and exhibits defects of the vertebral column. Homozygous mutant animals have compressed tail vertebrae and wedge-shaped intervertebral discs, resulting in a meandering tail. Delayed closure of lumbar neural arches and lack of processus spinosi have been observed; these defects become most prominent during the transition from cartilage to bone. The spina bifida was resistant to folic acid treatment, while retinoic acid administration caused severe skeletal defects in the mutant, but none in wild type control animals. The transgene integrated at chromosome 11 band D, in an area of high gene density. The insertion site was located between the transcription start sites of the Rpl23 and Lasp1 genes. LASP1 (an actin binding protein involved in cell migration and survival) was found to be produced in resting and hypertrophic chondrocytes in the vertebrae. In mutant vertebrae, temporal and spatial misexpression of Lasp1 was observed, indicating that alterations in Lasp1 transcription are most likely responsible for the observed phenotype. These data reveal a yet unappreciated role of Lasp1 in chondrocyte differentiation during cartilage to bone transition.

Structure, formation and role of cartilage canals in the developing bone.

In the long bones, endochondral bone formation proceeds via the development of a diaphyseal primary ossification centre (POC) and an epiphyseal secondary ossification centre (SOC). The growth plate, the essential structure for longitudinal bone growth, is located between these two sites of ossification. Basically, endochondral bone development depends upon neovascularization, and the early generation of vascularized cartilage canals is an initial event, clearly preceding the formation of the SOC. These canals form a discrete network within the cartilaginous epiphysis giving rise to the formation of the marrow space followed by the establishment of the SOC. These processes require excavation of the provisional cartilaginous matrix which is eventually replaced by permanent bone matrix. In this review, we discuss the formation of the cartilage canals and the importance of their cells in the ossification process. Special attention is paid to the enzymes required in disintegration of the cartilaginous matrix which, in turn, will allow for the invasion of new vessels. Furthermore, we show that the mesenchymal cells of the cartilage canals express bone-relevant proteins and transform into osteocytes. We conclude that the canals are essential for normal epiphyseal bone development, the establishment of the growth plate and ultimately longitudinal growth of the bones.