Search

Antimicrobial peptides are multifunctional in innate immunity and wound repair of multicellular organisms. We were the first to discover that histatins, a family of salivary antimicrobial peptides, enhance epithelial cell migration, suggesting a role in oral wound healing. It is unknown whether histatins display innate-immunity activities, similar to other antimicrobial peptides such as LL-37. Therefore, we compared the effect of Histatin-2 and LL-37 on several activities within the context of wound healing and innate immunity. We found that Histatin-2 enhances fibroblast migration, but only weakly induces proliferation. LL-37 enhances both fibroblast migration and proliferation, but only at a narrow concentration optimum (approximately 1 μm). At higher concentrations LL-37 causes cell death, whereas Histatin-2 is not cytotoxic. Both peptides do not alter fibroblast-to-myofibroblast differentiation. Histatin-2 does not alter interleukin-8 (IL-8) expression and lipopolysaccharide (LPS)-elevated cytokine and chemokine expression. In contrast, LL-37 induces IL-8 expression, but dampens the LPS-induced immune response. Neither Histatin-2 nor LL-37 affects human-neutrophil migration. Histatins are, unlike other antimicrobial peptides, not cytotoxic or proinflammatory. It seems that they are important for the initial stage of wound healing in which fast wound coverage is important for healing without infection, inflammation, or fibrosis development. Interestingly, these characteristics are more typical for the mouth than for skin.

Udgivelsesdato: 2010-Feb For engineering bone tissue, mechanosensitive cells are needed for bone (re)modelling. Local bone mass and architecture are affected by mechanical loading, which provokes a cellular response via loading-induced interstitial fluid flow. We studied whether human dental pulp-derived mesenchymal stem cells (PDSCs) portraying mature (PDSC-mature) or immature (PDSC-immature) bone cell characteristics are responsive to pulsating fluid flow (PFF) in vitro. We also assessed bone formation by PDSCs on hydroxyapatite-tricalcium phosphate granules after subcutaneous implantation in mice. Cultured PDSC-mature exhibited higher osteocalcin and alkaline phosphatase gene expression and activity than PDSC-immature. Pulsating fluid flow (PFF) stimulated nitric oxide production within 5 min by PDSC-mature but not by PDSC-immature. In PDSC-mature, PFF induced prostaglandin E(2) production, and cyclooxygenase 2 gene expression was higher than in PDSC-immature. Implantation of PDSC-mature resulted in more osteoid deposition and lamellar bone formation than PDSC-immature. We conclude that PDSCs with a mature osteogenic phenotype are more responsive to pulsating fluid shear stress than osteogenically immature PDSCs and produce more bone in vivo. These data suggest that PDSCs with a mature osteogenic phenotype might be preferable for bone tissue engineering to restore, for example, maxillofacial defects, because they might be able to perform mature bone cell-specific functions during bone adaptation to mechanical loading in vivo.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth-supporting tissues in human periodontitis. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are functionally different cell types in the periodontium that can participate in the host immune response in periodontitis. This study aimed to investigate the effects of viable P. gingivalis on the expression of genes associated with inflammation and bone degradation by these fibroblast subsets.MATERIAL AND METHODS: Primary human GF and PDLF from six healthy donors were challenged in vitro with viable P. gingivalis W83 for 6 h. Gene expression of inflammatory cytokines in GF and PDLF was analyzed using real-time PCR, and protein expression was analyzed using ELISA.RESULTS: Viable P. gingivalis induced a strong in vitro inflammatory response in both GF and PDLF. We found increased gene expression of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor-alpha, monocyte chemotactic protein-1 and regulated upon activation, normal T-cell expressed and secreted (RANTES). Macrophage colony-stimulating factor was induced and the expression of osteoprotegerin was decreased in GF, but not in PDLF. In nonchallenged cells, a higher level of expression of IL-6 was observed in GF than in PDLF. Between individual donors there was large heterogeneity in responsiveness to P. gingivalis. Also, in each individual, either GF or PDLF was more responsive to P. gingivalis.CONCLUSION: Considerable heterogeneity in responsiveness to P. gingivalis exists both between GF and PDLF and between individuals, which may be crucial determinants for the susceptibility to develop periodontitis.

Seeds of Arabidopsis thaliana were sent to space and germinated in orbit. Seedlings grew for 4 d and were then fixed in-flight with paraformaldehyde. The experiment was replicated on the ground in a Random Positioning Machine, an effective simulator of microgravity. In addition, samples from a different space experiment, processed in a similar way but fixed in glutaraldehyde, including a control flight experiment in a 1g centrifuge, were also used. In all cases, comparisons were performed with ground controls at 1g. Seedlings grown in microgravity were significantly longer than the ground 1g controls. The cortical root meristematic cells were analyzed to investigate the alterations in cell proliferation and cell growth. Proliferation rate was quantified by counting the number of cells per millimeter in the specific cell files, and was found to be higher in microgravity-grown samples than in the control 1g. Cell growth was appraised through the rate of ribosome biogenesis, assessed by morphological and morphometrical parameters of the nucleolus and by the levels of the nucleolar protein nucleolin. All these parameters showed a depletion of the rate of ribosome production in microgravity-grown samples versus samples grown at 1g. The results show that growth in microgravity induces alterations in essential cellular functions. Cell growth and proliferation, which are strictly associated functions under normal ground conditions, appeared divergent after gravity modification; proliferation was enhanced, whereas growth was depleted. We suggest that the cause of these changes could be an alteration in the cell cycle regulation, at the levels of checkpoints regulating cell cycle progression, leading to a shortened G2 period. © 2009 Elsevier GmbH., This work was supported by grants from the Spanish “Plan Nacional de I+D+i”, Refs. nos. ESP2001-4522-PE and ESP2006-13600-C02-02, and the use of RPM, by Grant no. MG-057 (SRON, NL).

Background aims. Human (h) adipose tissue-derived mesenchymal stromal cells (ASC) constitute an interesting cellular source for bone tissue engineering applications. Wnts, for example Wnt5a, are probably important regulators of osteogenic differentiation of stem cells, but the role of Wnt5a in hASC lineage commitment and the mechanisms activated upon Wnt5a binding are unknown. We examined whether Wnt5a induces osteogenic and/or adipogenic differentiation of hASC. Methods. hASC were incubated for 7 days with or without Wnt5a, rho-associated kinase (ROCK)-activity inhibitor Y27632 or Wnt3a. Cells were lysed for total RNA isolation, DNA content and alkaline phosphatase (ALP) activity. Mineralized nodule formation and gene expression of osteogenic markers osteocalcin and runt-related protein-2 (RUNX2), and adipogenic markers peroxisome proliferator activator receptor-γ (PPARγ) and transcription factor apetala-2 (aP2), were analyzed. hASC were incubated with Wnt5a or Wnt3a to determine activation of canonical and/or non-canonical Wnt signaling pathways, and protein kinase C activity (PKC), total β-catenin content and gene expression of connexin 43 and cyclin D1 were quantified. Results. Wnt5a increased ALP activity and RUNX2 and osteocalcin gene expression, and down-regulated adipogenic markers through ROCK activity. Wnt5a also induced mineralized nodule formation. Wnt3a only enhanced RUNX2 and osteocalcin gene expression, and did not induce osteogenic differentiation. Wnt5a activated the non-canonical Wnt signaling pathway by increasing PKC activity, while Wnt3a mildly activated the Wnt canonical pathway by increasing total β-catenin content and connexin 43 and cyclin D1 gene expression. Conclusions. Our data illustrate the importance of Wnt5a as a stimulator of hASC osteogenic differentiation, and show that changes in actin cytoskeleton controlled by ROCK are determinants for Wnt5a-induced osteogenic differentiation of hASC.

Bone mechanotransduction is vital for skeletal integrity. Osteocytes are thought to be the cellular structures that sense physical forces and transform these signals into a biological response. The Wnt/β-catenin signaling pathway has been identified as one of the signaling pathways that is activated in response to mechanical loading, but the molecular events that lead to an activation of this pathway in osteocytes are not well understood. We assessed whether nitric oxide, focal adhesion kinase, and/or the phosphatidyl inositol-3 kinase/Akt signaling pathway mediate loading-induced β-catenin pathway activation in MLO-Y4 osteocytes. We found that mechanical stimulation by pulsating fluid flow (PFF, 0.7 ± 0.3 Pa, 5 Hz) for 30 min induced β-catenin stabilization and activation of the Wnt/β-catenin signaling pathway. The PFF-induced stabilization of β-catenin and activation of the β-catenin signaling pathway was abolished by adding focal kinase inhibitor FAK inhibitor-14 (50 μM), or phosphatidyl inositol-3 kinase inhibitor LY-294002 (50 μM). Addition of nitric oxide synthase inhibitor l-NAME (1.0 mM) also abolished PFF-induced stabilization of β-catenin. This suggests that mechanical loading activates the β-catenin signaling pathway by a mechanism involving nitric oxide, focal adhesion kinase, and the Akt signaling pathway. These data provide a framework for understanding the role of β-catenin in mechanical adaptation of bone. © 2009 Elsevier Inc. All rights reserved.

Stem cells offer an interesting tool for tissue engineering, but the clinical applications are limited by donor-site morbidity and low cell number upon harvest. Recent studies have identified an abundant source of stem cells in subcutaneous adipose tissue. Adipose stem cells (ASCs) present in adipose tissue are able to differentiate to several lineages and express multiple growth factors, which makes them suitable for clinical application. Buccal fat pad (BFP), an adipose-encapsulated mass found in the oral cavity, could represent an easy access source for dentists and oral surgeons. The stromal vascular fraction obtained from fresh BFP-derived adipose tissue and passaged ASCs were analyzed to detect and quantify the percentage of ASCs in this tissue. Here we show that BFP contains a population of stem cells that share a similar phenotype with ASCs from abdominal subcutaneous fat tissue, and are also able to differentiate into the chondrogenic, adipogenic, and osteogenic lineage. These results define BFP as a new, rich, and accessible source of ASCs for tissue engineering purposes.

Contains fulltext : 87804.pdf (Publisher’s version ) (Closed access) Osteoblasts respond to mechanical stimulation by changing morphology, gene expression and matrix mineralization. Introducing surface topography on biomaterials, independently of mechanical loading, has been reported to give similar effects. In the current study, using a nanotextured surface, and mechanical loading, we aimed to develop a multi-factorial model in which both parameters interact. Mechanical stimulation to osteoblast-like cells was applied by longitudinal stretch in parallel direction to the nanotexture (300 nm wide and 60 nm deep grooves), with frequency of 1 Hz and stretch magnitude varying from 1% to 8%. Scanning electron microscopy showed that osteoblast-like cells subjected to mechanical loading oriented perpendicularly to the stretch direction. When cultured on nanotextured surfaces, cells aligned parallel to the texture. However, the parallel cell direction to the nanotextured surface was lost and turned to perpendicular when parallel stretch to the nanotexture, greater than 3% was applied to the cells. This phenomenon could not be achieved when a texture with micro-sized dimensions was used. Moreover, a significant synergistic effect on upregulation of fibronectin and Cfba was observed when dual stimulation was used. These findings can lead to a development of new biomimetic materials that can guide morphogenesis in tissue repair and bone remodeling. 01 oktober 2010

In regions of high bone loading, the mechanoresponsive osteocytes inhibit osteoclastic bone resorption by producing signaling molecules. One possible candidate is matrix extracellular phosphoglycoprotein (MEPE) because acidic serine- and aspartate-rich MEPE-associated motif peptides upregulate osteoprotegerin (OPG) gene expression, a negative regulator of osteoclastogenesis. These peptides are cleaved from MEPE when relatively more MEPE than PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome) is present. We investigated whether mechanical loading of osteocytes affects osteocyte-stimulated osteoclastogenesis by involvement of MEPE. MLO-Y4 osteocytes were mechanically loaded by 1-h pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) or kept under static control conditions. Recombinant MEPE (0.05, 0.5, or 5 μg/ml) was added to some static cultures. Mouse bone marrow cells were seeded on top of the osteocytes to determine osteoclastogenesis. Gene expression of MEPE, PHEX, receptor activator of nuclear factor kappa-B ligand (RANKL), and OPG by osteocytes was determined after PFF. Osteocytes supported osteoclast formation under static control conditions. Both PFF and recombinant MEPE inhibited osteocyte-stimulated osteoclastogenesis. PFF upregulated MEPE gene expression by 2.5-fold, but not PHEX expression. PFF decreased the RANKL/OPG ratio at 1-h PFF treatment. Our data suggest that mechanical loading induces changes in gene expression by osteocytes, which likely contributes to the inhibition of osteoclastogenesis after mechanical loading of bone. Because mechanical loading upregulated gene expression of MEPE but not PHEX, possibly resulting in the upregulation of OPG gene expression, we speculate that MEPE is a soluble factor involved in the inhibition of osteoclastogenesis by osteocytes.

Adipose tissue-derived mesenchymal stem cells (AT-MSCs) in combination with bone morphogenetic protein-2 (BMP-2) or transforming growth factor-beta1 (TGF-beta1) are under evaluation for bone tissue engineering. Posttranslational modification of type I collagen is essential for functional bone tissue with adequate physical and mechanical properties. We investigated whether BMP-2 (10-100 ng/mL) and/or TGF-beta1 (1-10 ng/mL) affect gene expression of alpha2(I) procollagen and collagen-modifying enzymes, that is, lysyl oxidase and lysyl hydroxylases 1, 2, and 3 (encoded by PLOD1, 2, and 3), by human AT-MSCs. BMP-2, but not TGF-beta1, increased alkaline phosphatase activity after 28 days, indicating osteogenic differentiation of AT-MSCs. At day 4, both BMP-2 and TGF-beta1 upregulated alpha2(I) procollagen and PLOD1, which was downregulated at day 28. TGF-beta1, but not BMP-2, downregulated PLOD3 at day 28. Lysyl oxidase was upregulated by TGF-beta1 at day 4 and by BMP-2 at day 7. Neither BMP-2 nor TGF-beta1 affected PLOD2. In conclusion, these results suggest that AT-MSCs differentially respond to BMP-2 and TGF-beta1 with changes in gene expression of collagen-modifying enzymes. AT-MSCs may thus be able to appropriately modify type I collagen to form a functional bone extracellular matrix for tissue engineering, dependent on the growth factor added.

To explore the functions of the anion exchanger 2 (Ae2) in the development of bones and teeth we examined the distribution of Ae2 in cells involved in the formation of teeth and surrounding bone in young hamsters, mice and rats. In all three species strongest immunostaining for Ae2 was obtained in basolateral membranes of maturation ameloblasts and in osteoclasts resorbing bone. In hamsters a weaker staining was also seen in the Golgi apparatus of secretory ameloblasts, young osteoblasts and osteocytes, odontoblasts and fibroblasts of the forming periodontal ligament. In adult Ae2(a,b)(-/-) mice, in which Ae2-targeted disruption precluded the expression of Ae2a, Ae2b1 and Ae2b2 isoforms, the immunostaining for Ae2 in ameloblasts and osteoclasts was totally abolished. The enamel formation was abnormal but teeth erupted, osteoclasts in jaw bone were functional and structure of dentin and bone was normal. In another mouse model, Ae2(-/-) mice in which the expression of all five Ae2 isoforms was disrupted, teeth failed to erupt and the alveolar bone proved poorly formed with giant but apparently functional osteoclasts. Our data indicate that basolaterally located Ae2a, Ae2b1 or Ae2b2 (or a combination of these) is present in maturation ameloblasts critical for the cells' normal functioning. Although isoforms of Ae2 were also present in basolateral membranes of osteoclasts, they proved to be not critical to osteoclast resorption of orofacial bone. Poorly formed bone and the failure of teeth to erupt seen in the Ae2(-/-) mice with gene disruption affecting all isoforms may result from secondary (systemic) changes that are different from Ae2(a,b)(-/-) mice. J. Exp. Zool. (Mol. Dev. Evol.) 312B:375-387, 2009. (c) 2009 Wiley-Liss, Inc

PURPOSE. The vitreous body of the human eye undergoes progressive morphologic changes with aging. Since the enzymatic collagen cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are known to be important for the integrity of the collagen matrix, the presence in the vitreous on aging was studied.METHODS. Vitreous bodies (VBs; n = 143) from 119 donors (age 4-80 years; mean +/- SD, 54.3 +/- 17.0 years) were carefully dissected. After weighing and freeze-drying, all samples were analyzed by high performance liquid chromatography. Left and right eyes of 24 donors were compared and, for age-related phenomena, 119 single eyes were used.RESULTS. Within one donor, no significant differences were found between left and right eyes. On aging, VB wet weight (4.42 +/- 0.84 g) accumulates until 35 years and decreases thereafter. Collagen content (0.30 +/- 0.14 mg), HP per triple helix (TH; 0.55 +/- 0.18), and (HP plus LP)/TH (0.61 +/- 0.19) increase until 50 years followed by a decrease, whereas LP/TH (0.057 +/- 0.018) accumulates until 50 years and remains constant thereafter. The ratio between HP and LP (range, 0.42 - 31.0; median, 10.0) is constant over time.CONCLUSIONS. The accumulation of enzymatic collagen cross-links until 50 years is consistent with collagen maturation and possible collagen synthesis in the human vitreous body. The decline of collagen cross-links after 50 years is consistent with collagen breakdown. (Invest Ophthalmol Vis Sci. 2009; 50: 1041-1046) DOI: 10.1167/iovs.08-1714

Recent literature indicates that osteoclast formation in vitro from peripheral blood of patients with diseases associated with bone loss such as rheumatoid arthritis, osteoporosis, periodontitis and bone metastatic cancer may occur spontaneously being independent of addition of osteoclast formation stimulating factors such as macrophage colony forming factor (M-CSF) and receptor activator of NFκB ligand (RANKL). This could provide important clues to our understanding on how osteoclast precursors are primed within the circulation whilst commuting to the site of ultimate osteoclast differentiation. When comparing the various bone-lytic diseases, the common view emerges that accessory cells, such as T- and B-lymphocytes provide osteoclastogenesis stimulating factors. The roles of B- and T-cells in providing osteoclastogenesis-positive signals such RANKL, TNF-α and IL-7 are discussed. Immune cells present in the circulation may play an important role in preparing the osteoclast precursor for its ultimate destiny, contributing to the bone resorbing multinucleated cell at the required bone site.

How gravity influences the growth form and flavor components of plants is of interest to the space program because plants could be used for food and life support during prolonged missions away from the planet, where that constant feature of Earth's environment does not prevail. We used plant growth hardware from prior experiments on the space shuttle to grow Brassica rapa and Arabidopsis thaliana plants during 16-d or 11-d hypergravity treatments on large-diameter centrifuge rotors. Both species showed radical changes in growth form, becoming more prostrate with increasing g-loads (2-g and 4-g). In Brassica, height decreased and stems thickened in a linear relationship with increasing g-load. Glucosinolates, secondary compounds that contribute flavor to Brassica, decreased by 140% over the range of micro to 4-g, while the structural secondary compound, lignin, remained constant at ∼15% (w/w) cell wall dry mass. Stem thickening at 4-g was associated with substantial increases in cell size (47%, 226%, and 33% for pith, cortex, and vascular tissue), rather than any change in cell number. The results, which demonstrate the profound effect of gravity on plant growth form and secondary metabolism, are discussed in the context of similar thigmostresses such as touch and wind.

Lysyl oxidase (LOX) catalyzes cross-linking of elastin and collagen, which is essential for the structural integrity and function of bone tissue. The present study examined the role of Lox gene deficiency for the osteoblast phenotype in primary calvarial osteoblasts from E18.5 Lox knockout (Lox −/− ) and wild type (wt) (C57BL/6) mice. Next to Lox gene depletion, mRNA expression of Lox isoforms, LOXL1–4, was significantly downregulated in Lox −/− bone tissue. A significant decrease of DNA synthesis of Lox −/− osteoblasts compared to wt was found. Early stages of osteoblastic apoptosis studied by annexin-V binding as well as later stages of DNA fragmentation were not affected. However, mineral nodule formation and osteoblastic differentiation were markedly decreased, as revealed by significant downregulation of osteoblastic markers, type I collagen, bone sialoprotein, and Runx2/Cbfa1.

Contains fulltext : 81153.pdf (Publisher’s version ) (Open Access) In this study we present a new three-dimensional (3D) model to study effects of mechanical loading on tendon/ligament formation in vitro. The model mimics a functional periodontal ligament (PDL), which anchors dental roots to the jaw bone and transfers the axial load of mastication to the jaw bone. A collagen gel containing human PDL fibroblasts was seeded in a PDL space between an artificial root and bone surface. The effects of 3-day loading on the fibroblasts were studied in vitro by axial and intermittent displacement of the root to which the gel was attached. Cell responses were recorded by measuring expression of three sets of genes: (i) cyclooxygenase 1 and 2 (COX-1, COX-2) producing prostaglandins (signaling molecules); (ii) Runx2, a transcription factor for the osteogenic lineage; and (iii) the extracellular matrix proteins osteopontin, dentin matrix protein 1, and collagen type I (COL1). Loading for 3 days resulted in magnitude-dependent changes in the expression of COX-2 and COL1. A low loading magnitude significantly decreased COX-2 expression, an intermediate magnitude increased its expression, while a high magnitude increased COL1 expression. We concluded that the 3D loading model provides a useful, well-controlled method to examine ligament fibroblast responses to mechanical loading. The model may serve to explore the application of mechanical loading as an anabolic factor for ligament reconstruction.

Stochastic resonance is exhibited by many biological systems, where the response to a small stimulus is enhanced with the aid of noise. This intriguing possibility provides a novel paradigm for understanding previously reported osteogenic benefits of low amplitude dynamic loading. However, it is unknown whether bone cell mechanosensitivity is enhanced by noise as an alternative mechanism for an amplified response to small stresses. We studied whether noise of varying intensities enhanced the mechanosensitivity of MC3T3-E1 cells. Nitric oxide (NO) production was measured as the parameter for bone cell activation. Dynamic fluid shear stress stimulated bone cells provided an initial-stress kick was implemented. Without the initial stress-kick bone cells did not release a significant amount of NO demonstrating an essential non-linearity to bone cell responses to stress and the possibility of stochastic resonance in bone cell mechanosensitivity. The rapid NO response of MC3T3-E1 cells to a small periodic fluid shear stress was increased with the addition of noise compared to the response to stress with only noise. This confirms the possibility of stochastic resonance enhancement of NO production by bone cells. Since NO regulate bone formation as well as resorption, our results suggest that noise enhances the activity of bone cells in driving the mechanical adaptation of bone.

Collagen type V is highly expressed during tissue development and wound repair, but its exact function remains unclear. Cell binding to collagen V affects various basic cell functions and increased collagen V levels alter the structural organization and the stiffness of the ECM. We studied the combined effects of collagen V and substrate stiffness on the morphology, focal adhesion formation, and actin organization of fibroblasts. We found that a hybrid collagen I/V coating impairs fibroblast spreading on soft substrates (

6 páginas, 2 figuras, 1 tabla -- PAGS nros. 299-304, In the GENE experiment performed during an 11-day Soyuz Mission to the International Space Station (ISS), we intended to determine if microgravity affects Drosophila metamorphosis processes. Control experiments were performed including a 1g ground control parallel to the ISS flight samples and a Random Position Machine microgravity simulated control. A preliminary analysis of the results indicates that five hundred to one thousand genes change their expression profiles depending on the cut-off levels selected. Especially affected among them are the mitochondrial ones (an example with the respiratory chain is presented). We show here that there is a synergic effect of the constraints introduced to meet the requirements of the space experiment (mainly, a cold step and the use of hermetically closed Type-I containers). The cold transport step to the launch site was introduced to slow down the pupal development. The hermetically closed Type I containers were required to ensure the containment of the fixative (acetone) in the experiment. As shown here, the oxygen concentration inside the container was not optimal but fully compatible with pupal development. It is highly likely that such combined environmental effects will become a common finding in these types of studies as they become more complicated and extensive. They could open the way to understand how the gene expression patterns and the actual phenotypes can adjust to the environment. These findings indicate the importance of a vigorous ground based program in support of real microgravity experiments. Only then we can utilize the ISS in order to understand the consequences of the modified environment in outer space on living organisms, This work was supported by Grants from The Spanish "Plan Nacional de Investigación Científica y Desarrollo Tecnológico" Ref. nos. ESP2001-4522-PE, and ESP2003-09475-C02-01 and from The Netherlands Institute for Space Researchs, NW0-SROM, MG-057

Bone has the capacity to regenerate in response to injury. During distraction osteogenesis, the renewal of bone is enhanced by gradual stretching of the soft connec- tive tissues in the gap area between two separated bone segments. This procedure has received much clinical atten- tion as a way to correct congenital growth retardation of bone tissue or to generate bone to fill skeletal defects. The process of bone regeneration involves a complex system of biological changes whereby mechanical stress is converted into a cascade of signals that activate cellular behavior resulting in (enhanced) formation of bone. Over the last decade, significant progress has been made in understand- ing the bone regeneration process during distraction osteo- genesis. The mechanical and biological factors that are important for the success of the distraction treatment have been partially characterized and are discussed in this review.

Sclerostin is an inhibitor of bone formation expressed by osteocytes. We hypothesized that sclerostin is expressed by cells of the same origin and also embedded within mineralized matrices. In this study, we analyzed (a) sclerostin expression using immunohistochemistry, (b) whether the genomic defect in individuals with van Buchem disease (VBD) was associated with the absence of sclerostin expression, and (c) whether this was associated with hypercementosis. Sclerostin was expressed by cementocytes in mouse and human teeth and by mineralized hypertrophic chondrocytes in the human growth plate. In individuals with VBD, sclerostin expression was absent or strongly decreased in osteocytes and cementocytes. This was associated with increased bone formation, but no overt changes in cementum thickness. In conclusion, sclerostin is expressed by all 3 terminally differentiated cell types embedded within mineralized matrices: osteocytes, cementocytes, and hypertrophic chondrocytes.

Tumor cells in the bone microenvironment are able to initiate a vicious cycle of bone degradation by mobilizing osteoclasts, multinucleated cells specialized in bone degradation. c-Src is highly expressed both in tumors and in osteoclasts. Therefore, drugs like AZD0530, designed to inhibit Src activity, could selectively interfere with both tumor and osteoclast activity. Here we explored the effects of AZD0530 on human osteoclast differentiation and activity. The effect on osteoclasts formed in vivo was assessed in mouse fetal calvarial explants and in isolated rabbit osteoclasts, where it dose-dependently inhibited osteoclast activity. Its effect on formation and activity of human osteoclasts in vitro was determined in cocultures of human osteoblasts and peripheral blood mononuclear cells. AZD0530 was most effective in inhibiting osteoclast-like cell formation when present at the onset of osteoclastogenesis, suggesting that Src activity is important during the initial phase of osteoclast formation. Formation of active phosphorylated c-Src, which was highly present in osteoclast-like cells in cocultures and in peripheral blood mononuclear cell monocultures, was significantly reduced by AZD0530. Furthermore, it reversibly prevented osteoclast precursor migration from the osteoblast layer to the bone surface and subsequent formation of actin rings and resorption pits. These data suggest that Src is pivotal for the formation and activity of human osteoclasts, probably through its effect on the distribution of the actin microfilament system. The reversible effect of AZD0530 on osteoclast formation and activity makes it a promising candidate to temper osteoclastic bone degradation in bone diseases with enhanced osteoclast activity such as osteolytic metastatic bone disease. (Mol Cancer Res 2009;7(4):476–88)

IntroductionBones are subjected to a variety of mechanical loads duringdaily activities. In the nineteenth century, Julius Wolffproposed that bones adapt their mass and 3D structure tothe loading conditions in order to optimize their load-bearing capacity, and that this process is driven bymechanical stress [1]. For the past centuries, an increasingnumber of theoretical and experimental results reveal thatosteocytes are the pivotal cells orchestrating this bio-mechanical regulation of bone mass and structure, whichis accomplished by the process of bone remodeling [2–5]Osteocytes are terminally differentiated cells of theosteogenic lineage that are derived from mesenchymalprecursor cells. A number of molecules have been iden-tified as important markers of osteocytes, such as matrixextracellular phosphoglycoprotein [6] sclerostin [7], dentinmatrix protein-1 [8], and phex protein [8]. The osteocytesare the most abundant cells in adult bone and are constantlyspaced throughout the mineralized matrix. Mature osteo-cytes have a characteristic dendritic cell shape, with pro-cesses radiating from the cell body through the canaliculi indifferent directions. These processes form an intercellularnetwork through gap and adherent junctions with surround-ing osteocytes, the cells lining the bone surface and bonemarrow. Through this unique 3D network, osteocytes areanatomically placed in a prime position not only to sensedeformations driven by stresses placed upon bone, butalso to respond with passage of signals to the neighboringcells [9].For more than a decade now, it is known that theosteocytes are very sensitive to stress applied to intact bonetissue [10–16]. Computer simulation models have shownthat mechanosensors lying at the surface of bone, asosteoblasts and bone lining cells do, would be less sensitiveto changes in the loading pattern than the osteocytes, lyingwithin the calcified matrix [3]. Interestingly, targetedablation of osteocytes in mice disturbs the adaptation ofbone to mechanical loading [16].Osteocytes as key players in the process of bonemechanotransductionIt is currently believed that when bones are loaded, theresulting deformation will drive the thin layer of interstitialfluid surrounding the network of osteocytes to flow fromregions under high pressure to regions under low pressure[17, 18]. This flow of fluid is sensed by the osteocyteswhich in turn produce signaling molecules that can regulatebone resorption through the osteoclasts, and bone formationthrough the osteoblasts, leading to adequate bone remodel-ing [17, 18]. This concept is known as the fluid flowhypothesis. Evidence has been increasing for the flow ofcanalicular interstitial fluid as the likely factor that informsthe osteocytes about the level of bone loading [2, 5, 17, 18].Nevertheless, Vatsa and colleagues [19, 20] proposed thatif osteocytes could sense matrix strains directly, the cellshape, cytoskeletal alignment and distribution of adhesionsites in osteocytes in situ would bear alignment to themechanical loading patterns. Indeed, it was shown that the

Although morbidity of the Iliac crest after grafting has been reported to occur frequently, It Is the most widely used donor site. A proper regeneration of the defect could decrease morbidity. The purpose of this study was to evaluate the efficacy of two types of reconstruction of standardized critical size defects of the iliac crest after 3, 6, and 12 months. Standardized critical defects in iliac crests were bilaterally reconstructed in 28 goats randomly with (1) no reconstruction (group A); (2) a bioresorbable polylactide niesh (group B); or (3) 60/40 biphasic calcium phosphate (BCP) granules placed within a bioresorbable mesh (group C). At follow up, the Iliac crests were harvested for histologic, histomorphometrlc, and radlologic analyses. The defects treated with "mesh-BCP" (group C) showed a significantly (p = 0.03) larger area with diffuse bone formation, while only a small area of high density trabecular bone ingrowth was observed in the "no reconstruction" (group A) and "mesh group" (group B). However, no difference in the total volume of bone was observed; in group C, the bone was more diffusely spread over the defect. Substitution of the BCP granules by trabecular bone did not start before 6 months. At the 12 months time point, extensive resorptlon of BCP was found because of phagocytic activity of numerous multlnucleated giant cells. We confirm the positive influence of BCP on bone formation but due to a slow rate of resorption of BCP, regeneration takes a relatively long period of time. © 2008 Wiley Periodicals, Inc.

Sensing gravity by ‘non-specialized’ cells is still puzzling. We don’t know where or by which mechanism such cells sense gravity. These questions in ‘gravisensing’ are not much different from questions in general mechanobiology. Numerous studies have been reported in this field in the last couple of decades. What are the mechanical properties of a cell? Are there differences in mechanical properties between cell types and if so why? How are forces perceived and transduced to a meaningful biological event. Novel techniques such as optical and magnetic tweezers, atomic force microscopy, magnetophoresis and computer modeling make the field of mechano-sensing or perhaps physicomics accessible. A similar approach should also be applied for gravity-related research. This paper addresses the current techniques used in mechanosensing and exemplifies how a cell could sense the relatively weak force of gravity.

How tightly land plants are adapted to the gravitational force (g) prevailing on Earth has been of interest because unlike many other environmental factors, g presents as a constant force. Ontogeny of mature angiosperms begins with an embryo that is formed after tip growth by a pollen tube delivers the sperm nucleus to the egg. Because of the importance to plant fitness, we have investigated how gravity affects these early stages of reproductive development. Arabidopsis thaliana (L.) Heynh. plants were grown for 13 days prior to being transferred to growth chambers attached to a large diameter rotor, where they were continuously exposed to 2-g or 4-g for the subsequent 11 days. Plants began flowering 1 day after start of the treatments, producing hundreds of flowers for analysis of reproductive development. At 4-g, Arabidopsis flowers self-pollinated normally but did not produce seeds, thus derailing the entire life cycle. Pollen viability and stigma esterase activity were not compromised by hypergravity; however, the growth of pollen tubes into the stigmas was curtailed at 4-g. In vitro pollen germination assays showed that 4-g average tube length was less than half that for 1-g controls. Closely related Brassica rapa L., which produces seeds at 4-g, required forces in excess of 6-g to slow in vitro tube growth to half that at 1-g. The results explain why seed production is absent in Arabidopsis at 4-g and point to species differences with regard to the g-sensitivity of pollen tube growth.

5 páginas -- PAGS nros. 293-297, Changes have been reported in the pattern of gene expression in Arabidopsis on exposure to microgravity. Plant cell growth and proliferation are functions that are potentially affected by such changes in gene expression. In the present investigation, the cell proliferation rate, the regulation of cell cycle progression and the rate of ribosome biogenesis (this latter taken to estimate cell growth) have been studied using morphometric markers or parameters evaluated by light and electron microscopy in real microgravity on the International Space Station (ISS) and in ground-based simulated microgravity, using the Random Positioning Machine and the Magnetic Levitation Instrument. Results showed enhanced cell proliferation but depleted cell growth in both real and simulated microgravity, indicating that the two processes are uncoupled, unlike the situation under normal gravity on Earth in which they are strictly co-ordinated events. It is concluded that microgravity is an important stress condition for plant cells compared to normal ground gravity conditions

Contains fulltext : 71030.pdf (Publisher’s version ) (Closed access) This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and microgrooved substrata (groove depth: 0.5 mum, width: 1 mum), which were subjected to simulated microgravity. The aim of the study was to clarify which of these parameters was more dominant to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell alignment. Expression of collagen type I, and alpha1-, beta1-, beta3-integrin were investigated by QPCR. Finally, immunoblotting was applied to visualise MAPK signalling pathways. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata, cells had spread out in a random fashion. The alignment of cells cultured on grooved surfaces under simulated microgravity, after 48 h of culturing appeared similar to those cultured at 1g, although cell shape was different. Analysis of variance proved that all main parameters: topography, gravity force, and time were significant. In addition, gene levels were reduced by simulated microgravity particularly those of beta3-integrin and collagen, however alpha-1 and beta-1 integrin levels were up-regulated. ERK1/2 was reduced in RPM, however, JNK/SAPK and p38 remained active. The members of the small GTPases family were stimulated under microgravity, particularly RhoA and Cdc42. The results are in agreement that application of microgravity to fibroblasts promotes a change in their morphological appearance and their expression of cell-substratum proteins through the MAPK intracellular signalling pathways.

We investigated whether low-intensity pulsed ultrasound (LIPUS) stimulates chondrocyte proliferation and matrix production in explants of human articular cartilage obtained from donors suffering from unicompartimental osteoarthritis of the knee, as well as in isolated human chondrocytes in vitro. Chondrocytes and explants were exposed to LIPUS (30 mW/cm2; 20 min/day, 6 days). Stimulation of [35S]-sulphate incorporation into proteoglycans by LIPUS was 1.3-fold higher in degenerative than in collateral monolayers as assessed biochemically and 1.9-fold higher in explants as assessed by autoradiography. LIPUS decreased the number of cell nests containing 1-3 chondrocytes by 1.5 fold in collateral and by 1.6 fold in degenerative explants. LIPUS increased the number of nests containing 4-6 chondrocytes by 4.8 fold in collateral and by 3.9 fold in degenerative explants. This suggests that LIPUS stimulates chondrocyte proliferation and matrix production in chondrocytes of human articular cartilage in vitro. LIPUS might provide a feasible tool for cartilage tissue repair in osteoarthritic patients, since it stimulates chondrocyte proliferation and matrix production.

Nano-scale pattern templates have been manufactured in order to study the differences in cell behaviour between fibroblasts cultured on smooth and on grooved substrata. The pattern templates were made on silicon wafers using electron beam lithography in hydrogen silsesquioxane (HSQ) and subsequent reactive ion etching (RIE). These masters were replicated in polystyrene cell culture material using solvent casting. The replicas were assessed with atomic force microscopy (AFM). After seeding with fibroblasts, morphological characteristics were investigated using scanning electron microscopy (SEM) and light microscopy, in order to obtain qualitative and quantitative information on cell alignment. It appears that both groove depth and width determine the cellular alignment on patterns with a ridge/groove ratio of 1:1. On smooth substrata, cells always spread out in a random fashion. There appears to be a threshold groove barrier size of around 70-80 nm, above which random cell spreading is not possible anymore and contact guidance occurs. It is speculated that this threshold size may be associated with the size of contact molecules at the cell extensions, which grow and find anchoring spots preceding cell spread out and cell alignment.

Whether low intensity pulsed ultrasound therapy stimulates osteogenesis in mandibular distraction was investigated in a double-blind trial. Nine patients underwent a vertical mandibular distraction over a distance of 5.1 +/- 1.2 mm. Ultrasound or placebo therapy was started daily from the first day of distraction. After 46 +/- 8.1 days consolidation, two endosseous implants were inserted and a transmandibular biopsy was taken. Ultrasonographs were taken regularly to follow osteogenesis inside the gap. There were no complications during the 44 +/- 7.1 months of follow-up. Microradiographic measurements of the biopsies revealed no differences in the area of mineralized tissue in the distraction gap. The cranially distracted bone segment appeared significantly more radiolucent than the caudal bone. Histological examination showed large lacunae inside the cranially distracted bone segment, filled with clusters of osteoclasts and surrounded by clear tetracycline double labels. Within the distraction gap, woven bone was present, with no apparent differences between the treatment groups. Ultrasonographic follow-up revealed that osteogenesis inside the distraction gap progresses from 4 to 20 weeks post distraction, with no differences between the ultrasound and the placebo groups. In summary, ultrasound treatment does not appear to stimulate bone formation in the severely resorbed vertical distracted mandible.

Almost all tissues in our body contain specific cells associated with the tissue itself, and an extracellular matrix (ECM) that consists of a variety of proteins of which the bulk is formed by different types of collagens, glycoproteins and proteoglycans. TheECMplays a pivotal role in numerous processes not only related to the mechanical properties of a tissue, but also in modulating cellular activity. For a proper functioning of a tissue remodeling of the ECM is essential. Some connective tissues are characterized by a very rapid turnover (e.g. periodontal ligament) whereas others hardly show signs of turnover (e.g. cartilage). In all situations degradation of the ECM constituents occur. Under certain conditions, especially during a pathological situation, a high level of degradation may take place. In other situations matrix synthesis and deposition outstrips breakdown, leading to a fibrosis. In order to obtain information on the level of degradation of the different ECMcomponents, various methods have been employed. A number of these methods will be discussed in this article.

Transforming growth factor beta (TGF beta) is often used in cartilage tissue engineering to increase matrix formation by cells with various phenotypes. However, adverse effects of TGF beta, such as extensive cross-linking in cultured fibroblasts, have also been reported. Our goal was to study effects of TGF beta on collagen cross-linking and evaluating the role of cellular phenotype and physical environment. We therefore used four different cell populations in two very different physical environments: primary and expanded chondrocytes and fibroblasts embedded in alginate gel and attached to tissue culture plastic. Matrix production, collagen cross-linking, and alpha-smooth muscle actin (alpha SMA) were analyzed during 4 weeks with or without 2.5 ng/mL TGF beta 2. TGF beta 2 did not affect collagen deposition by primary cells. In expanded cells, TGF beta 2 increased collagen deposition. Chondrocytes and fibroblasts in monolayer produced more collagen cross-links with TGF beta 2. In alginate, primary and expanded cells displayed an unexpected decrease in collagen cross-linking with TGF beta 2. aSMA was not present in alginate cultures and barelay upregulated by TGF beta 2. Organized alpha SMA fibers were present in all monolayer cultures and became more pronounced with TGF beta 2. This study demonstrates that the physical environment determined by the substrate used co-determines the response of cells to TGF beta. The presence of mechanical stress, determined with alpha SMA-staining, is probably responsible for the increase in collagen cross-linking upon addition of TGF beta.

Renal impairment in children is associated with tooth defects that include enamel pitting and hypoplasia. However, the specific effects of uremia on tooth formation are not known. In this study, we used rat mandibular incisors, which continuously erupt and contain all stages of tooth formation, to characterize the effects of uremia on tooth formation. We also tested the hypothesis that uremia aggravates the fluoride (F)-induced changes in developing teeth. Rats were subjected to a two-stage 5/6 nephrectomy or sham operation and then exposed to 0 (control) or 50 ppm NaF in drinking water for 14 days. The effects of these treatments on food intake, body growth rate, and biochemical serum parameters for renal function and calcium metabolism were monitored. Nephrectomy reduced food intake and weight gain. Intake of F by nephrectomized rats increased plasma F levels twofold and further decreased food intake and body weight gain. Uremia affected formation of dentin and enamel and was more extensive than the effect of F alone. Uremia also significantly increased predentin width and induced deposition of large amounts of osteodentin-like matrix-containing cells in the pulp chamber. In enamel formation, the cells most sensitive to uremia were the transitional-stage ameloblasts. These data demonstrate that intake of F by rats with reduced renal function impairs F clearance from the plasma and aggravates the already negative effects of uremia on incisor tooth development.

Inflammatory periodontal diseases constitute one of the most common infections in humans, resulting in the destruction of the supporting structures of the dentition. Circulating neutrophils are an essential component of the human innate immune system. We observed that mice deficient for the major lysosomal-associated membrane protein-2 (LAMP-2) developed severe periodontitis early in life. This development was accompanied by a massive accumulation of bacterial plaque along the tooth surfaces, gingival inflammation, alveolar bone resorption, loss of connective tissue fiber attachment, apical migration of junctional epithelium, and pathological movement of the molars. The inflammatory lesions were dominated by polymorphonuclear leukocytes (PMNs) apparently being unable to efficiently clear bacterial pathogens. Systemic treatment of LAMP-2-deficient mice with antibiotics prevented the periodontal pathology. Isolated PMNs from LAMP-2-deficient mice showed an accumulation of autophagic vacuoles and a reduced bacterial killing capacity. Oxidative burst response was not altered in these cells. Latex bead and bacterial feeding experiments showed a reduced ability of the phagosomes to acquire an acidic pH and late endocytic markers, suggesting an impaired fusion of late endosomes-lysosomes with phagosomes. This study underlines the importance of LAMP-2 for the maturation of phagosomes in PMNs. It also underscores the requirement of lysosomal fusion events to provide sufficient antimicrobial activity in PMNs, which is needed to prevent periodontal disease.

Implantation of biomaterials into the body, e.g. for tissue engineering purposes, induces a material dependent inflammatory response called the foreign body reaction (FBR). A hallmark feature of this response is the formation of large multinucleated cells: foreign body giant cells (FBGCs). Biomaterials like cross-linked and non-cross-linked collagen often induce the formation of FBGCs. It is unknown whether different biomaterials result in the formation of different FBGCs. To investigate this, we implanted cross linked and non-cross-linked dermal sheep collagen subcutaneously in mice. After 21 days the implanted material was collected and prepared for ultrastructural analysis. More FBGCs formed on and between implants of cross-linked collagen compared to non-cross-linked material. The ultrastructural aspects of the FBGCs present on the two types of implants proved to be similar. On both materials, they formed long slender protrusions on the basolateral membrane, they were very rich in mitochondria, contained numerous nuclei, and showed signs of the presence of a clear zone facing the implanted material. Similar clear zones, that resemble osteoclastic features, were also seen in FBGCs generated in vitro on bone slices, but these cells did not form a ruffled border. However, similarities in ultrastructure such as the occurrence of slender protrusions and high mitochondrion content were also found in the FBGCs generated in vitro. These data indicate that FBGCs formed on different substrates share many morphological characteristics. The formation of long finger-like protrusions seemed typical for the FBGCs, in vivo as well as in vitro, however the function of these structures needs further analysis. (C) 2016 Elsevier Inc. All rights reserved.

Background: Receptors for the Fc part of IgG (Fc gamma RIIa) on polymorphonuclear leukocytes (PMN) mediate phagocytosis and cell activation. Previous results show that one of the genetic variants of the Fc gamma RIIa, the 131 H/H, is associated with more periodontal breakdown than the R/R. This may be due to hyper-reactivity of the H/H-PMNs upon interaction with bacteria.Aim: To study whether the Fc gamma RIIa genotype modifies the PMN reactivity in periodontitis patients.Material and Methods: A cohort of 98 periodontitis patients was genotyped. From these, 10 H/H and 10 R/R consented to participate. PMNs were incubated with immune serum-opsonized Actinobacillus actinomycetemcomitans (A.a.). Phagocytosis, degranulation (CD63 and CD66b expression), respiratory burst and elastase release were assessed.Results: Patients of the H/H genotype showed more bone loss than those with the H/R or R/R genotype (p=0.038). H/H-PMNs phagocytosed more opsonized A.a. than did R/R-PMNs (p=0.019). The H/H-PMNs also expressed more CD63 and CD66b than did the R/R-PMNs (p=0.004 and 0.002, respectively) and released more elastase (p=0.001).Conclusions: The genotyping results confirm previous reports that more periodontal destruction occurs in the H/H genotype than in the H/R or R/R genotype. The functional studies indicate a hyper-reactivity of the H/H-PMN in response to bacteria, which may be one of several pathways leading to more periodontal breakdown.

In an experiment conducted in the “Cervantes” Spanish Soyuz Mission, a 10-day flight to the International Space Station, Arabidopsis seeds were germinated, seedlings grew for 4 days at 22ºC, and they were fixed in flight with paraformaldehyde. A ground 1 g control experiment was replicated, and an additional experiment in simulated microgravity, using a Random Positioning Machine, was performed in the same conditions. Structural, morphometric and immunocytochemical data were compared. Glutaraldehyde-fixed seedlings similarly grown in the Space Shuttle (STS-84 Mission) were also used for ultrastructural and morphometric studies. Seedlings grown for 4 days in real or simulated weightlessness showed a longer size than the ground 1 g control. Root meristematic cells showed an enhanced proliferating rate, but ribosome biogenesis was reduced, as inferred from the nucleolar size and from the levels of the nucleolar protein nucleolin. This could be the consequence of the acceleration of the cell cycle, with shortening of its phases. Weightlessness induces stress in the plant, influencing cellular processes decisive for development and morphogenesis. This stress may endanger the plant and would require the action of compensating specialized defence mechanisms

The perspective of an innovative new concept integrating tissue-engineering techniques with an established surgical technique is described. The focus is primarily on a one-step surgical procedure using adipose tissue-derived mesenchymal stem cells, a calcium phosphate scaffold as a carrier, and a bioresorbable polymer cage to facilitate spinal interbody fusion. We address the harvesting and processing of clinically relevant quantities of adipose tissue-derived mesenchymal stem cells, triggering of these stem cells toward lineage-specific differentiation, seeding of the triggered stem cells on a bioresorbable scaffold, and implantation of the resulting tissue-engineered construct. The integrated steps can be accomplished within one surgical procedure in a surgical theater. Although the proposed concept has been developed for spinal fusion, potential application in other surgical disciplines is presumed realistic.

Infantile malignant osteopetrosis (IMO) is a fatal disease caused by lack of functional osteoclasts, and the only available treatment is hematopoietic stem cell (HSC) transplantation. In the majority of patients, the TCIRG1 gene, coding for a subunit of a proton pump essential for bone resorption, is mutated. Oc/oc mice have a deletion in the homologue gene (tcirg1) and die at 3 to 4 weeks, but can be rescued by neonatal transplantation of HSCs. Here, HSC-targeted gene therapy of osteopetrosis in the oc/oc mouse model was developed. Oc/oc fetal liver cells depleted of Ter119-expressing erythroid cells were transduced with a retroviral vector expressing tcirg1 and GFP, and subsequently transplanted intraperitoneally to irradiated neonatal oc/oc mice. Eight of 15 mice survived past the normal life span of oc/oc mice. In vitro osteoclastogenesis revealed formation of GFP-positive osteoclasts and bone resorption, albeit at a lower level than from wild-type cells. The skeletal phenotype was analyzed by X-ray and histopathology and showed partial correction at 8 weeks and almost normalization after 18 weeks. In summary, osteopetrosis in oc/oc mice can be reversed by neonatal transplantation of gene-modified HSCs leading to long-term survival. This represents a significant step toward the development of gene therapy for osteopetrosis.

Bone remodelling is a dynamic process that requires the coordinated interaction of osteocytes, osteoblasts, and osteoclasts, collaborating in basic multicellular units (BMUs). Communication between these cells can be by extracellular soluble molecules as well as directly propagating intercellular signalling molecules. Key to the understanding of bone remodelling is osteocyte mechanosensing and chemical signalling to the surrounding cells, since osteocytes are believed to be the mechanosensors of bone, responding to mechanical stresses. Nitric oxide (NO) is an important parameter to study osteocyte activation following mechanical loading. It is a small short-lived molecule, which makes its real-time, quantitative monitoring difficult. However, recently we demonstrated that DAR-4M AM chromophore can be used for real-time quantitative monitoring of intracellular NO production in individual cells following mechanical loading. Here we studied if a single mechanically stimulated osteocyte communicates with, and thus activates its surrounding cells via extracellular soluble factors. We monitored quantitatively intracellular NO production in the stimulated osteocyte and in its surrounding osteocytes, which were not interconnected. Mechanical stimulation by microneedle of a single-MLO-Y4 osteocyte-like cell upregulated the average intracellular NO production by 94% in the stimulated cell, and by 31-150% in the surrounding osteocytes. In conclusion, a single osteocyte can disseminate a mechanical stimulus to its surrounding osteocytes via extracellular soluble signalling factors. This reinforces the putative mechanosensory role of osteocytes, and demonstrates a possible mechanism by which a single mechanically stimulated osteocyte can communicate with other cells in a BMU, which might help to better understand the intricacies of intercellular interactions in BMUs and thus bone remodelling.

The purpose of this study was to evaluate the efficacy of recombinant human osteogenic protein-1 (rhOP-1) with a carboxymethylcellulose-stabilized collagenous carrier as a bone graft substitute for instrumented lumbar spinal fusion in an established goat model. Twenty goats received a resorbable poly-L-lactic acid (PLLA) interbody cage packed with either rhOP-1 and its carrier or autologous bone graft. The carrier material was bovine collagen type-1 stabilized with carboxymethylcellulose. The fusion segments were retrieved at 3 or 6 months postimplantation and evaluated by radiographic and histologic analyses. The rhOP-1 graft substitute, used in combination with the resorbable PLLA cage, showed inferior results as compared to autologous bone graft in the goat lumbar fusion model. Whereas four out of five segments from the autograft group were fused after 6 months, none of the four segments receiving the rhOP-1 graft substitute were fused at this time point. Bone ingrowth into the cage was delayed or absent in the experimental group, whereas all autograft specimens showed advanced bone ingrowth (3 months) or fusion (6 months). We suggest that the fusion process was inhibited, because cells were unable to penetrate the rhOP-1 graft material. This led to delayed bone formation and in some cases inadequate tissue formation.

We have generated mice with targeted inactivation of the Plod1 gene for lysyl hydroxylase 1 (LH1). Its human mutations cause Ehlers-Danlos syndrome VIA (EDS VIA) characterized by muscular hypotonia, joint laxity, and kyphoscoliosis. The Plod1(-/-) mice are flaccid and have gait abnormalities. About 15% of them died because of aortic rupture and smooth muscle cells in non-ruptured Plod1(-/-) aortas showed degenerative changes. Collagen fibrils in the Plod1(-/-) aorta and skin had an abnormal morphology. The LH activity level in the Plod1(-/-) skin and aorta samples was 35-45% of that in the wild type. The hydroxylysine content was decreased in all the Plod1(-/-) tissues, ranging from 22% of that in the wild type in the skin to 75 and 86% in the femur and lung. The hydroxylysylpyridinoline crosslinks likewise showed decreases in all the Plod1(-/-) tissues, ranging from 28 and 33% of that in the wild type in the aorta and cornea to 47 and 59% in femur and tendon, while lysylpyridinolines were increased. The hydroxylysines found in the Plod1(-/-) collagens and their cross-links were evidently synthesized by the other two LH isoenzymes. Few data are available on abnormalities in EDS VIA tissues other than the skin. Plod1(-/-) mice offer an in vivo model for systematic analysis of the tissue-specific consequences of the lack of LH1 activity and may also provide a tool for analyzing the roles of connective tissue in muscle function and the complex interactions occurring in the proper assembly of the extracellular matrix.

We tested the hypothesis that mechanical loading of human bone increases expression of the transcription factor RUNX2 and bone matrix proteins osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein-1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE). We examined this in tissue sections of atrophic mandibular bone taken from edentulous patients who had undergone distraction osteogenesis. In undistracted bone, weak to moderate staining for OPN and BSP was found in osteoblasts and bone matrix of immature woven bone. RUNX2 was also detectable in osteoblasts and in cells of the periosteum. In woven bone, but not in lamellar bone, a small number of osteocytes stained for all proteins tested. After distraction, staining intensity had increased in the existing old bone and staining was seen in more bone cells than before distraction. We also found a high expression of DMP1 and MEPE in many osteocytes embedded in woven bone and in some osteocytes of lamellar bone not seen before distraction. New bone trabeculae were forming in the fibrous tissue of the distraction gap containing all stages of intramembranous bone formation. Moderate to strong staining was seen for all five proteins tested in osteocytes located in woven bone of these trabeculae and for RUNX2, OPN, and BSP in osteoblasts lining the trabecular surfaces. We conclude that loading of atrophic human jawbone by distraction activates matrix synthesis of bone cells in and around existing bone. Increased staining of DMP1 and MEPE in osteocytes after loading is in line with the concept that these proteins may be involved in signaling the effector cells to adapt the bone structure to its mechanical demands.

The catabolic effects of microgravity on mineral metabolism in bone organ cultures might be explained as resulting from an exceptional form of disuse. It is possible that the mechanosensitivity of bone cells is altered under near weightlessness conditions, which likely contributes to disturbed bone metabolism observed in astronauts. In the experiment “FLOW”, we tested whether the production of early signaling molecules that are involved in the mechanical load-induced osteogenic response by bone cells is changed under microgravity conditions. FLOW was one of the Biological experiment entries to the Dutch Soyuz Mission “DELTA” (Dutch Expedition for Life Science, Technology and Atmospheric Research). FLOW was flown by the Soyuz craft, launched on April 19, 2004, on its way to the International Space Station. Primary osteocytes, osteoblasts, and periosteal fibroblasts were incubated in plunger boxes, developed by Centre for Concepts in Mechatronics, using plunger activation events for single pulse fluid shear stress stimulations. Due to unforeseen hardware complications, results from in-flight cultures are considered lost. Ground control experiments showed an accumulative increase of NO in medium for osteocytes (as well as for osteoblasts and periosteal fibroblasts). Data from the online-NO sensor showed that the NO produced in medium by osteocytes increased sharply after pulse shear stress stimulations. COX-2 mRNA expression revealed high levels in osteoblasts compared to the other cell types tested. In conclusion, preparations for the FLOW experiment and preliminary ground results indicate that the FLOW setup is viable for a future flight opportunity.

STUDY DESIGN: A study of lumbar interbody fusion using polylactic acid-based bioresorbable fusion cages in a goat model.OBJECTIVE: To evaluate the effect of polylactic acid polymer composition, and internal stabilization on the rate and quality of interbody fusion.SUMMARY OF BACKGROUND DATA: A spinal cage should provide an appropriate biomechanical environment to facilitate interbody fusion. Previous studies have shown that bioresorbable polylactic acid-based cages can provide adequate stability for spinal fusion. However, at present and to our knowledge, the best bioresorbable materials, optimal cage stiffness, and desired period over which the cage should biodegrade are unknown.METHODS: Interbody fusions were performed at L3-L4 level in 35 skeletally mature Dutch milk goats. Titanium and poly-L-lactide-CO-D,L-lactide (PLDLLA) cages were implanted at random as stand-alone cages. In addition, PLDLLA cages were implanted with anterior fixation. The goats were euthanized at 3, 6, or 12 months. Radiographic, magnetic resonance imaging, histologic, and histomorphometric analyses were performed on retrieved segments. Chemical analysis was used to assess degradation of the retrieved PLDLLA cages. Beforehand, chemical and mechanical degradation of the PLDLLA cages were assessed in vitro.RESULTS: At 3 months, bone graft was almost completely remodeled. Endochondral bone formation was observed in all specimens. At 6 months, 50% of the PLDLLA stand-alone cages and 83% of the PLDLLA anterior fixation cages were fused. At 12 months, 38% of the PLDLLA stand-alone and 83% of the titanium cages realized fusion. A very mild and dispersed foreign body reaction was seen in all PLDLLA specimens. E-beam sterilized PLDLLA cages degraded more rapidly in vivo as compared to both, PLDLLA cages in vitro, and ethylene oxide sterilized poly-L-lactic acid cages in vivo.CONCLUSIONS: Within the 3-6-month period, PLDLLA stand-alone cages provided insufficient mechanical stability, which manifested as cracking and deformation of the cages and lower fusion rates. This result implies that within this time, additional stabilization is required; supplemental internal fixation proved sufficient to obtain successful fusion. In all cases, only a mild host response was seen, indicating good biocompatibility.

Bone resorption by osteoclasts depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Next to these enzymes, tartrate-resistant acid phosphatase (TRAP) is considered to participate in this process. TRAP is synthesized as an inactive proenzyme, and in vitro studies have shown its activation by cysteine proteinases. In the present study, the possible involvement of the latter enzyme class in the in vivo modulation of TRAP was investigated using mice deficient for cathepsin K and/or L and in bones that express a high (long bone) or low (calvaria) level of cysteine proteinase activity. The results demonstrated, in mice lacking cathepsin K but not in those deficient for cathepsin L, significantly higher levels of TRAP activity in long bone. This higher activity was due to a higher number of osteoclasts. Next, we found considerable differences in TRAP activity between calvarial and long bones. Calvarial bones contained a 25-fold higher level of activity than long bones. This difference was seen in all mice, irrespective of genotype. Osteoclasts isolated from the two types of bone revealed that calvarial osteoclasts expressed higher enzyme activity as well as a higher level of mRNA for the enzyme. Analysis of TRAP-deficient mice revealed higher levels of nondigested bone matrix components in and around calvarial osteoclasts than in long bone osteoclasts. Finally, inhibition of cysteine proteinase activity by specific inhibitors resulted in increased TRAP activity. Our data suggest that neither cathepsin K nor L is essential in activating TRAP. The findings also point to functional differences between osteoclasts from different bone sites in terms of participation of TRAP in degradation of bone matrix. We propose that the higher level of TRAP activity in calvarial osteoclasts compared to that in long bone cells may partially compensate for the lower cysteine proteinase activity found in calvarial osteoclasts and TRAP may contribute to the degradation of noncollagenous proteins during the digestion of this type of bone.

Bone morphogenetic proteins (BMPs) initiate, promote, and maintain chondrogenesis and osteogenesis. We hypothesize that BMP-2 induces an osteogenic, and BMP-7 a chondrogenic phenotype in adipose tissue-derived mesenchymal stem cells (AT-MSCs). We compared the effects of a short 15min BMP-2 or BMP-7 (10ng/ml) treatment on osteogenic and chondrogenic differentiation of AT-MSCs. Gene expression was studied 4 and 14 days after BMP-treatment. At day 4 BMP-2, but not BMP-7, stimulated runx-2 and osteopontin gene expression, and at day 14 BMP-7 down-regulated expression of these genes. At day 4 BMP-2 and BMP-7 stimulated biglycan gene expression, which was down-regulated by BMP-7 at day 14. BMP-7 stimulated aggrecan gene expression at day 14. Our data indicate that BMP-2 treatment for 15min induces osteogenic differentiation, whereas BMP-7 stimulates a chondrogenic phenotype of AT-MSCs. Therefore, AT-MSCs triggered for only 15min with BMP-2 or BMP-7 provide a feasible tool for bone and cartilage tissue engineering.

Periodontitis is a chronic destructive infection of the tooth-supportive tissues, which is caused by pathogenic bacteria such asActinobacillus actinomycetemcomitans. A severe form of periodontitis is found in Papillon-Lefèvre syndrome (PLS), an inheritable disease caused by loss-of-function mutations in the cathepsin C gene. Recently, we demonstrated that these patients lack the activity of the polymorphonuclear leukocyte (PMN)-derived serine proteinases elastase, cathepsin G, and proteinase 3. In the present study we identified possible pathways along which serine proteinases may be involved in the defense againstA. actinomycetemcomitans. Serine proteinases are capable to convert the PMN-derived hCAP-18 into LL-37, an antimicrobial peptide with activity againstA. actinomycetemcomitans. We found that the PMNs of PLS patients released lower levels of LL-37. Furthermore, because of their deficiency in serine proteases, the PMNs of PLS patients were incapable of neutralizing the leukotoxin produced by this pathogen, which resulted in increased cell damage. Finally, the capacity of PMNs from PLS patients to killA. actinomycetemcomitansin an anaerobic environment, such as that found in the periodontal pocket, seemed to be reduced. Our report demonstrates a mechanism that suggests a direct link between an inheritable defect in PMN functioning and difficulty in coping with a periodontitis-associated pathogen.

We examined the effect of distraction rate on blood vessel growth in intramembraneous ossification after vertical distraction osteogenesis in the human mandible. Six edentulous patients (aged 60+/-9 years) with a severely atrophic mandible underwent bone augmentation with distraction osteogenesis. Two distraction rates (0.5 and 1 mm/day) were compared and for each group three patients were analyzed. Vascular histomorphometry was carried out in two different areas in the distraction gap: (1) in the first and (2) in the second 1 mm area from the osteotomy line, representing the oldest and younger new-bone area, respectively. Correlation analysis was performed between blood vessel parameters and the amount of new bone formed during distraction. Histological analysis demonstrated the presence of blood vessels throughout the soft connective tissue in the distraction gap. The volume density of blood vessels between the two investigated areas was significantly lower in the 1 mm/day groups, suggesting a delay in angiogenesis in this group of patients. A positive correlation between blood vessel volume and bone volume density was found in the younger new-bone area but not in the oldest new-bone area. This correlation was due to a higher number of blood vessels rather than to a larger size of the blood vessels. Our data suggest that the lower blood vessel density found in the patients with 1 mm/day distraction rate may be related to disruption of angiogenesis in the soft connective tissue of the gap or to a less optimal mechanical stimulation of cells involved in angiogenesis. This probably results in the slower rate of osteogenesis seen at the 1 mm/day distraction rate compared with the 0.5 mm/day distraction rate. The data support the concept that a positive relationship exists between the density of blood vessels and the formation of bone. For distraction of the human mandible in elderly patients, a distraction rate of 0.5 mm/day seems beneficial.