Influence of the Immune Microenvironment Provided by Implanted Biomaterials on the Biological Properties of Masquelet-Induced Membranes in Rats: Metakaolin as an Alternative Spacer.

Macrophages play a key role in the inflammatory phase of wound repair and foreign body reactions-two important processes in the Masquelet-induced membrane technique for extremity reconstruction. The macrophage response depends largely on the nature of the biomaterials implanted. However, little is known about the influence of the macrophage microenvironment on the osteogenic properties of the induced membrane or subsequent bone regeneration. We used metakaolin, an immunogenic material, as an alternative spacer to standard polymethylmethacrylate (PMMA) in a Masquelet model in rats. Four weeks after implantation, the PMMA- and metakaolin-induced membranes were harvested, and their osteogenic properties and macrophage microenvironments were investigated by histology, immunohistochemistry, mass spectroscopy and gene expression analysis. The metakaolin spacer induced membranes with higher levels of two potent pro-osteogenic factors, transforming growth factor-ß (TGF-ß) and bone morphogenic protein-2 (BMP-2). These alternative membranes thus had greater osteogenic activity, which was accompanied by a significant expansion of the total macrophage population, including both the M1-like and M2-like subtypes. Microcomputed tomographic analysis showed that metakaolin-induced membranes supported bone regeneration more effectively than PMMA-induced membranes through better callus properties (+58%), although this difference was not significant. This study provides the first evidence of the influence of the immune microenvironment on the osteogenic properties of the induced membranes.

Precision Phenotyping of Nectar-Related Traits Using X-ray Micro Computed Tomography.

Flower morphologies shape the accessibility to nectar and pollen, two major traits that determine plant-pollinator interactions and reproductive success. Melon is an economically important crop whose reproduction is completely pollinator-dependent and, as such, is a valuable model for studying crop-ecological functions. High-resolution imaging techniques, such as micro-computed tomography (micro-CT), have recently become popular for phenotyping in plant science. Here, we implemented micro-CT to study floral morphology and honey bees in the context of nectar-related traits without a sample preparation to improve the phenotyping precision and quality. We generated high-quality 3D models of melon male and female flowers and compared the geometric measures. Micro-CT allowed for a relatively easy and rapid generation of 3D volumetric data on nectar, nectary, flower, and honey bee body sizes. A comparative analysis of male and female flowers showed a strong positive correlation between the nectar gland volume and the volume of the secreted nectar. We modeled the nectar level inside the flower and reconstructed a 3D model of the accessibility by honey bees. By combining data on flower morphology, the honey bee size and nectar volume, this protocol can be used to assess the flower accessibility to pollinators in a high resolution, and can readily carry out genotypes comparative analysis to identify nectar-pollination-related traits.

Peripheral denervation participates in heterotopic ossification in a spinal cord injury model.

We previously reported the development of a new acquired neurogenic HO (NHO) mouse model, combining spinal cord transection (SCI) and chemical muscle injury. Pathological mechanisms responsible for ectopic osteogenesis after central neurological damage are still to be elucidated. In this study, we first hypothesized that peripheral nervous system (PNS) might convey pathological signals from injured spinal cord to muscles in NHO mouse model. Secondly, we sought to determine whether SCI could lead to intramuscular modifications of BMP2 signaling pathways. Twenty one C57Bl6 mice were included in this protocol. Bilateral cardiotoxin (CTX) injection in hamstring muscles was associated with a two-stage surgical procedure, combining thoracic SCI with unilateral peripheral denervation. Volumes of HO (Bone Volume, BV) were measured 28 days after surgery using micro-computed tomography imaging techniques and histological analyses were made to confirm intramuscular osteogenesis. Volume comparisons were conducted between right and left hind limb of each animal, using a Wilcoxon signed rank test. Quantitative polymerase chain reaction (qPCR) was performed to explore intra muscular expression of BMP2, Alk3 and Id1. Nineteen mice survive the complete SCI and peripheral denervation procedure. When CTX injections were done right after surgery (n = 7), bilateral HO were detected in all animals after 28 days. Micro-CT measurements showed significantly increased BV in denervated paws (1.47 mm3 +/- 0.5) compared to contralateral sides (0.56 mm3 +/-0.4), p = 0.03. When peripheral denervation and CTX injections were performed after sham SCI surgery (n = 6), bilateral HO were present in three mice at day 28. Quantitative PCR analyses showed no changes in intra muscular BMP2 expression after SCI as compared to control mice (shamSCI). Peripheral denervation can be reliably added to spinal cord transection in NHO mouse model. This new experimental design confirms that neuro inflammatory mechanisms induced by central or peripheral nervous system injury plays a key role in triggering ectopic osteogenesis.

Seasonal influence over serum and urine metabolic markers in submariners during prolonged patrols.

Within the framework of earlier publications, we have consistently dedicated our investigations to eliciting the effects of both seasonal vitamin D deficiency and submarine-induced hypercapnia on serum parameters for acid-base balance and bone metabolism in submariners over a 2-month winter (WP) or summer (SP) patrols. The latest findings reported herein, contribute further evidence with regard to overall physiological regulations in the same submariner populations that underwent past scrutiny. Hence, urine and blood samples were collected in WP and SP submariners at control prepatrol time as well as on submarine patrol days 20, 41, and 58. Several urine and serum metabolic markers were quantified, namely, deoxypyridinoline (DPD), lactate, albumin, creatinine, nonesterified fatty acids (NEFA), and ionized sodium (Na(+)) or potassium (K(+)), with a view to assessing bone, muscle, liver, or kidney metabolisms. We evidenced bone metabolism alteration (urine DPD, calcium, and phosphorus) previously recorded in submarine crewmembers under prolonged patrols. We also highlighted transitory modifications in liver metabolism (serum albumin) occurring within the first 20 days of submersion. We further evidenced changes in submariners' renal physiology (serum creatinine) throughout the entire patrol time span. Measurements of ionic homeostasis (serum Na(+) and K(+)) displayed potential seasonal impact over active ionic pumps in submariners. Finally, there is some evidence that submersion provides beneficial conditions prone to fend off seasonal lactic acidosis (serum lactate) detected in WP submariners.

In vivo hypobaric hypoxia performed during the remodeling process accelerates bone healing in mice.

We investigated the effects of respiratory hypobaric hypoxia on femoral bone-defect repair in mice because hypoxia is believed to influence both mesenchymal stromal cell (MSC) and hematopoietic stem cell mobilization, a process involved in the bone-healing mechanism. To mimic conditions of non-weight-bearing limb immobilization in patients suffering from bone trauma, our hypoxic mouse model was further subjected to hind-limb unloading. A hole was drilled in the right femur of adult male C57/BL6J mice. Four days after surgery, mice were subjected to hind-limb unloading for 1 week. Seven days after surgery, mice were either housed for 4 days in a hypobaric room (FiO2 at 10%) or kept under normoxic conditions. Unsuspended control mice were housed in either hypobaric or normoxic conditions. Animals were sacrificed on postsurgery day 11 to allow for collection of both contralateral and lesioned femurs, blood, and spleen. As assessed by microtomography, delayed hypoxia enhanced bone-healing efficiency by increasing the closing of the cortical defect and the newly synthesized bone volume in the cavity by +55% and +35%, respectively. Proteome analysis and histomorphometric data suggested that bone-repair improvement likely results from the acceleration of the natural bone-healing process rather than from extended mobilization of MSC-derived osteoprogenitors. Hind-limb unloading had hardly any effect beyond delayed hypoxia-enhanced bone-healing efficiency.

Effects of seasonal vitamin D deficiency and respiratory acidosis on bone metabolism markers in submarine crewmembers during prolonged patrols.

The aim of the study was to determine the seasonal influence of vitamin D status on bone metabolism in French submariners over a 2-mo patrol. Blood samples were collected as follows: prepatrol and patrol days 20, 41, and 58 on crewmembers from both a winter (WP; n = 20) and a summer patrol (SP; n = 20), respectively. Vitamin D status was evaluated for WP and SP. Moreover, extended parameters for acid-base balance (Pco(2), pH, and bicarbonate), bone metabolism (bone alkaline phosphatase and COOH-terminal telopeptide of type I collagen), and mineral homeostasis (parathyroid hormone, ionized calcium and phosphorus) were scrutinized. As expected, SP vitamin D status was higher than WP vitamin D status, regardless of the considered experimental time. A mild chronic respiratory acidosis (CRA) was identified in both SP and WP submariners, up to patrol day 41. Such an occurrence paired up with an altered bone remodeling coupling (decreased bone alkaline phosphatase-to-COOH-terminal telopeptide of type I collagen ratio). At the end of the patrol (day 58), a partial compensation of CRA episode, combined with a recovered normal bone remodeling coupling, was observed in SP, not, however, in WP submariners. The mild CRA episode displayed over the initial 41-day submersion period was mainly induced by a hypercapnia resulting from the submarine-enriched CO(2) level. The correlated impaired bone remodeling may imply a physiological attempt to compensate this acidosis via bone buffering. On patrol day 58, the discrepancy observed in terms of CRA compensation between SP and WP may result from the seasonal influence on vitamin D status.

Effects of high-phosphorus and/or low-calcium diets on bone tissue in trained male rats.

PURPOSE: The aim of the present study was to investigate if wheel running exercise could offset the detrimental influences of independent or combined high-phosphorus and low-calcium diets on bone tissue in rats. METHODS: Forty male dark Agouti rats were randomly assigned to eight groups of five animals. Four sedentary groups (SED) and four voluntary trained groups (TR) were fed over 6 wk of either a standard food or a modified diet, namely, high phosphorus (HP), low calcium (LCa), or high phosphorus combined with low calcium (HP/LCa). After sacrifice, blood samples were collected to determine parathyroid hormone, Ca(2+), and Pi levels. Both tibiae were removed for bone mass determination and extended histomorphometric analyses. RESULTS: In SED rats, all unbalanced diets induced a sizeable bone volume decrease, up to 56%. Interestingly, steady training partially compensates for this bone volume loss, regardless of the considered modified diets. At the cellular level, only independent LCa diet induced a 38% decrease in osteoblastic surface in both SED and TR rat groups, generating thereby a reduction in bone neosynthesis. In terms of osteoclastic surface, an increase in this parameter was evidenced only in HP diets (both HP and HP-LCa), implying heightened bone resorption. The major effects of unbalanced diets are mainly observed on bone tissue because serum parameters (parathyroid hormone, Ca(2+), and Pi levels) remained only slightly modified. CONCLUSIONS: Training induced a positive effect on unbalanced diet-altered bone tissue formation but remained inadequate to reach standard bone mass measured in SED rats fed with balanced food. Further, we suggest that the nature of the diet influences the balance between bone formation and resorption: LCa diet decreases bone formation, whereas HP and HP-LCa increase bone resorption.

Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications.

Mesenchymal stem cells (MSCs) are considered as emergent "universal" cells and various tissue repair programs using MSCs are in development. In vitro expansion of MSCs is conventionally achieved in medium containing fetal calf serum (FCS) and is increased by addition of growth factors. However, for widespread clinical applications, contact of MSCs with FCS must be minimized since it is a putative source of prion or virus transmission. Therefore, because platelets are a natural source of growth factors, we sought to investigate in vitro MSC expansion in response to platelet lysates (PL) obtained from platelet-rich plasma. Human MSCs were expanded in FCS (+/-bFGF)- or PL-supplemented medium through a process of subculture. We demonstrated that PL-containing medium is enriched by growth factors (platelet-derived growth factors (PDGFs), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-beta), insulin-like growth factor-1 (IGF-1) ...) and showed that PL is able to promote MSC expansion, to decrease the time required to reach confluence, and to increase CFU-F size, as compared to the FCS medium. Furthermore, we demonstrated that MSCs cultured in the presence of PL maintain their osteogenic, chondrogenic, and adipogenic differentiation properties and retain their immunosuppressive activity. Therefore, we propose that PL may be a powerful and safe substitute for FCS in development of tissue- and cellular-engineered products in clinical settings using MSCs.

The Arabidopsis nuclear DAL gene encodes a chloroplast protein which is required for the maturation of the plastid ribosomal RNAs and is essential for chloroplast differentiation.

Altered pigmentation is an easily scored and sensitive monitor of plastid function. We analyzed in detail a yellow colored transposon-tagged mutant (dal1-2) that is allelic to the dal mutant previously identified (Babiychuk et al., 1997). Mesophyll cells of mutant plants possess abnormal nucleoids and more but smaller plastids than wild type cells. Plastid development in dal1-2 is not altered in the dark but is arrested at the early steps of thylakoid assembly. The amino acid sequence of the protein deduced from our cDNA clone is 21 amino acids longer than the previously published DAL sequence (Babiychuk et al., 1997) and allowed us to show that DAL codes for a chloroplast protein. The dal1-2 mutation has a global negative effect on plastid RNA accumulation and on expression of nuclear encoded photosynthetic genes. We show that the plastid RNA polymerases, the nuclear-encoded NEP and the plastid-encoded PEP, are functional in the mutant. Precursor 16S and 23S rRNA species specifically accumulate at a high level in the mutant but the 5'-end and the long 3'-end trailer are not modified. We suggest that the dal mutation is involved in plastid rRNA processing and consequently in translation and early chloroplast differentiation.