Comparing "intra operative" tissue engineering strategies for the repair of craniofacial bone defects.

BACKGROUND: In craniofacial reconstruction, the gold standard procedure for bone regeneration is the autologous bone graft (BG). However, this procedure requiring bone harvesting is a source of morbidity. Bone substitutes, such as biphasic calcium phosphate (BCP), represent an interesting alternative but are not sufficient for bone healing in hypoplastic conditions. In such conditions, osteoprogenitors are essential to provide osteoinduction. Previous studies have shown that BCP associated with total bone marrow (TBM) provides same bone reconstruction as bone graft in a rat model of calvaria defect. Furthermore, adipose tissue stromal vascular fraction (SVF) seems to be another promising source of osteoprogenitor cells that can be used intra-operatively. This study aimed to combine, intra-operative BCP-based bone tissue engineering strategies with TBM or SVF from human sources. METHODS: 5 mm critical-size calvaria defects were performed in 18 nude rat. The defects were filled with intra-operative bone tissue engineering procedures: human BG, human TBM + BCP, human SVF + BCP and, rat TBM + BCP. Animals were sacrificed 8 weeks after implantation and calvaria were processed for histological and radiological examinations. Implanted cells were labelled with a fluorochrome. RESULTS: Micro-CT analysis revealed partial repair of bone defect. Only hBG significantly succeeded in healing the defect (43.1%). However, low rate of newly formed bone tissue was observed in all tissue engineering conditions (hTBM, hSVF, ratTBM). DISCUSSION: The lack of bone formation observed in this study could possibly be attributed to the model. CONCLUSION: This study combined with a literature analysis show the stringency of the nude rat calvaria model in term of bone regeneration.

Expression of Phosphate Transporters during Dental Mineralization.

The importance of phosphate (Pi) as an essential component of hydroxyapatite crystals suggests a key role for membrane proteins controlling Pi uptake during mineralization in the tooth. To clarify the involvement of the currently known Pi transporters (Slc17a1, Slc34a1, Slc34a2, Slc34a3, Slc20a1, Slc20a2, and Xpr1) during tooth development and mineralization, we determined their spatiotemporal expression in murine tooth germs from embryonic day 14.5 to postnatal day 15 and in human dental samples from Nolla stages 6 to 9. Using real-time polymerase chain reaction, in situ hybridization, immunohistochemistry, and X-gal staining, we showed that the expression of Slc17a1, Slc34a1, and Slc34a3 in tooth germs from C57BL/6 mice were very low. In contrast, Slc34a2, Slc20a1, Slc20a2, and Xpr1 were highly expressed, mostly during the postnatal stages. The expression of Slc20a2 was 2- to 10-fold higher than the other transporters. Comparable results were obtained in human tooth germs. In mice, Slc34a2 and Slc20a1 were predominantly expressed in ameloblasts but not odontoblasts, while Slc20a2 was detected neither in ameloblasts nor in odontoblasts. Rather, Slc20a2 was highly expressed in the stratum intermedium and the subodontoblastic cell layer. Although Slc20a2 knockout mice did not show enamel defects, mutant mice showed a disrupted dentin mineralization, displaying unmerged calcospherites at the mineralization front. This latter phenotypical finding raises the possibility that Slc20a2 may play an indirect role in regulating the extracellular Pi availability for mineralizing cells rather than a direct role in mediating Pi transport through mineralizing plasma cell membranes. By documenting the spatiotemporal expression of Pi transporters in the tooth, our data support the possibility that the currently known Pi transporters may be dispensable for the initiation of dental mineralization and may rather be involved later during the tooth mineralization scheme.

Habitat-related variation in the plasticity of a UV-sensitive photoreceptor over a small spatial scale in the palmate newt.

Plastic phenotypes are expected to be favoured in heterogeneous environments compared with stable environments. Sensory systems are interesting to test this theory because they are costly to produce and support, and strong fitness costs are expected if they are not tuned to the local environment. Consistently, the visual system of several species changes with the conditions experienced during early development. However, there is little information on whether the amplitude of the change, that is the reaction norm, differs between visual environments. Given the rapid change of many ecosystems, especially eutrophication for aquatic habitats, it is crucial to determine down to which spatial scale, change in the reaction norm occurs. We addressed this issue by quantifying the between-habitat variation in the expression of a UV-sensitive opsin in a newt. In western France, this species breeds in ponds of small forest patches, where water filters out UV, and in agricultural ponds where UV transmission is variable. We raised larvae from both habitats with or without exposure to UV. Opsin expression was reduced in larvae from agricultural habitats when raised without UV, whereas it was low in larvae from forest ponds under all lighting conditions. Thus, the variation in the reaction norm of opsin expression was lower in stable filtering environments and higher in heterogeneous environments. Its variation occurred between habitats across a small spatial scale. We discuss the hypotheses for this pattern and for the maintenance of residual opsin expression in forest populations.

Enriching a cellulose hydrogel with a biologically active marine exopolysaccharide for cell-based cartilage engineering.

The development of biologically and mechanically competent hydrogels is a prerequisite in cartilage engineering. We recently demonstrated that a marine exopolysaccharide, GY785, stimulates the in vitro chondrogenesis of adipose stromal cells. In the present study, we thus hypothesized that enriching our silated hydroxypropyl methylcellulose hydrogel (Si-HPMC) with GY785 might offer new prospects in the development of scaffolds for cartilage regeneration. The interaction properties of GY785 with growth factors was tested by surface plasmon resonance (SPR). The biocompatibility of Si-HPMC/GY785 towards rabbit articular chondrocytes (RACs) and its ability to maintain and recover a chondrocytic phenotype were then evaluated in vitro by MTS assay, cell counting and qRT-PCR. Finally, we evaluated the potential of Si-HPMC/GY785 associated with RACs to form cartilaginous tissue in vivo by transplantation into the subcutis of nude mice for 3 weeks. Our SPR data indicated that GY785 was able to physically interact with BMP-2 and TGFß. Our analyses also showed that three-dimensionally (3D)-cultured RACs into Si-HPMC/GY785 strongly expressed type II collagen (COL2) and aggrecan transcripts when compared to Si-HPMC alone. In addition, RACs also produced large amounts of extracellular matrix (ECM) containing glycosaminoglycans (GAG) and COL2. When dedifferentiated RACs were replaced in 3D in Si-HPMC/GY785, the expressions of COL2 and aggrecan transcripts were recovered and that of type I collagen decreased. Immunohistological analyses of Si-HPMC/GY785 constructs transplanted into nude mice revealed the production of a cartilage-like extracellular matrix (ECM) containing high amounts of GAG and COL2. These results indicate that GY785-enriched Si-HPMC appears to be a promising hydrogel for cartilage tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.

Direct comparison of current cell-based and cell-free approaches towards the repair of craniofacial bone defects - A preclinical study.

For craniofacial bone defect repair, several alternatives to bone graft (BG) exist, including the combination of biphasic calcium phosphate (BCP) biomaterials with total bone marrow (TBM) and bone marrow-derived mesenchymal stromal cells (MSCs), or the use of growth factors like recombinant human bone morphogenic protein-2 (RhBMP-2) and various scaffolds. Therefore, clinicians might be unsure as to which approach will offer their patients the most benefit. Here, we aimed to compare different clinically relevant bone tissue engineering methods in an "all-in-one" study in rat calvarial defects. TBM, and MSCs committed or not, and cultured in two- or three-dimensions were mixed with BCP and implanted in bilateral parietal bone defects in rats. RhBMP-2 and BG were used as positive controls. After 7 weeks, significant de novo bone formation was observed in rhBMP-2 and BG groups, and in a lesser amount, when BCP biomaterials were mixed with TBM or committed MSCs cultured in three-dimensions. Due to the efficacy and safety of the TBM/BCP combination approach, we recommend this one-step procedure for further clinical investigation. STATEMENT OF SIGNIFICANCE: For craniofacial repair, total bone marrow (BM) and BM mesenchymal stem cell (MSC)-based regenerative medicine have shown to be promising in alternative to bone grafting (BG). Therefore, clinicians might be unsure as to which approach will offer the most benefit. Here, BM and MSCs committed or not were mixed with calcium phosphate ceramics (CaP) and implanted in bone defects in rats. RhBMP-2 and BG were used as positive controls. After 7 weeks, significant bone formation was observed in rhBMP-2 and BG groups, and when CaP were mixed with BM or committed MSCs. Since the BM-based procedure does not require bone harvest or cell culture, but provides de novo bone formation, we recommend consideration of this strategy for craniofacial applications.

Evaluation of new bone formation in irradiated areas using association of mesenchymal stem cells and total fresh bone marrow mixed with calcium phosphate scaffold.

The consequences of the treatment of the squamous cell carcinomas of the upper aerodigestive tract (bone removal and external radiation therapy) are constant. Tissue engineering using biphasic calcium phosphate (BCP) and mesenchymal stem cells (MSC) is considered as a promising alternative. We previously demonstrated the efficacy of BCP and total fresh bone marrow (TBM) in regenerating irradiated bone defect. The aim of this study was to know if adding MSC to BCP + TBM mixture could improve the bone formation in irradiated bone defects. Twenty-four Lewis 1A rats received a single dose of 20 Gy to the hind limbs. MSC were sampled from non-irradiated donors and amplified in proliferative, and a part in osteogenic, medium. 3 weeks after, defects were created on femurs and tibias, which were filled with BCP alone, BCP + TBM, BCP + TBM + uncommitted MSC, or BCP + TBM + committed MSC. 3 weeks after, samples were removed and prepared for qualitative and quantitative analysis. The rate of bone ingrowth was significantly higher after implantation of BCP + TBM mixture. The adding of a high concentration of MSC, committed or not, didn't improve the bone regeneration. The association BCP + TBM remains the most efficient material for bone substitution in irradiated areas.

Membrane-targeted HrpNEa can modulate apple defense gene expression.

Fire blight caused by Erwinia amylovora is the major bacterial disease of tribe Maleae, including apple. Among the proteins secreted by this bacterium, HrpNEa, also called harpin, is known to induce hypersensitive response in nonhost plants and to form amyloid oligomers leading to pore opening in the plasma membrane and alteration of membrane homeostasis. To better understand the physiological effects of HrpNEa in the host plant, we produced transgenic apple plants expressing HrpNEa with or without a secretion signal peptide (SP). HrpNEa expressed with a SP was found to be associated within the membrane fraction, in accordance with amyloidogenic properties and the presence of transmembrane domains revealed by in silico analysis. Expression analysis of 28 apple defense-related genes revealed gene modulations in the transgenic line expressing membrane-targeted HrpNEa. While apple transgenic trees displaying a high constitutive expression level of SP-HrpNEa showed a slight reduction of infection frequency after E. amylovora inoculation, there was no decrease in the disease severity. Thus HrpNEa seems to act as an elicitor of host defenses, when localized in the host membrane.

An in vitro study of two GAG-like marine polysaccharides incorporated into injectable hydrogels for bone and cartilage tissue engineering.

Natural polysaccharides are attractive compounds with which to build scaffolds for bone and cartilage tissue engineering. Here we tested two non-standard ones, HE800 and GY785, for the two-dimensional (2-D) and three-dimensional (3-D) culture of osteoblasts (MC3T3-E1) and chondrocytes (C28/I2). These two glycosaminoglycan-like marine exopolysaccharides were incorporated into an injectable silylated hydroxypropylmethylcellulose-based hydrogel (Si-HPMC) that has already shown its suitability for bone and cartilage tissue engineering. Results showed that, similarly to hyaluronic acid (HA) (the control), HE800 and GY785 significantly improved the mechanical properties of the Si-HPMC hydrogel and induced the attachment of MC3T3-E1 and C28/I2 cells when these were cultured on top of the scaffolds. Si-HPMC hydrogel containing 0.67% HE800 exhibited the highest compressive modulus (11kPa) and allowed the best cell dispersion, especially of MC3T3-E1 cells. However, these cells did not survive when cultured in 3-D within hydrogels containing HE800, in contrast to C28/I2 cells. The latter proliferated in the microenvironment or concentrically depending on the nature of the hydrogel. Among all the constructs tested the Si-HPMC hydrogels containing 0.34% HE800 or 0.67% GY785 or 0.67% HA presented the most interesting features for cartilage tissue engineering applications, since they offered the highest compressive modulus (9.5-11kPa) while supporting the proliferation of chondrocytes.

High-level resistance to oxidative stress in Lactococcus lactis conferred by Bacillus subtilis catalase KatE.

Lactococcus lactis, a lactic acid bacterium widely used for food fermentations, is often exposed to damaging stress conditions. In particular, oxidative stress leads to DNA, protein and membrane damages that can be lethal. As L. lactis has no catalase, the impact of production of the Bacillus subtilis haem catalase KatE on its oxidative stress resistance was tested. This cytoplasmic catalase was engineered for extracellular expression in L. lactis with an optimization strategy based on fusion to the nisin-inducible promoter and a lactococcal signal peptide (SP(Usp45)). The production of KatE by L. lactis conferred an 800-fold increase in survival after 1 h exposure to 4 mM hydrogen peroxide, and a 160-fold greater survival in long-term (3 days) survival of aerated cultures in a cydA mutant, which is unable to respire. The presence of KatE protected DNA from oxidative damage and limited its degradation after long-term aeration in a cydA/recA mutant, defective in DNA repair. L. lactis is thus able to produce active catalase that can provide efficient antioxidant activity.

Respiration capacity of the fermenting bacterium Lactococcus lactis and its positive effects on growth and survival.

Oxygen is a major determinant of both survival and mortality of aerobic organisms. For the facultative anaerobe Lactococcus lactis, oxygen has negative effects on both growth and survival. We show here that oxygen can be beneficial to L. lactis if heme is present during aerated growth. The growth period is extended and long-term survival is markedly improved compared to results obtained under the usual fermentation conditions. We considered that improved growth and survival could be due to the capacity of L. lactis to undergo respiration. To test this idea, we confirmed that the metabolic behavior of lactococci in the presence of oxygen and hemin is consistent with respiration and is most pronounced late in growth. We then used a genetic approach to show the following. (i) The cydA gene, encoding cytochrome d oxidase, is required for respiration and plays a direct role in oxygen utilization. cydA expression is induced late in growth under respiration conditions. (ii) The hemZ gene, encoding ferrochelatase, which converts protoporphyrin IX to heme, is needed for respiration if the precursor, rather than the final heme product, is present in the medium. Surprisingly, survival improved by respiration is observed in a superoxide dismutase-deficient strain, a result which emphasizes the physiological differences between fermenting and respiring lactococci. These studies confirm respiratory metabolism in L. lactis and suggest that this organism may be better adapted to respiration than to traditional fermentative metabolism.

The origin of the azygos venous system, as deduced from an anatomical and radiological study employing a corrosion technique.

The aim of this study is not only to describe the origin of the human azygos venous system by performing a 3-D reconstruction of a CT scan but also to evaluate the value of the techniques employed in investigating the topographical anatomy of a venous system in the body. Following perfusion with saline to wash away the blood, we injected an ALTUFIX/MINIUM mixture into the azygos vein of a cadaver. The head and trunk were subsequently corroded with hydrochloric acid (HCl). A CT scan of the trunk was obtained both before and after corrosion. According to the spatial resolution of the CT scan, the thinnest identifiable detail was measurable as 0.5 mm. The vertebral lumbar venous system was described, specifying the nomenclature of the lumbar veins (the lumbar veins being designated according to the vertebral body along which they run). On the right side, the lumbar veins at L2 and at L3 formed the lateral root of the azygos vein. On the left side, the vein at L2 formed the reno-azygo-lumbar arch (of Lejars). The lumbar veins, and the origin of the azygos system, were described and compared with previous studies. The 3-D reconstruction showed the importance of veins associated with the posterior paravertebral muscles. This description poses the problem of the metamerisation of the veins, but further evidence is required. Comparisons of the CT scans, 3-D reconstructions, and the ALTUFIX models of the veins obtained from the corrosion technique allowed verification of the 3-D reconstruction and correction of the errors inherent in a computer reconstruction. It is concluded that the description, and understanding, of such a complex system as the vertebral venous system is more valid when the results obtained using different techniques are compared.

Lactococcus lactis, a bacterial model for stress responses and survival.

The dairy organism, Lactococcus lactis, is continuously exposed to stress conditions generated during industrial processes. To identify the mechanisms that confer resistance to the lethal effects of oxygen and thermal stress, we isolated resistant strains by insertional mutagenesis. Mutated genes were identified and mutations were shown to confer resistance to multiple stresses (including non-selected stresses such as carbon starvation). Our results revealed that metabolic flux plays an important role in L. lactis stress response, and suggested that phosphate and guanine pools may be intracellular stress sensors. As previously shown, we also observed an increase of stress resistance during the stationary phase. We have evidence that stationary phase actually initiates very early during growth. Taken together, these data show that the stationary phase is a very complex system with multiple participants interacting altogether. These results reinforce the idea of the interdependence of stress response and the intimate relation between metabolic flux and stress responses in L. lactis.

[The lumbar vein at L2 and the reno-azygo-lumbar arch: anatomic and radiologic studies]. / La veine lombaire en L2 et l'arc réno-azygo-lombaire: étude anatomique et radiologique.

The lumbar vein at L2 was described by C. Gillot and B. Singer (1974). On the right side, after drawing off the 12th intercostal vein, it forms the lateral root of the azygos vein. Its way is as a frame, transverse going along the body of the 2nd lumbar vertebra, then upward along the spine after having integrated the veins of the L2-L3 intervertebral foramen. In its typical form, the vein is at L2 but it can be at L1 or L3. It takes the name of lateral root of the azygos vein only after receiving the 12th intercostal vein. Because of its diameter (5 mm), it forms a cavo-caval anastomosis via the azygos vein. The renal azygo-lumbar arch of Lejars is the equivalent on the left side of the right vein at L2. This arch contributes to the formation of the lateral root of the hemi-azygos vein. The right vein at L2 and the reno-azygo-lumbar arch were studied by dissections and by radiologic protocols. The radiologic studies (CT, MRI, 3D reconstructions) were carried out after injections of gelatin-gadolinium-minimum and altufix-minimum mixtures. The results showed the numerous variations of origin of the azygos system. The use of multiple and complementary technics are very helpful to describe these variations.

Identification of the Chi site of Haemophilus influenzae as several sequences related to the Escherichia coli Chi site.

The Escherichia coli Chi site 5'-GCTGGTGG-3' modulates the activity of the powerful dsDNA exonuclease and helicase RecBCD. Genome sequence analyses revealed that Chi is frequent on the chromosome and oriented with respect to replication on the E. coli genome. Chi is also present much more frequently than predicted statistically for a random 8-mer sequence. Although it is assumed that Chi is ubiquitous, there is virtually no proof that its features are conserved in other microorganisms. We therefore identified and analysed the Chi sequence of an organism for which the full genome sequence was available, Haemophilus influenzae. The biological test we used is based on our finding that rolling circle plasmids provide a specific substrate for RecBCD analogues in different microorganisms. Unexpectedly, several related sequences, corresponding to 5'-GNTGGTGG-3' and 5'-G(G/C)TGGAGG-3', showed Chi activity. As in E. coli, the H. influenzae Chi sites are frequent on the genome, which is in keeping with the need for frequent Chi sites for dsDNA break repair of chromosomal DNA. Although statistically over-represented, this feature is less marked than that of the E. coli Chi site. In contrast to E. coli, the H. influenzae Chi motifs are only slightly oriented with respect to the replication strand. Thus, although Chi appears to have a highly conserved biological role in attenuating exonuclease activity, its sequence characteristics and statistical representation on the genome may differ according to the particular features of the host.

recA gene involvement in oxidative and thermal stress in Lactococcus lactis.

The recA gene is best known for its effects on homologous recombination and DNA repair via SOS induction. There is gathering evidence that recA also affects expression of genes associated with different types of stress. We studied recA properties in Lactococcus lactis by generating a recA-disrupted mutant of MG1363 and comparing it with the wild type strain. recA appears to have an important role in cell survival upon oxygen or thermal stress, in addition to its conserved role in DNA repair. Oxygen toxicity appears to be due to the production of hydroxyl radicals via the Fenton reaction; recA would be involved in the repair of DNA damage generated by these radicals. Surprisingly, the recA strain stops growing at elevated temperature (37 degrees C). Immunological tests indicate that amounts of three heat shock proteins are reduced in the recA strain compared to the wild type strain. In contrast, the amount of heat shock regulator HflB is markedly increased, even at low temperature. HflB is known to degrade heat shock transcription factor sigma 32 in Escherichia coli. We propose that heat shock response is reduced in the recA mutant due to overproduction of HflB.