Les pertes de substance osseuses cranio-maxillo-faciales nécessitent une réhabilitation des fonctions manducatrices, l’esthétique et le psychisme. La greffe osseuse autologue (GOA) reste le « gold standard » mais peut-être associer à une morbidité du site donneur; d’où la nécessité d’une alternative. L’utilisation de produits d’ingénierie tissulaire osseuse (ITO) demeure un challenge pour cliniciens et les chercheurs. Leur succès dépend essentiellement de l’interaction entre le biomatériau support, les morphogènes utilisés. Les stratégies d’ITO,médiées par la BMP2, présentent des limites. Pour y remédier, différents systèmes de délivrance des protéines sont étudiés ou testés. Ce travail contribue au développement d’un substitut osseux auto-réticulant (SOAR), améliorant l’efficacité locale de BMP2 et la régénération de perte de substance osseuse. Pour ce faire, nous développons un biomatériau composite constitué de phosphate de calcium biphasé (BCP) et d’un hydrogel d’hydroxypropyle-méthylecellulose silanisé (HPMC-Si). L’hypothèse est que le relargage progressif de BMP2 induirait la régénération osseuse sans engendrer d’effets indésirables. Les résultats obtenus permettent de considérer ce biomatériau composite comme une alternative à la greffe.des investigations supplémentaires, pour une meilleure efficacité et sécurité, s’avèrent nécessaires en vue d’un transfert en clinique humaine. / Cranio-maxillofacial bone loss requires rehabilitation of the manducatory functions and aesthetics. Autologous bone graft (ABG) remains the gold standard but may be associated with donor site morbidity; hence the need for an alternative. The use of bone tissue engineering (BTE) products remains a challenge for clinicians and researchers.Their success depends essentially on the interaction between the biomaterial and the morphogens used. BTE strategies, mediated by BMP2, have limitations. To remedy this, different protein delivery systems are studied or tested. This work contributes to the development of a self-crosslinking bone substitute (SCBS), improving the local efficiency of BMP2 and the regeneration of bone loss. Therefor, we develop a composite biomaterial consisting of two phases, calcium phosphate granules (BCP) and a silanized hydroxypropyl-methylcellulose hydrogel (Si-HPMC). The hypothesis is that the gradual release of BMP2 induces bone regeneration without causing any undesirable effects. The results obtained make it possible to consider this composite biomaterial as a promising alternative to grafting. Additional investigations, for better efficiency and safety, are needed for a transfer to human clinic.

1 / Rongrong Zhang

Humans and mice lack the broad regenerative capacity of Urodele amphibians, capable only of partial regeneration of the terminal phalanx (P3), i.e., amputation mid-way through P3 results in essentially complete regeneration of the digit tip mediated via blastema formation and subsequent direct bone formation, culminating in distal bone growth, patterning, and function. Conversely, amputation injuries occurring proximal to the mid-point of P3 result in scar formation. Here, in part, we studied the endochondral bone healing response following amputation of the middle phalanx (P2). We showed the endochondral ossification healing response post amputation of P2 is analogous to the fracture healing response of P2 and other long bones of the body, ultimately proving useful in yielding insight into effectively inducing regeneration of the amputated digit. We showed the periosteal-derived chondrocytes of P2 play an integral role in the bone healing process in that they provide a template for subsequent bone formation following amputation injury. We also showed the periosteal-derived cells can be targeted through the temporal application of BMP2 to accumulate and proliferate at the distal digit tip and thus induce regeneration of the amputated bone. Our studies indicated that P2 amputation injuries of various time points, i.e. previously healed injuries, can be induced to regenerate via re-wounding of the periosteal tissue and subsequent BMP2 application, and thus is immeasurably promising from a translational therapeutic perspective. Lastly, we studied the fracture healing response in conjunction with the intramembranous regeneration response of P3. Following fracture of the digit, we showed the relative lack of periosteal callus formation, the lack of periosteal chondrogenesis, and a novel endosteal/marrow chondrogenic response. Unlike P2, the periosteal tissue of the fractured P3 bone does not respond to BMP2-treatment via endochondral bone growth, instead the bone heals via intramembranous ossification, possibly via intrinsic differentiation limitations and extrinsic factors. Notably, we showed that in the absence of the periosteal tissue of the amputated P3 bone, the regeneration response was greatly attenuated. Taken together, our work blending regeneration and fracture repair may prove useful in enhancing regeneration studies with methods and ideas not previously considered. / acase@tulane.edu

The formation of a continuous tubular network in the mammalian urinary system requires the interconnection of two epithelial populations with distinct cellular origins. The proximal component of the renal network is the nephron--a complex tubule responsible for much of the physiological action of the kidney. Nephrons connect to a collecting duct network to transport urine from the kidney to the bladder, via the ureter. I have used high-resolution image analysis of genetically labeled nephron and collecting duct networks together with apical and luminal markers to characterize the epithelial interconnection process in the developing kidney. Morphological protrusions at the distal end of the nephron precursor, adjacent to the tip of the collecting duct epithelium, precede luminal interconnection at the S-shaped body stage. Distal cells in the nephron precursor do not display clear epithelial junction complexes and show upregulation of phospho-myosin light chain, suggestive of a quasi-mesenchymal cell behavior. The close apposition of this group of cells with the collecting duct epithelium is facilitated by the absence on an intervening basal lamina. Live imaging of explanted kidneys suggests that distal cells break through into the lumen of the collecting duct epithelium and undergo cell death. No interconnection is observed upon Notch-mediated proximalization of distal cell fates. Furthermore, distal factor bone morphogenetic protein 2 (Bmp2) expression is lost in proximalized nephron precursor derivatives. Finally, I demonstrate that mice with specific loss of Bmp2 in nephron precursors and their derivatives results in a fraction of disconnected mature nephrons that later results in nephron atrophy and compromised renal function at juvenile stage compared to control mice. These data support a model in which the establishment of distal identity in nephron precursor cells closest to the nascent collecting duct epithelium leads to an active cell invasion that establishes a patent tubular interconnection between the nephron and collecting duct.

Diamond is a promising material with unique chemical properties. In this thesis, nano-scale diamond quantum size effects were investigated using several chemical property indicators. The results show that the chemical properties are strongly dependent on size for film thicknesses smaller than 1 nm (1D), and for nanodiamond particle diameters less than 2 nm (3D). When the sizes exceed these ranges there are no longer any quantum effects. The influence of surface termination coverage on the surface chemical properties has been calculated for the 2×1 reconstructed diamond (100) surface and for the diamond (111) surface. The terminating species included COOH and NH2 groups, which both are beneficial for the immobilization of biomolecules. The results of the calculations show that it is energetically possible to terminate the diamond surfaces up to 100% with NH2, while it is only possible to cover the surfaces up to 50% with COOH species. The reason for the latter result is most probably the larger sterical hindrance amongst the adsorbates. Both types of termination species were shown to influence the diamond surface electronic properties (e.g., HOMO/LUMO levels). In order to extend the diamond utility for biomedical applications, especially implant design, interactions of various growth factors with the diamond surfaces were also simulated. For non-solvent diamond-biomolecule systems, the results show that adhesion affinities are strongly dependent on biomolecule molecular weights. When including a water based solvent in the systems, the results show good physisorption affinities between proteins and diamond. Proteins structures, before and after physisorption, were visualized, and further investigated with respect to electrostatic properties and functional groups. By comparing the biomolecular structural changes during the adhesion processes, it can be concluded that both the general structures, as well as the binding pocket structures, were kept intact after the adhesion to the diamond surfaces (regardless of the adhesion affinities). In addition, the surface electronic potential distributions were maintained, which indicate preserved biomolecule functionalities. / Vascubone

ABSTRACT I CHARACTERIZATION OF A NOVEL NUCLEAR VARIANT OF BMP2Bone morphogenetic protein 2 (Bmp2) is a signaling protein that was first detected by its ability to induce cartilage and bone formation. It has since been implicated in broad variety of developmental, patterning, and disease processes. To date, Bmp2 has only been known to function as an extracellular signaling molecule. However, we have obtained clear evidence for a nuclear form of Bmp2. This nuclear variant, nBmp2, contains a bipartite NLS that overlaps the site of proteolytic cleavage. The NLS remains intact and functional when translation of Bmp2 initiates from a downstream alternative start codon. The resulting protein lacks the signal peptide and is therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing and secretion. Instead, the uncleaved protein containing the intact NLS is translocated to the nucleus. Preliminary functional analyses in zebrafish indicate that nBmp2 is critical for proper heart development. To determine if this function is conserved in mammals, we have also generated mice harboring a null allele for nBmp2. ABSTRACT II COORDINATE REGULATION OF COL11A2 AND COL27A1 BY THE TRANSCRIPTION FACTOR LC-MAF During skeletal development, long bones of the body develop from a cartilage template that is progressively replaced by bone. This process of endochondral ossification requires precisely coordinated expression of extracellular matrix proteins such as the cartilage-specific collagens. In this study, enhancer/reporter assays demonstrated that the transcription factor Lc-Maf inhibits the transcriptional activity of a cartilage-specific Col11a2 enhancer element while a cartilage-specific COL27A1 enhancer element was strongly activated by Lc-Maf. Site-directed mutagenesis identified the binding region within the COL27A1 enhancer, and it was found to be unlike any known consensus Maf family binding site. The in vivo significance of these results was examined using immunohistochemistry and in situ hybridization in mouse limbs undergoing endochondral ossification. Taken together, these results suggest that Lc-Maf participates in the developmental transition from proliferating to hypertrophic chondrocytes during endochondral ossification by coordinately downregulating Col11a2 and upregulating Col27a1 collagen gene expression.

Bone morphogenetic protein 2 (BMP2) is a secreted growth factor that is essential for proper embryonic development and proliferation. Our laboratory discovered a nuclear variant of BMP2 (nBMP2) which is produced when translation is initiated at an alternative start codon within the BMP2 gene. When translation occurs at the downstream start codon, the resulting protein lacks the ER signal peptide, thereby allowing cytoplasmic translation and nuclear localization. Our aim is to distinguish the role of this nuclear localized variant from secreted BMP2. Overexpression of nBMP2 in HEK293 and HT29 cell lines resulted in a higher percentage of cells in proliferative phases of the cell cycle. We determined that nBMP2 does not regulate cell cycle progression by inducing hyperphosphorylation of retinoblastoma protein (Rb), but it may regulate the cell cycle by interacting with ROC1. In order to examine the role of nBMP2 in vivo, we have generated a mouse model in which a mutation of the nuclear localization signal (NLS) disrupts nuclear localization of nBMP2. Aberrant crypts were more abundant in nBmp2NLStm azoxymethane (AOM) treated mice than in wild type mice. Furthermore, H&E staining of colonic tissue showed that mutant mice have increased levels of dysplasia and aberrant crypt foci. This work suggests that nBMP2 is involved in regulating cell cycle progression and proliferation, and therefore may play a role in tumorigenesis.

Nuclear bone morphogenetic protein 2 (nBMP2) is a nuclear variant of the secreted growth factor BMP2. Experiments in nBmp2NLStm mutant mice, which lack nBMP2 in the nucleus, have shown that nBMP2 affects intracellular calcium transport in skeletal muscle and hippocampal neurons. The objective of this study was to determine whether nBMP2 affects the immune system, since activation of lymphocytes and other immune cells depends on intracellular calcium transport. We found that spleens in nBmp2NLStm mutant mice were 24% smaller than in wild type mice. The white pulp of the spleen contains many immune cells, particularly B and T lymphocytes and reduced spleen size in the nBmp2NLStm mutant mice could be caused by a reduced number of lymphocytes migrating to the spleen. When mutants and wild types were challenged with an intravenous infection of 10^7 CFU of S. aureus, they showed similar immune responses. Samples of blood, liver, spleen, kidney and lymph nodes cultured three days after infection showed no difference in post infection bacterial load between mutant and wild type. Likewise, post-infection weight loss and percent survival were similar between mutant and wild type, suggesting that the innate immune response is functional in nBmp2NLStm mice. However, when mice were challenged with a secondary infection, immune response and spleen function were severely impaired. Mutant mice showed higher levels of bacteria remaining in the blood and had lower rate of survival to day 3 after secondary infection. In addition, CD4+ and CD8+ T-cell levels within mutant lymph nodes were significantly reduced, indicating that nBMP2 is involved in the secondary immune response.

No description available.

Das Bone Morphogenetic Protein 2 (BMP2) gehört zur TGF-beta Superfamilie und findet seinen Fokus in der osteogenen Aktivierung und in der Anwendung bei der Frakturheilung. Es wird angenommen, dass weitere, bisher unbekannte Verbindungen existieren, die die BMP2-Signalübertragung und die osteogene Aktivität verbessern und somit zu einer verbesserten klinischen Wirksamkeit von BMP2 führen. Für den Stickstoffoxid (NO)-Signalweg ist bereits bekannt, dass im endothelialen Kontext eine Verbindung zum BMP2-Signalweg existiert. Ziel dieser Arbeit war es daher, eine Verbindung zwischen dem NO- und BMP2-Signalweg bezüglich der Regulierung des BMP2-abhängigen Signalwegs und der Osteoinduktion aufzuzeigen. Dies erfolgte durch Anwendung von Inhibitoren (LNAME, ODQ und LY83583) und Aktivatoren (L-Arginin, Deta NONOate, SNAP und YC-1) des NO-Signalwegs, in Kombination mit BMP2. Eine mögliche Verbindung zwischen dem BMP2- und NO-Signalweg, über eine Protein Kinase A (PKA) Brücke, wurde durch die Anwendung des PKA Inhibitors H89 untersucht. Zusammenfassend zeigen diese Ergebnisse, dass der NO-Stoffwechselweg den BMP2-vermittelten Signalweg und die osteoinduktive Aktivität modulieren kann, wobei PKA beide Signalwege im Rahmen der BMP Signalübertragung verbindet, jedoch nicht zu einer BMP2-vermittelten Osteoinduktion führt. / Bone Morphogenetic Protein 2 (BMP2) is a TGF-beta superfamily member, with a major focus on osteogenic activity and application in fracture healing. In order to improve efficiency of BMP2 in the clinic, it is assumed that additional, yet unknown compounds can improve BMP2 signalling and osteogenic activity. The Nitric Oxide (NO) pathway has previously shown to be connected with the BMP2 pathway in an endothelial context. Therefore, it was the aim of this study to unravel connections between the NO and BMP2 pathway in regulating BMP2 mediated signalling and osteoinduction. This was carried out through the application of inhibitors (LNAME, ODQ and LY83583) and activators (L-Arginine, Deta NONOate, SNAP and YC-1) of the NO pathway in combination with BMP2. A proposed connection between BMP2 and NO pathways via a Protein Kinase A (PKA) bridge was investigated by application of H89 inhibitor. In summary, these results show that the NO pathway can modulate BMP2 mediated signalling and osteoinductive activity. The PKA bridge connects NO and BMP2 only for the process of BMP2 signalling, but not for BMP2 mediated osteoinduction.