Bone morphogenetic protein-2 delivered by hyaluronan-based hydrogel induces massive bone formation and healing of cranial defects in minipigs.

BACKGROUND: Reconstruction of large craniofacial bone defects is a challenge using bone transplants or alloplastic materials. The use of bone morphogenetic protein (BMP)-2 together with a suitable carrier is an attractive option that may facilitate new bone formation. The authors have developed a hydrogel that is formed in situ by the cross-linking of multifunctional hyaluronic acid and polyvinyl alcohol derivatives mixed with hydroxyapatite nanoparticles, in the presence of BMP-2. The aim of this study was to evaluate the suitability of the hydrogel as a carrier for BMP-2 in repairing critical size cranial defects in a minipig model. METHODS: Cranial defects (2 x 4 cm) were created in 14 minipigs. The experimental groups were as follows: group 1, craniotomy and application of 5 ml of hydrogel with 1.25 mg of BMP-2 (n = 6); group 2, craniotomy and application of 5 ml of hydrogel without BMP-2 (n = 6); and group 3, craniotomy with no further treatment (n = 2). RESULTS: After 3 months, computed tomographic and histologic examinations were performed. There was spontaneous ossification in the untreated group, but the healing was incomplete. The hydrogel alone demonstrated no further effects. The addition of 1.25 mg of BMP-2 to the hydrogel induced a greater than 100 percent increase in bone volume (p = 0.003) and complete healing of the defects. Histologic examination revealed compact lamellar bone in the BMP group without intertrabecular fibrous tissue, as was seen in the other groups. The hydrogel was resorbed completely within 3 months and, importantly, caused no inflammatory reaction. CONCLUSION: The injectable hydrogel may be favorable as a BMP-2 carrier for bone reconstruction.

RNA cleavage by 2,9-diamino-1,10-phenanthroline PNA conjugates.

We report on the synthesis of 2,9-diamino-1,10-phenanthroline PNA conjugates as well as on their action in cleavage of a target RNA. Synthesis of the PNA conjugates are performed on solid support and the phenanthroline derivative is conjugated either to the amino-end or to a centrally positioned diaminopropionic acid in the PNA via a urea linker. Cleavage of the target RNA is achieved and compared to cleavage with the corresponding 2,9-dimethyl-1,10-phenanthroline and glycine conjugates.

Investigation of potential RNA bulge stabilizing elements.

As a part of our interest in recognition and cleavage of RNA we carried out thermal melting studies with the aim of screening a number of simple oligonucleotide modifications for their potential as modifying elements for RNA bulge stabilizing oligonucleotides. A specific model system from our studies on oligonucleotide-based artificial nuclease (OBAN) systems was chosen and the bulge size was varied from one to five nucleotides. Introduction of single 2'-modified nucleoside moieties (2'-O-methyl, 2'-deoxy and 2'-deoxy-2'-amino) with different conformational preferences adjacent to the bulge revealed that a higher preference for the north conformers gave more stable bulges across the whole range of bulge sizes. Changing a bulge closing a G-U wobble base pair to a G-C pair resulted in the interesting observation that, although the fully complementary complex and small bulges were highly stabilized, there was little difference in the stability of the larger bulges. The wobble base pair even gave a slight stabilization of the 5 nt bulge system. Introduction of a uridine C-5 linker with a single ammonium group was clearly bulge stabilizing (DeltaT(m) + 4.6 to + 5.4 degrees C for the three most stabilized bulges), although with limited selectivity for different bulge sizes since the fully complementary duplex was also stabilized. Introduction of a naphthoyl group on a 2'-aminolinker mostly gave a destabilizing effect, while introduction of a 5-aminoneocuproine moiety on the same linker resulted in stabilization of all bulges, in particular those with two or four unpaired nucleotides (DeltaT(m) + 3.6 and + 2.9 degrees C respectively). The aromatic groups destabilize the fully complementary duplex, resulting in higher selectivity towards stabilization of bulges. A combination of the studied partial element should be suitable for future designs of modified oligonucleotides that, apart from standard base pairing, can also provide additional non-Watson-Crick recognition of RNA.

Synthesis of the DNA-[Ru(tpy)(dppz)(CH(3)CN)](2+) conjugates and their photo cross-linking studies with the complementary DNA strand.

We here report our studies on the conjugation of photoreactive Ru(2+) complex to oligonucleotides (ODNs), which give a stable duplex with the complementary target DNA strand. These functionalized DNA duplexes bearing photoreactive Ru(2+) complex can be specifically photolyzed to give the reactive aqua derivative, [Ru(tpy)(dppz)(H(2)O)](2+)-ODN (tpy = 2,2':6',2' '-terpyridine; dppz = dipyrido[3,2-a:2',3'-c]phenazine), in situ, which successfully cross-links to give photoproduct(s) in the duplex form with the target complementary DNA strand. Thus, the stable precursor of the aquaruthenium complex, the monofunctional polypyridyl ruthenium complex [Ru(tpy)(dppz)(CH(3)CN)](2+), has been site-specifically tethered to ODN, for the first time, by both solid-phase synthesis and postsynthetic modifications. (i) In the first approach, pure 3'-[Ru(tpy)(dppz)(CH(3)CN)](2+)-ODN conjugate has been obtained in 42% overall yield (from the monomer blocks) by the automated solid-phase synthesis on a support labeled with [Ru(tpy)(dppz)Cl](+) complex with subsequent liberation of the crude conjugate from the support under mild conditions and displacement of the Cl(-) ligand by acetonitrile in the coordination sphere of the Ru(2+) label. (ii) In the second approach, the single-modified (3'- or 5'- or middle-modified) or 3',5'-bis-modified Ru(2+)-ODN conjugates were prepared in 28-50% yield by an amide bond formation between an active ester of the metal complex and the ODNs conjugated with an amino linker. The pure conjugates were characterized unambiguously by ultraviolet-visible (UV-vis) absorption spectroscopy, enzymatic digestion followed by HPLC quantitation, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry (MALDI-TOF as well as by ESI). [Ru(tpy)(dppz)(CH(3)CN)](2+)-ODNs form highly stabilized ODN.DNA duplexes compared to the unlabeled counterpart (DeltaT(m) varies from 8.4 to 23.6 degrees C) as a result of intercalation of the dppz moiety; they undergo clean and selective photodissociation of the CH(3)CN ligand to give the corresponding aqua complex, [Ru(tpy)(dppz)(H(2)O)](2+)-ODNs (in the aqueous medium), which is evidenced from the change of their UV-vis absorption properties and the detection of the naked Ru(2+)-ODN ions generated in the course of the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis. Thus, when [Ru(tpy)(dppz)(CH(3)CN)](2+)-ODN conjugate was hybridized to the complementary guanine (G)-rich target strand (T), and photolyzed in a buffer (pH 6.8), the corresponding aqua complex formed in situ immediately reacted with the G residue of the opposite strand, giving the cross-linked product. The highest yield (34%) of the photo cross-linked product obtained was with the ODN carrying two reactive Ru(2+) centers at both 3'- and 5'-ends. For ODNs carrying only one Ru(2+) complex, the yield of the cross-linked adduct in the corresponding duplex is found to decrease in the following order: 3'-Ru(2+)-ODN (22%) > 5'-Ru(2+)-ODN (9%) > middle-Ru(2+)-ODN (7%). It was also found that the photo cross-coupling efficiency of the tethered Ru(2+) complex with the target T strand decreased as the stabilization of the resulting duplex increased: 3'-Ru(2+)-ODN (VI.T) (DeltaT(m)(b) = 7 degrees C) < 5'-Ru(2+)-ODN (V.T) (DeltaT(m)(b) = 16 degrees C) < middle-Ru(2+)-ODN (VII.T) (DeltaT(m)(b) = 24.3 degrees C, Table 2). This shows that, with the rigidly packed structure, as in the duplex with middle-Ru(2+)-ODN, the metal center flexibility is considerably reduced, and consequently the accessibility of target G residue by the aquaruthunium moiety becomes severely restricted, which results in a poor yield in the cross-coupling reaction. The cross-linked product was characterized by PAGE, followed by MALDI-TOF MS.