Bone Morphogenetic Protein-2 Hybridized with Nano-Anchored Oligonucleotides on Titanium Implants Enhances Osteogenic Differentiation In Vivo.

PURPOSE: Previous in vitro studies have shown that DNA oligonucleotides (ODN) can be successfully used as anchor strands for the binding and retarded release of biologically active recombinant human bone morphogenetic protein 2 (rhBMP-2). The aim of the present study was to test the hypothesis that rhBMP-2 bound to the surface of titanium implants through hybridization with nano-anchored ODN strands is biologically active and can enhance the induction of osteogenic markers in peri-implant bone in vivo. MATERIALS AND METHODS: Custom-made, surface acid-etched (SAE) titanium discs and implants were coated with ODN anchor strands and subsequently hybridized with complementary ODN strands conjugated to rhBMP-2 (AS_CS_BMP-2). Discs/implants with SAE surface, ODN-coated surface (AS), and ODN-coated surface with nonconjugated rhBMP-2 (AS_BMP-2) served as controls. Release of rhBMP-2 from the coated discs was evaluated in vitro using enzyme-linked immunosorbent assay (ELISA), and bone-specific activity was assessed through pNPP turnover by induced alkaline phosphatase (AP) up to a period of 56 days. In vivo expression of bone-specific markers was analyzed after bilateral placement of coated implants into the tibiae of 36 Wistar rats (72 tibiae total). Immunostaining for AP and runt-related transcription factor 2 (Runx2) was carried out after 1, 4, and 13 weeks. RESULTS: Release from the AS_CS_ BMP-2-coated titanium surfaces was significantly retarded compared to surfaces loaded with AS_BMP-2. The in vitro biologic activity of the released rhBMP-2 conjugates measured by AP induction was equivalent to released nonconjugated rhBMP-2. Immunostaining revealed a significant increase in the in vivo induction of AP around AS_CS_BMP-2 implants compared to the controls after 1 and 4 weeks. CONCLUSION: Titanium AS_CS_BMP-2 implants can significantly enhance osteogenic differentiation in vivo in peri-implant bone in early periods of osseointegration.

Application of Lateral and Distance Spacers in an Oligonucleotide Based Immobilization System for Bioactive Molecules onto Titanium Implants.

Immobilization of bioactive molecules (BAMs) on a nanometer scale is of great interest for functionalization of implant and scaffold surfaces in current biomaterials research. A system for immobilization of one or more compounds is described, which is based on nanomechanical fixation of single-stranded nucleic acids into an anodic titanium oxide layer and their subsequent hybridization with BAMs conjugated to the respective complementary strands. This paper focuses on further development and in depth understanding of the immobilization system, as some of the major findings established for common sensor applications for immobilization of single-stranded DNA onto gold surfaces cannot be transferred to the TiO2 surface. The first approach concerning the influence of the internal spacer sequence revealed the best performance for a polyadenine based sequence out of four homologous spacer sequences (A30, T30, C30, and G30). This overall best performance of the A30 spacer is attributed to an increased contour length by nucleotide staggering, which resulted in the best protection of the hybridization sequence from unfavorable interactions with the surface or damaging attacks by reactive oxygen species. The second approach comprises the implementation of a lateral spacer, also based on a homologous sequence of A30. Simultaneous as well as sequential adsorption of anchor strands and spacer strands were performed, and it could be shown that a preadsorption with high density of the spacer was most effective to increase hybridization efficiency.

Grafting of Neonatal marmoset monkey testicular single-cell suspensions into immunodeficient mice leads to ex situ testicular cord neomorphogenesis.

Single-cell suspensions derived from immature rodent and ungulate testes can reconstitute testicular cords upon grafting into immunodeficient mice. In the present study, neonatal common marmoset monkey (Callithrix jacchus) testes were digested to a single-cell suspension, which was transplanted subcutaneously into immunodeficient mice. After 9 or 18 weeks of incubation, the derivatives of the grafted single-cell suspensions were retrieved and analyzed histologically and immunohistochemically. Three of 4 (75%) neonatal grafts exhibited reconstituted seminiferous cords strongly resembling seminiferous cords of the intact neonatal testis. The cords consisted of Sertoli cells, germ cells and peritubular myoid cells, which was confirmed by immunohistochemical marker analysis. Three-dimensional reconstruction models of the grafts revealed elongated tubules. Some of the tubules were branched, which occurs also in vivo, as we show here for the marmoset monkey. Importantly, no teratoma formation by immature pluripotency factor-expressing germ cells was observed. In summary, the reconstituted testicular cords were almost indistinguishable from the cords formed in situ, thereby impressively demonstrating a very high reconstructive potential of a single-cell suspension obtained from the neonatal marmoset monkey testis. To our knowledge, this is the first study demonstrating testicular cord neomorphogenesis for a primate species ex situ.

Reactions of the melatonin metabolite N(1)-acetyl-5-methoxykynuramine with carbamoyl phosphate and related compounds.

N-[2-(6-methoxyquinazolin-4-yl)-ethyl] acetamide (MQA) is a compound formed from the melatonin metabolite N(1)-acetyl-5-methoxykynuramine (AMK). We followed MQA production in reaction systems containing various putative reaction partners, in the absence and presence of hydrogen peroxide and/or copper(II). Although MQA may be formally described as a condensation product of either N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) with ammonia, or AMK with formamide, none of these combinations led to substantial quantities of MQA. However, MQA formation was observed in mixtures containing AMK, hydrogen peroxide, hydrogen carbonate and ammonia, or AMK, hydrogen peroxide, copper(II) and potentially carbamoylating agents, such as potassium cyanate or, more efficiently, carbamoyl phosphate. In the presence of hydrogen peroxide, copper(II) and carbamoyl phosphate, MQA was the major product obtained from AMK, but the omission of copper(II) mainly led to another metabolite, 3-acetamidomethyl-6-methoxycinnolinone (AMMC). This was caused by nitric oxide (NO) generated under oxidative conditions from carbamoyl phosphate, as shown by an NO spin trap. MQA formation with carbamoyl phosphate was not due to the possible decomposition product, formamide. The reaction of AMK with carbamoyl phosphate under oxidative conditions, in which inorganic phosphate and water are released and which differs from the typical process of carbamoylation via isocyanate, may be considered as a new physiological route of MQA formation.