The present study examined the impact of implant surface modifications on osseointegration in an osteoporotic rodent model. Sandblasted, acid-etched titanium implants were either used directly (control) or were further modified by surface conditioning with NaOH or by coating with one of the following active agents: collagen/chondroitin sulphate, simvastatin, or zoledronic acid. Control and modified implants were inserted into the proximal tibia of aged ovariectomised (OVX) osteoporotic rats (n = 32/group). In addition, aged oestrogen competent animals received either control or NaOH conditioned implants. Animals were sacrificed 2 and 4 weeks post-implantation. The excised tibiae were utilised for biomechanical and morphometric readouts (n = 8/group/readout). Biomechanical testing revealed at both time points dramatically reduced osseointegration in the tibia of oestrogen deprived osteoporotic animals compared to intact controls irrespective of NaOH exposure. Consistently, histomorphometric and microCT analyses demonstrated diminished bone-implant contact (BIC), periimplant bone area (BA), bone volume/tissue volume (BV/ TV) and bone-mineral density (BMD) in OVX animals. Surface coating with collagen/chondroitin sulphate had no detectable impact on osseointegration. Interestingly, statin coating resulted in a transient increase in BIC 2 weeks post-implantation; which, however, did not correspond to improvement of biomechanical readouts. Local exposure to zoledronic acid increased BIC, BA, BV/TV and BMD at 4 weeks. Yet this translated only into a non-significant improvement of biomechanical properties. In conclusion, this study presents a rodent model mimicking severely osteoporotic bone. Contrary to the other bioactive agents, locally released zoledronic acid had a positive impact on osseointegration albeit to a lesser extent than reported in less challenging models.

Carboxymethyl dextran (CMD) with varying degrees of substitution was prepared and used as biocompatible coating for magnetic iron oxide nanoparticles. An oligonucleotide (19-mer) was coupled to the CMD-coated particles as a model compound for DNA fragments. Transmission electron microscopy investigations on the cellular uptake of the particles by different tumor cell lines demonstrated that both the CMD-coated and the oligonucleotide-coupled particles are internalized by the cells and deposited in cellular endosomes. The nanoparticles prepared have potential applications in tumor diagnosis and therapy.

DNA polymerase ␣-primase consists of four subunits, p180, p68, p58, and p48, and comprises two essential enzymatic functions. To study the primase activity of the complex, we expressed cDNAs encoding for the human p58 and p48 subunits either as single proteins or together using Escherichia coli expression vectors. Coexpression of both primase subunits allowed the purification of a heterodimer in high yields that revealed stable primase activity. Purified recombinant p48 subunit showed enzyme activity, whereas purified p58 did not. In contrast to the heterodimer, the primase activity of p48 was unstable. The activity of p48 could be stabilized by the addition of the divalent cations Mg 2؉ and Mn 2؉ but not Zn 2؉ . On a poly(dC) template the primase activity was hardly influenced by the monovalent cation potassium. However, by using poly(dT) as a template the recombinant p48 activity was sensitive to salt, whereas recombinant p58-p48 and the bovine DNA polymerase ␣-primase purified from thymus were less sensitive to the addition of monovalent cations. A complex of bacterially expressed primase and baculovirus-expressed p180 and p68 was assembled in vitro and shown to support replication of simian virus 40 DNA in a cell-free system.

Osseointegration of dental implants can be promoted by implant-surface modifications using bisphosphonate coatings. In addition, it is of clinical interest to promote peri-implant bone formation and to restore bony structure in low bone-mass patients. The present study evaluated a combination of an anti-resorptive zoledronic acid (ZOL) implant-coating and a systemically applied sclerostin antibody, a known bone anabolic treatment principle, versus sole sclerostin antibody treatment or ZOL implant-coating in a rat osteoporosis model. Uncoated reference surface implants or ZOL-coated implants (n = 64/group) were inserted into the proximal tibia of aged osteoporotic rats three months following ovariectomy. 32 animals of each group received once weekly sclerostin antibody therapy. Osseointegration was assessed 2 or 4 weeks post-implantation by ex vivo µCT, histology and biomechanical testing. Overall implant survival rate was 97 %. Histomorphology revealed pronounced bone formation along the entire implant length of ZOL-coated implants. At 4 weeks following implant insertion, bone-implant contact, cancellous bone mineral density and bone volume/tissue volume were significantly increased for the combination of ZOL and sclerostin antibody as compared to sclerostin antibody or ZOL implant-coating alone. Removal torque was also significantly increased in the combination therapy group relative to animals receiving only sclerostin antibody therapy or ZOL-coated implants. In an osteoporotic rat model, the combination of anti-resorptive ZOL implant-coating and systemically applied sclerostin antibody led to significantly increased peri-implant bone formation. Therefore, the combination of ZOL and the osteoanabolic sclerostin antibody was more effective than either agent alone.

The human polyomavirus JC virus (JCV) establishes persistent infections in most individuals and is the etiologic agent of progressive multifocal leukoencephalopathy. In this report, we describe the establishment of a soluble cell-free system that is capable of replicating exogenous plasmid DNA containing the JCV origin of replication. Replication in this system is completely dependent on the addition of JCV large T antigen (TAg). To prepare JCV TAg for replication analysis, a recombinant baculovirus containing the JCV TAg-coding sequence was generated. TAg expressed in insect cells was purified by metal chelate chromatography. JCV TAg supported initiation of JCV DNA replication in the presence of DNA polymerase ␣-primase, replication protein A, and topoisomerase I in a dose-dependent manner and was also capable of supporting DNA replication in crude human cell extracts. Point mutation of TAg-binding site I strongly diminished TAg binding and concomitantly reduced JCV DNA replication in vivo and in vitro by approximately 50%. Point mutation of TAg-binding site II or deletion of the early palindrome completely abolished replication of JCV origincontaining plasmid DNA in vivo and in vitro, marking these sequences as essential components of the JCV core origin. A comparison of several TAgs showed that simian virus 40 TAg, but not mouse polyomavirus (PyV) TAg, supported replication of a plasmid containing a JCV origin. These findings provide evidence that replication in the cell-free system faithfully mimics JCV DNA replication in vivo. Therefore, it may be a useful tool for future analysis of interactions between JCV and its host cell.

DNA polymerase ␣-primase (pol-prim), a complex consisting of four subunits, is the major species-specific factor for mouse polyomavirus (PyV) and simian virus 40 (SV40) DNA replication. Although p48 is the most conserved subunit of pol-prim, it is required for in vitro PyV DNA replication but can inhibit cell-free SV40 DNA replication. Production of chimeric human-mouse p48 revealed that different regions of p48 are involved in supporting PyV DNA replication and inhibiting SV40 DNA replication. The N and C-terminal parts of p48 do not have species-specific functions in cell-free PyV DNA replication, but the central part (amino acids [aa] 129 to 320) controls PyV DNA replication in vitro. However, PyV T antigen physically binds to mouse, human, and chimeric pol-prim complexes independently, whether they support PyV DNA replication or not. In contrast to the PyV system, the inhibitory effects of mouse p48 on SV40 DNA replication are mediated by N-and C-terminal regions of p48. Thus, a chimeric p48 containing human aa 1 to 128, mouse aa 129 to 320, and human aa 321 to 418 is active in both PyV and SV40 DNA replication in vitro.Papovaviruses are small DNA tumor viruses (47). For their DNA replication these viruses contribute a viral origin of replication (ori) and the large tumor antigens (Tag) of mouse polyomavirus (PyV) and simian virus 40 (SV40) or the E1 and E2 proteins of papillomaviruses; all other replication factors are supplied by the host (6,42,47,49). Therefore, papovaviral DNA replication in vivo and in vitro has served as a model system to study virus-host interactions and the mechanisms of DNA replication in mammalian cells (37,47,49).These studies allowed the development of a model for eukaryotic DNA replication which relies on unwinding of doublestranded (ds) DNA and stepwise assembly of multiprotein complexes mediated by the viral initiator of DNA synthesis, large Tag. After Tag has recognized and formed a double hexamer at the replication origin, specific distortions of the dsDNA occur (10, 25). The following steps of DNA replication require the specific recruitment of host replication proteins, such as DNA polymerase ␣-primase (pol-prim), eukaryotic single-stranded (ss) DNA-binding protein, replication protein A (RPA), and topoisomerase I (topo I), to the origin of replication (7,11,12,14,26,27,39,40,41). The interaction of Tag with pol-prim stimulates ori binding by Tag (29). Melting and unwinding of ori dsDNA by Tag's helicase activity requires stabilization of the ssDNA by RPA. The melting of ori DNA by Tag also needs the relaxation of supercoiled DNA by topo I. The first RNA primer is then synthesized by the primase activity of pol-prim. The preinitiation and initiation steps of viral DNA synthesis at the origin of DNA replication require, in addition to the DNA-binding and enzymatic activities of the replication factors, specific physical contacts between these protein complexes (11,48,50,51). The newly synthesized RNA is elongated by the DNA polymerase activity of pol-prim. After a replica...

Although p48 is the most conserved subunit of mammalian DNA polymerase ␣-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein.Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.The small DNA tumor viruses simian virus 40 (SV40) and mouse polyomavirus (PyV) have provided excellent model systems to study the mechanisms and regulation of eukaryotic DNA replication (reviewed in references 7, 9, 55, and 67). Their DNA replication in vivo and in vitro largely depends on the replication apparatus of their hosts, as only one protein, namely, the multifunctional viral large T antigen (Tag), is supplied by the virus (20,26,49).The establishment of cell-free SV40 and PyV DNA replication systems was a major step towards the purification and characterization of essential host replication factors (67). These studies allowed the understanding of the molecular mechanisms involved in the assembly and progression of replication forks (7,28,67). Along with genetic and other biochemical approaches, these DNA replication systems provided insights into the processes of initiation and elongation (67). Interestingly, several parallels between eukaryotic and prokaryotic DNA duplication emerged which show that central processes of DNA metabolism are in part conserved in all kingdoms (33,60,61). The studies of the polyomaviral systems have allowed the development of a general model for eukaryotic DNA replication. In an initial step, Tag recognizes the replication origin (ori) and forms a double hexameric complex, which causes specific distortions of the double-stranded (ds) DNA (4,15,20,37,49). Subsequently, dsDNA is unwound, and multiprotein complexes are assembled by Tag and host replication proteins, such as eukaryotic single-stranded (ss) DNA-binding protein (replication protein A [RPA]), topoisomerase I (topo I), and DNA polymerase ␣-primase (polprim), are recruited (10,11,17,23,38,53,54). After binding and distortion of the dsDNA, the helicase function of Tag melts and unwinds the ds ori DNA (4, 59). For this function, Tag depends on RPA as a cofactor to stabilize the ssDNA resulting from the helicase activity of Tag (7,29,72). Th...

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing.