Evaluation of osseointegration of titanium alloyed implants modified by plasma polymerization.

By means of plasma polymerization, positively charged, nanometre-thin coatings can be applied to implant surfaces. The aim of the present study was to quantify the adhesion of human bone cells in vitro and to evaluate the bone ongrowth in vivo, on titanium surfaces modified by plasma polymer coatings. Different implant surface configurations were examined: titanium alloy (Ti6Al4V) coated with plasma-polymerized allylamine (PPAAm) and plasma-polymerized ethylenediamine (PPEDA) versus uncoated. Shear stress on human osteoblast-like MG-63 cells was investigated in vitro using a spinning disc device. Furthermore, bone-to-implant contact (BIC) was evaluated in vivo. Custom-made conical titanium implants were inserted at the medial tibia of female Sprague-Dawley rats. After a follow-up of six weeks, the BIC was determined by means of histomorphometry. The quantification of cell adhesion showed a significantly higher shear stress for MG-63 cells on PPAAm and PPEDA compared to uncoated Ti6Al4V. Uncoated titanium alloyed implants showed the lowest BIC (40.4%). Implants with PPAAm coating revealed a clear but not significant increase of the BIC (58.5%) and implants with PPEDA a significantly increased BIC (63.7%). In conclusion, plasma polymer coatings demonstrate enhanced cell adhesion and bone ongrowth compared to uncoated titanium surfaces.

Ring-Opening Metathesis Polymerization-Based Synthesis of CaCO(3) Nanoparticle-Reinforced Polymeric Monoliths for Tissue Engineering.

Porous monolithic materials have been prepared via ring-opening metathesis polymerization from norborn-2-ene and a 7-oxanorborn-2-ene-based cross-linker in the presence of porogenic solvents (i.e., 2-propanol and toluene) and norborn-2-enephosphonate surface-modified CaCO(3) nanoparticles, using the 3(rd) -generation Grubbs-initiator RuCl(2) (Py)(2) (IMesH(2) )(CHPh). The experimental setup and the conditions chosen allowed for the manufacturing of polymeric monoliths characterized by a homogeneous distribution of the inorganic nanoparticles throughout the polymeric monolith. Depending on the nanoparticle content, the macropore diameters could be varied in the 30-120 µm regime. Noteworthy, the addition of nanoparticles did not affect the phase separation-triggered formation of the monolithic matrix nor the meso- and microporosity as evidenced by N(2) -adsorption experiments.

ROMP-Derived cyclooctene-based monolithic polymeric materials reinforced with inorganic nanoparticles for applications in tissue engineering.

Porous monolithic inorganic/polymeric hybrid materials have been prepared via ring-opening metathesis copolymerization starting from a highly polar monomer, i.e., cis-5-cyclooctene-trans-1,2-diol and a 7-oxanorborn-2-ene-derived cross-linker in the presence of porogenic solvents and two types of inorganic nanoparticles (i.e., CaCO3 and calcium hydroxyapatite, respectively) using the third-generation Grubbs initiator RuCl2(Py)2(IMesH2)(CHPh). The physico-chemical properties of the monolithic materials, such as pore size distribution and microhardness were studied with regard to the nanoparticle type and content. Moreover, the reinforced monoliths were tested for the possible use as scaffold materials in tissue engineering, by carrying out cell cultivation experiments with human adipose tissue-derived stromal cells.

Osteogenic capacity of nanocrystalline bone cement in a weight-bearing defect at the ovine tibial metaphysis.

The synthetic material Nanobone(®) (hydroxyapatite nanocrystallines embedded in a porous silica gel matrix) was examined in vivo using a standardized bone defect model in the ovine tibial metaphysis. A standardized 6 × 12 × 24-mm bone defect was created below the articular surface of the medial tibia condyles on both hind legs of 18 adult sheep. The defect on the right side was filled with Nanobone(®), while the defect on the contralateral side was left empty. The tibial heads of six sheep were analyzed after 6, 12, and 26 weeks each. The histological and radiological analysis of the defect on the control side did not reveal any bone formation after the total of 26 weeks. In contrast, the microcomputed tomography analysis of the defect filled with Nanobone(®) showed a 55%, 72%, and 74% volume fraction of structures with bone density after 6, 12, and 26 weeks, respectively. Quantitative histomorphological analysis after 6, and 12 weeks revealed an osteoneogenesis of 22%, and 36%, respectively. Hematoxylin and eosin sections demonstrated multinucleated giant cells on the surface of the biomaterial and resorption lacunae, indicating osteoclastic resorptive activity. Nanobone(®) appears to be a highly potent bone substitute material with osteoconductive properties in a loaded large animal defect model, supporting the potential use of Nanobone(®) also in humans.

Lateral augmentation of the mandible in minipigs with a synthetic nanostructured hydroxyapatite block.

The purpose of this study was to evaluate biomaterial degradation and new bone formation after implantation of a nanostructured hydroxyapatite (HA) grafting block. Furthermore, physical characteristics of the biomaterial were measured. The biomaterial consists of nanostructured HA embedded in a porous matrix of silica (SiO(2) ) gel. The blocks with two different contents of silica (group A: 24 wt % and group B: 39 wt %) were fixed with titanium screws at the lateral aspect of the mandible of minipigs (n = 5). The specific surface areas of both blocks were measured using Brunauer-Emmett-Teller (BET) equation and mercury intrusion. In all animals, the wound healing was uneventful. After 5 weeks, the biomaterial percentage was 51.5% ± 12.1% for group A and 33.2% ± 5.9% for group B (p = 0.017). New bone formation accounted to 7.6% ± 6.0% for group A and 15.3% ± 8.3% for group B (p = 0.126) after 5 weeks. After 10 weeks, further resorption of the biomaterial led to percentages of 30.6% ± 10.0% for group A and 12.1% ± 6.7% for group B (p = 0.000). After 10 weeks, new bone formations were measured to be 34.1% ± 10.8% in group A and 39.9% ± 13.5% in group B (p = 0.383). The rate of degradation of the biomaterial is controlled by the composition of the material. A higher content of silica gel matrix leads to faster degradation of the biomaterial. The formation of new bone failed to show a significant difference between both groups.

A preliminary study in osteoinduction by a nano-crystalline hydroxyapatite in the mini pig.

To test the probable osteoinductive properties of NanoBone, a new highly non-sintered porous nano-crystalline hydroxylapatite bone substitute embedded into a silica gel matrix, granules were implanted subcutaneously and intramuscularly into the back region of 18 mini pigs. After periods of 5 and 10 weeks as well as 4 and 8 months, implantation sites were investigated using histological and histomorphometric procedures. Signs of early osteogenesis could already be detected after 5 weeks. The later periods were characterized by increasing membranous osteogenesis in and around the granules leading to the formation of bone-like structures showing periosteal and tendon-like structures with bone marrow and focal chondrogenesis. Bone formation was better in the subcutaneous than in the intramuscular implantation sites. This ectopic osteogenesis is discussed with regard to the nanoporosity and microporosity of the material, physico-chemical interactions at its surface, the differentiation of osteoblasts, the role of angiogenesis and the probable involvement of growth factors. The results of this preliminary study indicate that this biomaterial has osteoinductive potential and induces the formation of bone structures, mainly in subcutaneous adipose tissue in the pig.

Early matrix change of a nanostructured bone grafting substitute in the rat.

A nanocrystalline bone substitute embedded in a highly porous silica gel matrix (NanoBone) has previously been shown to bridge bone defects by an organic matrix. As the initial host response on the bone graft substitute might be a determinant for subsequent bone formation, our present purpose was to characterize the early tissue reaction on this biomaterial. After implantation of 80 mg of NanoBone into the adipose neck tissue of a total of 35 rats, grafts were harvested for subsequent analysis at days 3, 6, 9, 12, and 21. The biomaterial was found encapsulated by granulation tissue which partly penetrated the implant at day 3 and completely pervaded the graft at day 12 on implantation. Histology revealed tartrate-resistant acid phosphatase (TRAP)-positive giant cells covering the biomaterial. ED1 (CD68) immunopositivity of these cells further indicated their osteoclast-like phenotype. Scanning electron microscopy revealed organic tissue components within the periphery of the graft already at day 9, whereas the central hematoma region still presented the silica-surface of the biomaterial. Energy dispersive X-ray spectroscopy further demonstrated that the silica gel was degraded faster in the peripheral granulation tissue than in the central hematoma and was replaced by organic host components by day 12. In conclusion, the silica gel matrix is rapidly replaced by carbohydrate macromolecules. This might represent a key step in the process of graft degradation on its way toward induction of bone formation. The unique composition and structure of this nanoscaled biomaterial seem to support its degradation by host osteoclast-like giant cells.

Macroscopical, histological, and morphometric studies of porous bone-replacement materials in minipigs 8 months after implantation.

OBJECTIVE: The aim of this investigation was to test the induction of bone formation and biodegradation of different biomaterials based on calcium phosphate (CaP). Up to now, hydroxyapatite and beta-tricalcium phosphate ceramics have routinely been sintered at temperatures of 1300 degrees C. The new CaP biomaterials tested are fabricated by a sol-gel process at only 700 degrees C. STUDY DESIGN: Critical-size defects (>5 cm(3)) in the mandible of 15 adult Goettingen minipigs were filled with 1 of the 2 new types of CaP biomaterials, or with 1 of 2 well-known old-type ceramics, or with a gelatin sponge (in the control group). Macroscopical, histological, and morphometric examination of the former defect areas were made 8 months postoperatively. RESULTS: Eight months after implantation of the new CaP biomaterials, complete bone formation was observed in the defect area, and at the same time, the foreign material was resorbed almost completely. After implantation of the classical types of ceramics, only incomplete bone formation and a lesser resorption rate of the foreign bodies were noted. The difference in the bone formation rate was significant: more than 93% for the new CaP biomaterials versus less than 58% for the classical types of ceramics (P < 0.01). CONCLUSION: The biological behavior of the new CaP biomaterials was better than that of the old-type sintered ceramic bone-grafting materials. These new CaP matrices are suitable for filling bone defects and are of interest for dentists, including implantologists, craniomaxillofacial and orthopedic surgeons, as well as traumatologists.

Expression of the bovine aromatase cytochrome P450 gene (Cyp19) is primarily regulated by promoter 2 in bovine follicles and by promoter 1.1 in corpora lutea.

Follicular development and differentiation are closely associated with increasing steroidogenesis. During the present study transcript concentration of Cyp19, Cyp11A1, and 3beta-hydroxysteroid dehydrogenase delta (3beta-HSD) encoding the steroidogenic enzymes P450(arom), P450(SCC), and 3beta-HSD were determined by real-time PCR in bovine granulosa cells (GC) as potential markers for follicular differentiation. Ovaries were collected from a local abattoir (experiment 1) and from synchronized animals at day 4 of estrus cycle (experiment 2). To study effects of luteinization, steroidogenic transcripts were also quantified in corpora lutea (CL) 4 and 20 days after fertilization. In most follicles, all three steroidogenic transcripts were detected, however, at very different concentration. Expression of 3beta-HSD and Cyp11A1 was highly significantly co-regulated and showed a significant correlation with follicular size. Contrary, Cyp19 expression was extremely variable even in follicles of similar size. Cyp19 transcripts were derived predominantly from promoter P2 and less abundant from promoters P1.1 and P1.5. After luteinization, the concentration of 3beta-HSD and Cyp11A1 transcripts increased (75-fold and fivefold, respectively) whereas the Cyp19 transcript level dropped (160-fold). Residual Cyp19 transcripts in CL were almost exclusively derived from P1.1. The data indicate that Cyp19 expression in GC is predominantly regulated by P2 and to a minor extend by P1.1, whereas P1.1 is almost exclusively responsible for residual Cyp19 expression in CL. Correlation analyses suggest that the expression of 3beta-HSD and Cyp11A1 primarily depend on the size of follicles whereas the concentration of P2 derived Cyp19 transcripts in GC is a marker for follicular differentiation towards selection and dominance.