17ß-estradiol and progesterone effect on human papillomavirus 16 positive cells grown as spheroid co-cultures.

Human papillomavirus (HPV) is the key epidemiologic factor of cervical cancer, but additional cofactors are mandatory. Estrogen has been considered as one of those. Here, the aim was to study the effects of steroid hormones on HPV16 E6-E7, estradiol receptors ERα and ERß, and progesterone receptor (PR) in HPV16-positive cervical carcinoma cell lines SiHa and CaSki grown as epithelial and fibroblast spheroid co-cultures. The spheroid co-cultures were exposured to 17ß-estradiol or progesterone from day 7 onwards. mRNA levels of HPV16 E6-E7, ERα, ERß and PR normalized against GAPDH were analyzed with quantitative reverse transcription-qPCR (RT-qPCR). 17ß-estradiol and progesterone decreased HPV16 E6-E7 mRNA expression in CaSki and increased in SiHA co-cultures. In CaSki co-cultures, ERß expression was blocked after 17ß-estradiol exposure while in SiHa cells it slightly increased ERß expression. PR expression was seen only in CaSki spheroids and it vanished after exposure to steroid hormones. Fibroblasts expressed all three hormone receptors as monolayers but ERß expression decreased and ERα and PR vanished after co-culturing. Cell culturing platform changes both oncogene and hormone receptor expression in HPV16 positive cervical cancer cell lines. This needs to be considered when in vitro results are extrapolated to in vivo situations.

Enhanced osteogenicity of bioactive composites with biomimetic treatment.

PURPOSE: This study aimed to explore if initiation of biomimetic apatite nucleation can be used to enhance osteoblast response to biodegradable tissue regeneration composite membranes. MATERIALS AND METHODS: Bioactive thermoplastic composites consisting of poly(ε-caprolactone/DL-lactide) and bioactive glass (BAG) were prepared at different stages of biomimetic calcium phosphate deposition by immersion in simulated body fluid (SBF). The modulation of the BAG dissolution and the osteogenic response of rat mesenchymal stem cells (MSCs) were analyzed. RESULTS: SBF treatment resulted in a gradual calcium phosphate deposition on the composites and decreased BAG reactivity in the subsequent cell cultures. Untreated composites and composites covered by thick calcium phosphate layer (14 days in SBF) expedited MSC mineralization in comparison to neat polymers without BAG, whereas other osteogenic markers--alkaline phosphatase activity, bone sialoprotein, and osteocalcin expression--were initially decreased. In contrast, surfaces with only small calcium phosphate aggregates (five days in SBF) had similar early response than neat polymers but still demonstrated enhanced mineralization. CONCLUSION: A short biomimetic treatment enhances osteoblast response to bioactive composite membranes.

Ectopic bone formation in and soft-tissue response to P(CL/DLLA)/bioactive glass composite scaffolds.

OBJECTIVES: To characterize biological response to subcutaneously implanted macroporous poly(ε-caprolactone/D,L-lactide)-based scaffolds, and to evaluate the effect of bioactive glass (BAG) filler and osteogenic cells to the tissue response and ectopic bone formation. MATERIAL AND METHODS: In the first part of this study, six different scaffold types were screened in a rat subcutaneous implantation model. The polymer scaffolds with 70/30 caprolactone/lactide ratio and corresponding composites with < 45 µm BAG filler size were chosen for the further ectopic bone formation assay. The scaffolds were loaded with differentiating bone marrow stromal cells and implanted subcutaneously in syngeneic rats. RESULTS: With plain scaffolds, only mild foreign body reaction with no signs of gross inflammation was observed after 4 weeks of implantation. Furthermore, the scaffolds were fully invaded by well-vascularized soft connective tissue. Overall, all the tested scaffold types showed an appropriate host response. With cell-seeded scaffolds, several loci of immature mineralizing tissue and small amounts of mature bone were observed after 4 weeks. The incidence of mature bone formation was two and four in polymer scaffolds and composites, respectively (n = 8). After twelve weeks, mature bone was observed in only one polymer scaffold but in seven composites (n = 8). Excluding bone formation, the host response was considered similar to that with cell-free scaffolds. CONCLUSIONS: Plain scaffolds supported the ingrowth of well-vascularized fibroconnective tissue. Furthermore, cell seeded composites with BAG filler showed enhanced ectopic bone formation in comparison with corresponding neat polymer scaffolds.

Nanoporous TiO(2) thin film on titanium oral implants for enhanced human soft tissue adhesion: a light and electron microscopy study.

BACKGROUND: Previous experimental studies have demonstrated direct soft tissue attachment for nanoporous titanium dioxide (TiO(2) ) thin film on implants, while implants without TiO(2) thin film have not shown this capability. PURPOSE: The aims were to evaluate and compare TiO(2) surface-modified experimental microimplants with unmodified microimplants with respect to tissue interaction of the human oral mucosa evaluated by light microscopy on ground sections and semithin sections and transmission electron microscopy on ultrathin sections, and to characterize the inflammatory response and the level of the marginal bone resorption. MATERIALS AND METHODS: The study was a single-center, randomized, comparative, clinical investigation with intrasubject comparison of implants with and without TiO(2) thin film in 15 patients. RESULTS: Two comparator microimplants showed mild erythema and expulsion of fluids. The surrounding tissues around all test implants were clinically healthy. The oral mucosa in contact with the abutment part of the microimplant was 72% for the test implants and 48% for the comparator implants, a statistically significant difference (p =.0268). No statistically significant difference was found in other histological variables. The marginal bone loss in 14 weeks was 0.5 mm for the stable test (n = 11) and 1.7 mm for the stable comparator implants (n = 9; p = .0248). CONCLUSIONS: The nanoporous TiO(2) surface modification has potential clinical benefits because of increased adherence of soft tissue and possible reduced bone resorption.

Fate of bone marrow-derived stromal cells after intraperitoneal infusion or implantation into femoral bone defects in the host animal.

The fate of intraperitoneally injected or implanted male rat bone marrow-derived stromal cells inside female sibling host animals was traced using Y-chromosome-sensitive PCR. When injected intraperitoneally, Y-chromosome-positive cells were found in all studied organs: heart muscle, lung, thymus, liver, spleen, kidney, skin, and femoral bone marrow with a few exceptions regardless of whether they had gone through osteogenic differentiation or not. In the implant experiments, expanded donor cells were seeded on poly(lactide-co-glycolide) scaffolds and grown under three different conditions (no additives, in osteogenic media for one or two weeks) prior to implantation into corticomedullar femoral defects. Although the impact of osteogenic in vitro cell differentiation on cell migration was more obvious in the implantation experiments than in the intraperitoneal experiments, the donor cells stay alive when injected intraperitoneally or grown in an implant and migrate inside the host. However, when the implants contained bioactive glass, no signs of Y-chromosomal DNA were observed in all studied organs including the implants indicating that the cells had been eliminated.

Adhesion of respiratory-infection-associated microorganisms on degradable thermoplastic composites.

The purpose of this study was to evaluate bacterial adhesion and early colonization on a composite consisting of bioactive glass (BAG) particles and copolymer of epsilon-caprolactone/D,L-lactide. Materials were incubated with suspensions of both type strains and clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae, and Pseudomonas aeruginosa for 30 minutes (adhesion) and 4 hours (colonization). Clear differences exist in the microorganisms' ability to adhere on the experimental materials. However, the presence of BAG particles does not inhibit bacterial adhesion, but early colonization of the materials with P. aeruginosa was inhibited by the addition of 90-315 mum BAG particles.

Bioactive glass induced in vitro apatite formation on composite GBR membranes.

The aim of this study was to investigate in vitro bioactivity of different thermoplastic biodegradable barrier membranes. Three experimental GBR membranes were fabricated using Poly(epsilon-caprolactone-co-D: ,L-lactide) P(CL/DL-LA) and particulate bioactive glass S53P4 (BAG; granule size 90-315 microm): (A) composite membrane with 60-wt.% of BAG, (B) membrane coated with BAG; and (C) copolymer membrane without BAG. Membranes were immersed in simulated body fluid (SBF), and their surfaces were characterized with SEM, XRD and EDS after 6 and 12 h and after 1, 3, 5, 7, and 14 days. Calcium phosphate (Ca-P) surface formation was observed on both composite membranes (A and B) but not on the copolymer membrane without bioactive glass (C). The Ca-P precipitation appeared to be initiated on the bioactive glass followed by growth of the layer along the polymer surface. In 6-12 h ion dissolution of the bioactive glass led to formation of the silica rich layer on the surface of the exposed glass granules on composite membrane B whereas only small amounts of silica was observed on the polymer surface of the composite membrane A. At 24 h nucleation of Ca-P precipitation was observed, and by 3-5 days membrane surface was covered with a uniform Ca-P layer transforming from amorphous to low crystalline structure. At 7 days composition and structure of the apatite surface resembled the apatite in bone. Once nucleated, the surface topography seemed to have significant effect on the growth of the apatite layer.

Peri-implant tissue response to TiO2 surface modified implants.

OBJECTIVES: The objective of this study was to evaluate peri-implant soft tissue attachment and alveolar bone height on nanoporous TiO(2) thin film on commercial titanium dental implants compared with unmodified standard implants. MATERIAL AND METHODS: In six adult beagle dogs, the mandibular premolars P2-P4 were extracted bilaterally. Sol-gel-derived nanoporous TiO(2) thin film was produced on smooth coronal part of standard ITI Straumann implants (4.1 mm x 8.0 mm) by dip coating method. After 3 months healing period of the extraction sockets modified (n=24) and unmodified (n=11) control implants were placed bilaterally. The animals were killed after 8 weeks and the samples were retrieved and processed for histologic/histomorfometric and TEM/SEM evaluations. RESULTS: Histological examination showed mild or absent inflammatory reaction in peri-implant connective tissues around the surface modified implants. Further, junctional epithelium (JE)/connective tissue (CT) appeared to be in immediate contact with the experimental implants. Of the experimental implants, 22% were judged to be detached from the implant surface while 45% of the untreated control implants were detached. Dense plaques of hemidesmosomes were found in TEM evaluation of the JE cell membrane facing the surface-treated implants. In the histomorfometric analysis, the distance between the implant margin and alveolar bone crest was significantly shorter in surface-treated implants than in the control implants (P<0.02). CONCLUSION: Nanoporous sol-gel-derived TiO(2) thin film on ITI Straumann dental implants improved soft tissue attachment in vivo.

Reconstruction of critical size calvarial bone defects in rabbits with glass-fiber-reinforced composite with bioactive glass granule coating.

UNLABELLED: The aim of this study was to evaluate glass-fiber-reinforced composite as a bone reconstruction material in the critical size defects in rabbit calvarial bones. The bone defect healing process and inflammatory reactions were evaluated histologically at 4 and 12 weeks postoperatively. Possible neuropathological effects on brain tissue were evaluated. The release of residual monomers from the fiber-reinforced composite (FRC) was analyzed by high performance liquid chromatograph (HPLC). RESULTS: At 4 weeks postoperatively, fibrous connective tissue ingrowth to implant structures was seen. Healing had started as new bone formation from defect margins, as well as woven bone islets in the middle of the defect. Woven bone was also seen inside the implant. Inflammation reaction was slight. At 12 weeks, part of the new bone had matured to lamellar-type, and inflammation reaction was slight to moderate. Control defects had healed by fibrous connective tissue. Histological examinations of the brain revealed no obvious damage to brain morphology. In HPLC analysis, the release of residual 1,4-butanedioldimethacrylate and methylmethacrylate from polymerized FRC was low. CONCLUSIONS: This FRC-implant was shown to promote the healing process of critical size calvarial bone defect in rabbits. After some modifications to the material properties, this type of implant has the potential to become an alternative for the reconstruction of bone defects in the head and neck area in the future.

Natural composite of wood as replacement material for ostechondral bone defects.

Deciduous wood, birch, pretreated by a technique combining heat and water vapor was applied for the reconstruction of bone defects in the knee joint of rabbits. It was observed that wood showed characteristic properties to be incorporated by the host bone during observation time of 4, 8, and 20 weeks. The natural channel structure of wood served as a porous scaffold, allowing host bone growth as small islets into the wood implants. The other properties of heat-treated wood, such as bioactivity, good handling properties, and sufficient biomechanical properties, might be additional favorable factors for the application of wood as a natural composite material for bone and cartilage repair. At the interface of the surfaces of wood and living bone, bonding occurred. The Chemical Interface Model for bonding bone to wood consists of the reactive ions, such as hydroxyl groups --OH, and covalent bonding as well as hydrogen bonding, which originate from both wood and bone. The bone tissue trauma, with its reactive Ca(2+) and PO(4) (3-) ions, proteins, and collagen, available for interaction at ionic and nanolevel, are associated with the complicated chemistry in the cellular response of the early bone healing process. It was concluded that heat-treated wood acted like a porous biomaterial scaffold, allowing ongrowth and ingrowth of bone and cartilage differentiation on its surface, and demonstrating osteoconductive contact, bonding at the interface.

Titania and titania-silica coatings for titanium: comparison of ectopic bone formation within cell-seeded scaffolds.

The aim of this study was to compare titania (TiO(2))-coated, titania-silica (TiSi)-coated, and uncoated (cpTi) titanium fiber meshes as scaffolds for bone engineering. The scaffolds were loaded with bone marrow stromal cells and implanted subcutaneously in rats. Ectopic bone formation after 1, 4, and 12 weeks of implantation was evaluated using histology and histomorphometry. After 1 week of implantation, multiple patches of unorganized mineralizing tissue were seen in all implants. The amount of this bone-like tissue clearly increased from 1 to 4 weeks. Bone apposition occurred in direct contact with coated meshes, while a thin layer of unmineralized fibrous tissue was often observed surrounding cpTi mesh fibers. After 12 weeks, the structure of bone, with bone marrow-like tissue, was further matured and mesh fibers were embedded in lamellar bone. No statistical differences in the amount of mineralized bone were observed between scaffold types at any point of time. Only TiSi scaffolds showed further increase in bone area from 4 to 12 weeks (p < 0.01). A notable difference was that the sol-gel coatings resulted in enhanced initial bone contact and distribution of bone tissue, whereas uncoated implants showed bone formation mainly in the center of the scaffolds. In conclusion, TiO(2)-based sol-gel coatings may be used in tissue engineering to gain more uniform distribution of bone throughout titanium fiber mesh scaffolds.

Injectable bioactive glass/biodegradable polymer composite for bone and cartilage reconstruction: concept and experimental outcome with thermoplastic composites of poly(epsilon-caprolactone-co-D,L-lactide) and bioactive glass S53P4.

Injectable composites (Glepron) of particulate bioactive glass S53P4 (BAG) and Poly(epsilon-caprolactone-co-D,L-lactide) as thermoplastic carrier matrix were investigated as bone fillers in cancellous and cartilagineous subchondral bone defects in rabbits. Composites were injected as viscous liquid or mouldable paste. The glass granules of the composites resulted in good osteoconductivity and bone bonding that occurred initially at the interface between the glass and the host bone. The bone bioactivity index (BBI) indicating bone contacts between BAG and bone, as well as the bone coverage index (BCI) indicating bone ongrowth, correlated with the amount of glass in the composites. The indices were highest with 70 wt % of BAG, granule size 90-315 microm and did not improve by the addition of sucrose as in situ porosity creating agent in the composite or by using smaller (<45 microm) glass granules. The percentage of new bone ingrowth into the composite with 70 wt % of BAG was 6-8% at 23 weeks. At the articular surface cartilage regeneration with chondroblasts and mature chondrocytes was often evident. The composites were osteoconductive and easy to handle with short setting time. They were biocompatible with low foreign body cellular reaction. Results indicate a suitable working concept as a filler bone substitute for subchondral cancellous bone defects.

In vitro evaluation of poly(epsilon-caprolactone-co-DL-lactide)/ bioactive glass composites.

In vitro bioactivity of composites of poly(epsilon-caprolactone-co-DL-lactide) P(CL/DL-LA) containing different amounts (40, 60 and 70 wt%) of bioactive glass, S53P4, was evaluated. Two ranges of granule size of bioactive glass (< 45 microm and 90-315 microm) were blended with P(CL/DL-LA) copolymer in a batch mixer. The composites were characterised by dynamic mechanical thermal analysis. The molecular weight and the melting temperature of the copolymer matrix were adjusted to enable the application of the composite material by injection below 50 degrees C. Formation of Ca-P deposition on the surface of the composites after dissolution in simulated body fluid at 37 degrees C was recorded by scanning electron microscopy. Degradation of the composite material was measured by water absorption and changes in the average molecular weights as a function of the dissolution time. In vitro bioactivity was found to be dependent on the weight fraction and granule size range of the bioactive glass used. The presence of the bioactive filler also accelerated the degradation compared with the neat polymer sample.