A new chemical etching process to improve endosseous implant osseointegration: in vitro evaluation on human osteoblast-like cells

The development of novel mechanical and chemical surface modification treatments to improve the osteointegration properties of osseointegrated dental implants is nowadays a topic of great applicative interest. The aim of the present study was to analyse the role of surface topography and chemistry of four different surface treatments on titanium by an in vitro human osteosarcoma immortalised cell line model (MG63). The surface treatments considered were (a) machined titanium, (b) chemical etched on machined titanium, (c) sandblasted titanium and (d) chemical etching on sandblasted titanium. Chemical and physical surface properties were investigated by Scanning Electron Microscopy, Thin Film-X ray Diffraction and by Laser Profilometry. The in vitro biological response was characterised using the MG63 cell line by elution cytotoxicity tests, cell morphology, adhesion, proliferation activity, alkaline phosphatase activity and total DNA content in order to show a relationship between osteoblast response and surface features. Chemical and physical characterisation showed that the considered treatments differently modify the surface morphology in the micro and sub-micrometric scale. Although some differences in alkaline phosphatase activity were observed in the biological characterisation, depending on the specific material's surface finishing, the results showed that cells were well responsive on all the tested materials and grew and differentiated with similar proliferation rate.     

Effects of transforming growth factor-beta 1 (TGF-beta1) on in vitro mineralization of human osteoblasts on implant materials

An in vitro cell-implant mineralization model system was used to study the effect of transforming growth factor-beta 1(TGF-beta1) on mineralization in human osteoblast cultures. SaOs-2 and primary human osteoblast (HOB) cells were cultured on Tivanium (Ti-6Al-4V) disks. Administration of different concentrations of TGF-beta1 (0.02, 0.01, 0.2, 1.0, 2.0ng/ml) to these cultures demonstrated a biphasic dose response with 0.2ng/ml TGF-beta1 maximally increasing the calcium content compared to control culture. Results with SaOs-2 and HOB cultures were similar. An optimal dose of TGF-beta1 (0.2ng/ml) was provided to the cultures either in one single dose or multiple doses. Continuous administration of 0.2ng/ml TGF-beta1 caused 77% (SaOs-2) and 60% (HOB) increases in calcification compared to the control and 0.2ng/ml single dose groups. Single administration of the accumulative dose at 1.6ng/ml had no significant effect on the calcium content in either cell culture compared to control. Two weeks continuous administration of 0.2ng/ml TGF-beta1 in both cell cultures resulted in significant increases in the expression of bone specific extracellular matrix proteins which included alkaline phosphatase, Type I collagen, and osteocalcin as measured by Northern blot analysis and RT-PCR. At 4 weeks, the mRNA level of Type I collagen was still significantly higher in the TGF-beta1 treatment group compared to control. In conclusion, TGF-beta1 enhances mineralization of HOB on implant materials.     

Response of rat osteoblast-like cells to microstructured model surfaces in vitro

The role of surface microtopography in combination with different surface wettability for rat calvaria cell differentiation was examined. Mineralization and alkaline phosphatase (ALP) activity of rat calvaria cells on flat polydimethylsiloxane (PDMS) or PDMS contained pyramids which were either hydrophilic or hydrophobic were compared. ALP expressing cells were more frequent on hydrophilic PDMS contained pyramids. ALP activity, peaked at day 9, was highest for hydrophilic pyramids followed by hydrophobic pyramids and flat hydrophilic PDMS surfaces. A similar pattern was obtained with respect to mineralized nodules. These observations showed that micro-sized surface features promote differentiation of rat calvaria cells. Further, hydrophilic surfaces are more prone to stimulate differentiation in comparison with hydrophobic surfaces. The results suggest that both material surface chemistry and topography affect osteoblast differentiation.     

Comparative in vitro study of the proliferation and growth of human osteoblast-like cells on various biomaterials

In vitro studies about the growth behavior of osteoblasts onto biomaterials is a basic knowledge and a screening method for the development and application of scaffolds in vivo. In this in vitro study human osteoblast-like (HOB) cells were cultured on seven different biomaterials used in dental and craniomaxillofacial surgery, respectively. The tested biomaterials were synthetic biodegradable (MacroPore, Ethisorb, PDS, Beriplast P) and nonbiodegradable polymers (Palacos) as well as calcium phosphate cement (BoneSource) and titanium. The cell proliferation and cell colonization were analyzed by scanning electron microscopy and EZ4U-test. Statistical analysis were performed. HOB-like cells cultivated on Ethisorb showed the highest proliferation rate. The proliferation rate was statistically significant compared with Palacos, MacroPore, and BoneSource. Whereas, Beriplast, PDS, and titanium yielded lower proliferation rates. However, there was no statistically significant difference compared with Palacos, MacroPore, and BoneSource. SEM analysis showed no significant difference in individual cell features and cell colonization. But an infiltration and a growth of HOB-like cells throughout the porous structure of Ethisorb, which is formed by crossing fibers, is a striking different feature (macrotopography). This feature can explain the highest proliferation rate of Ethisorb. The results showed that HOB-like cells appear to be sensitive to substrate composition and topography. Moreover, the basis for further studies with such biomaterial/osteoblast constructs in vivo are provided. Further focusing points are developing techniques to fabricate three-dimensional porous biomaterial/cell constructs, studying the tissue reaction and the bone regeneration of such constructs compared with the use of autologous bone.     

Intermittent versus continuous stretching effects on osteoblast-like cells in vitro

The objective of this study was to quantify and compare stretch-mediated responses of primary rat osteoblast-like cells to uniform cyclic strain applied intermittently or continuously. Primary rat osteoblast-like cells were seeded and cultured in silicone rubber dishes for 2 days. They were then subjected to 1000 microstrains at 1 Hz for periods of 60 consecutive minutes or to a series of 15-min stretch followed by 15-min rest, until a total stretch of 60 min. After stretching, cells were incubated and assayed on days 4, 8, 16, and 24 for DNA content, alkaline phosphatase (ALP) activity, and calcium (Ca) content. Additionally, qualitative information was obtained via scanning electron and confocal laser scanning micrographs. Significant increases in DNA were observed for cells stretched intermittently versus cells stretched continuously and versus controls. Results showed significant decreases (p < 0.05) in ALP for cells between stretched groups and between both stretched groups versus controls. Additionally, Ca content was greater in cells stretched intermittently versus controls on days 4 and 8 and versus cells stretched continuously on day 24. In conclusion, intermittently strained cells demonstrated significant decreases in ALP and increases in DNA and Ca versus cells strained continuously. This supports the theory that cells respond to mechanical loading in a "trigger-like" response.     

Effect of laser therapy on attachment, proliferation and differentiation of human osteoblast-like cells cultured on titanium implant material

The aim of this in vitro study was to investigate the effect of low-level laser therapy (LLLT) on the attachment, proliferation, differentiation and production of transforming growth factor-ss(1) (TGF-beta(1)) by human osteoblast-like cells (HOB). Cells derived from human mandibular bone were exposed to GaAlAs diode laser at dosages of 1.5 or 3 J/cm(2) and then seeded onto titanium discs. Non-irradiated cultures served as controls. After 1, 3 and 24h, cells were stained and the attached cells were counted under a light microscope. In order to investigate the effect of LLLT on cell proliferation after 48, 72 and 96 h, cells were cultured on titanium specimens for 24h and then exposed to laser irradiation for three consecutive days. Specific alkaline phosphatase activity and the ability of the cells to synthesize osteocalcin after 10 days were investigated using p-nitrophenylphosphate as a substrate and the ELSA-OST-NAT immunoradiometric kit, respectively. Cellular production of TGF-beta(1) was measured by an enzyme-linked immunosorbent assay (ELISA), using commercially available kits. LLLT significantly enhanced cellular attachment (P<0.05). Greater cell proliferation in the irradiated groups was observed first after 96 h. Osteocalcin synthesis and TGF-beta(1) production were significantly greater (P<0.05) on the samples exposed to 3 J/cm(2). However, alkaline phosphatase activity did not differ significantly among the three groups. These results showed that in response to LLLT, HOB cultured on titanium implant material had a tendency towards increased cellular attachment, proliferation, differentiation and production of TGF-beta(1), indicating that in vitro LLLT can modulate the activity of cells and tissues surrounding implant material.     

Influence of titanium surfaces on attachment of osteoblast-like cells in vitro

Implant surface topography influences osteoblastic proliferation, differentiation and extracellular matrix protein expressions. Studies on preliminary interactions of osteoblast-like cells on implant interface through in vitro systems, can give lucid insights to osseo-integrative efficacies of when in vivo implants. In the present investigation two titanium surfaces of dental implants, a sandblasted and acid-etched surface and an experimental grooved surface were compared through in vitro systems. The titanium implants were seeded with osteoblast-like primary cells and maintained for a period of 1-7 days. Expressions of fibronectin and osteonectin were assessed through immunogold labelling by scanning electron microscopy. The grooved surface, supported better osteoblastic cell adhesion and proliferation than the rough surfaces. Further, osteoblastic cells on the grooved surfaces also displayed a strong labelling for fibronectin at the cytoplasmic extensions coupled with intense osteonectin expression in comparison to the rough surfaced implants. In conclusion, grooved surfaces offered better cell attachment and proliferation than the other rough surfaces studied.     

Morphological behavior of osteoblast-like cells on surface-modified titanium in vitro.

In recent papers, we reported the results of a study on the graded porous titanium coatings on titanium by plasma spraying and amino-group ion implantation. The paper is to preliminarily evaluate the biocompatibility of surface-modified titanium through 2, 5 and 7 days cell culture in vitro. Cell morphology was observed by a scanning electron microscope. Cell proliferation and type I collagen synthesis were measured by 3(4.5-dimethyl-thiazole-2-yl)2,5-diphenyl tetrazolium bromide (MTT) and enzyme-linked immunosorbent assay (ELISA), respectively. Our experimental results showed that osteoblast-like cells attached and spread well on surface-modified titanium. Cells were observed to grow into the pores and form extracellular matrix. MTT and ELISA results showed no detrimental effect on the development of cell. These studies support the biocompatibility of surface-modified titanium.     

Three-dimensional,bioactive, biodegradable,polymer-bioactive glass composite scaffolds with improved mechanical properties support collagen synthesis and mineralization of human osteoblast-like cells in vitro

In the past decade, tissue engineering-based bone grafting has emerged as a viable alternative to biological and synthetic grafts. The biomaterial component is a critical determinant of the ultimate success of the tissue-engineered graft. Because no single existing material possesses all the necessary properties required in an ideal bone graft, our approach has been to develop a three dimensional (3-D), porous composite of polylactide-co-glycolide (PLAGA) and 45S5 bioactive glass (BG) that is biodegradable, bioactive, and suitable as a scaffold for bone tissue engineering (PLAGA-BG composite). The objectives of this study were to examine the mechanical properties of a PLAGA-BG matrix, to evaluate the response of human osteoblast-like cells to the PLAGA-BG composite, and to evaluate the ability of the composite to form a surface calcium phosphate layer in vitro. Structural and mechanical properties of PLAGA-BG were measured, and the formation of a surface calcium phosphate layer was evaluated by surface analysis methods. The growth and differentiation of human osteoblast-like cells on PLAGA-BG were also examined. A hypothesis was that the combination of PLAGA with BG would result in a biocompatible and bioactive composite, capable of supporting osteoblast adhesion, growth and differentiation, with mechanical properties superior to PLAGA alone. The addition of bioactive glass granules to the PLAGA matrix resulted in a structure with higher compressive modulus than PLAGA alone. Moreover, the PLAGA-BA composite was found to be a bioactive material, as it formed surface calcium phosphate deposits in a simulated body fluid (SBF), and in the presence of cells and serum proteins. The composite supported osteoblast-like morphology, stained positively for alkaline phosphatase, and supported higher levels of Type I collagen synthesis than tissue culture polystyrene controls. We have successfully developed a degradable, porous, polymer bioactive glass composite possessing improved mechanical properties and osteointegrative potential compared to degradable polymers of poly(lactic acid-glycolic acid) alone. Future work will focus on the optimization of the composite scaffold for bone tissue-engineering applications and the evaluation of the 3-D composite in an in vivo model.     

Stretch-mediated responses of osteoblast-like cells cultured on titanium-coated substrates in vitro

Cyclic stretching experiments on osteoblast-like cells have proven to be a useful tool in understanding the underlying mechanisms of load transduction at the bone-implant surface. However, most experimental setups use silicone rubber substrates, which are atypical for orthopedic and dental implant materials. Therefore, we investigated the responses of osteoblast-like cells to loading on titanium (Ti)-coated versus plain silicone substrates. Ti-coated substrates were made by a radio-frequency magnetron sputtering process, and characterized using Rutherford backscattering spectrometry, X-ray photoelectron spectroscopy, and contact-angle measurements. Osteoblast-like cells cultured from rat bone marrow were seeded on both types of substrates and stretched for 1 h continuously. Subsequently, cell proliferation, alkaline phosphatase activity, and calcium content were measured for up to 24 days after seeding. In addition light-, scanning electron-, and confocal laser scanning micrographs were made. The results showed that our Ti coating had a thickness of 50 nm and contained Ti/oxygen as 1:1. However, further characterization proved that the silicone material had a tendency to resurface through the coating. Osteoblast-like cells proliferated faster on the Ti-coated substrates, but differentiation was slower compared with the silicone substrates. It was concluded that that there was a definitive influence of the substrate material in mechanical stress models. Therefore, extrapolation of results obtained using silicone substrates cannot be translated directly toward the situation of metallic implant materials.     

Cytobiocompatibility of collagen and ePTFE membranes on osteoblast-like cells in vitro

In guided bone regeneration (GBR), a semipermeable membrane is placed over an osseous defect to create a secluded environment in which bone formation can proceed without ingrowth of connective tissue cells from the overlaying soft tissue. Although the cell-occlusive property of GBR membranes appear to be essential to new bone formation, the role of transmembrane tissue fluid diffusion is not known. The objective of this study was to evaluate the degree to which diffusion across commonly used GBR membranes can support functional properties of osteoblast-like cells in vitro. Cells from an established osteoblast-like line (SAOS-2) were cultured on membranes of cross-linked collagen, noncross linked collagen, and ePTFE. The membranes rested on metal grids which allowed the membranes to lightly contact the surface of the culture medium. As a control, cells were directly plated and cultured in control wells. At days 7 and 21, cells were harvested by scraping the membranes or culture wells and analyzed for expression of alkaline phosphatase (ALP), core binding factor 1 (cbfa-1), bone sialoprotein-2 (BSP-2), and osteocalcin (OC). Expression was determined by quantitative real-time PCR. Glucose-6-phosphate dehydrogenase (G6PD) served as a reference gene. The membranes were examined by transmission light microscopy. RT-PCR revealed up-regulation of ALP of up to 60-fold and of cbfa-1 and BSP of up to threefold relative to G6PDH. Expression of OC was less then onefold. The expression profile for each of the four genes tested demonstrated small variations among cells grown on different membranes. Microscopic observations revealed remnants of undisrupted osteoblast-like cells attached to both collagen membranes. Cell morphology and spatial arrangement indicated that vitality was maintained. Diffusion through the three membranes evaluated in this study was sufficient to support osteoblast-like cell differentiation.     

An in vitro culturing model for rabbit dural cells

The objectives of this study were (a) to construct an in vitro model of rabbit dural healing, (b) to test the influence of collagen, laminin, and poly-L-lysine on the migration and proliferation of dural cells, and (c) to study the healing mechanism of duraplasty. Rabbit dural pieces (1.5 cm x 1.5 cm) were perforated in their central part with a 2 mm punch to mimic a dural defect. The dural pieces were cultured in 24-well plates that had been coated with collagen, laminin, or poly-L-lysine, and the influence of different extracellular matrices on migration and proliferation of dural cells was observed. Cells were subcultured on slides for immunocytochemistry to study their characteristics; dural healing was observed by scanning electron microscopy. The results demonstrated that only the dural pieces that were cultured on collagen-coated wells showed migration of cells into the central defect after a period of 8 to 10 days and that healing of the dural defect occurred by 13 to 15 days. The cultured dural cells stained strongly positive with an antibody to vimentin, but negative with an antibody to factor VIII. New collagen fibers were observed in the dural defects. This report demonstrates that an in vitro model for dural healing was successfully constructed in collagen-coated wells; the results implicate cellular migration of fibroblasts from the dural defect margin as an important mechanism of wound healing following duraplasty.     

In vitro behavior of osteoblast-like cells on fluoridated hydroxyapatite coatings

In this work, fluoridated hydroxyapatite (Ca10(PO4)6Fx(OH)(2-x) or FHA) coatings are prepared by sol-gel method for study of the influence of F content on the behavior of osteoblast-like cells. The results show that the cells well attach and proliferate on the FHA coatings studied (Ca10(PO4)6F(0.67-2.00)(OH)(0-1.33)). With increasing F content in the FHA coatings, percentage of cell in S period increases, indicating F in the coating favors the proliferation process of the cells. On the other hand, the proliferation rate increases inversely with zeta potential of the coating surface. As tested from the MTT of the cells cultured in the leaching out solution, increase of F content in the FHA coatings results in a slight decrease in cell proliferation rate, which is most probably due to reduction in release of Ca2+ ions. As a compromise among cell attachment, cell proliferation, apatite deposition and ability to resist dissolution, it is suggested that FHA coatings (Ca10(PO4)6Fx(OH)(2-x)) with x in the range of 0.67-1.48, from the results of this study, may be most suitable for real case implantation.     

Growth promoting in vitro effect of synthetic cyclic RGD-peptides on human osteoblast-like cells attached to cancellous bone

In tissue engineering, the application of biofunctional compounds on biomaterials such as integrin binding RGD-peptides has gained growing interest. Anchorage-dependent cells like osteoblasts bind to these peptides thus ameliorating the integration of a synthetic implant. In case sterilized bone grafts are used as substitutes for reconstruction of bone defects, the ingrowth of the implanted bone is often disturbed because of severe pretreatment such as irradiation or autoclaving, impairing the biological and mechanical properties of the bone. We report for the first time on the in vitro coating of the surface of freshly resected, cleaned bone discs with synthetic, cyclic RGD-peptides. For this approach, two different RGD-peptides were used, one containing two phosphonate anchors, the other peptide four of these binding moieties to allow efficient association of these reactive RGD-peptides to the inorganic bone matrix. Human osteoblast-like cells were cultured on RGD-coated bone discs and the adherence and growth of the cells were analyzed. Coating of bone discs with RGD-peptides did not improve the adhesion rate of osteoblast-like cells to the discs but significantly (up to 40%) accelerated growth of these cells within 8 days after attachment. This effect points to pretreatment of bone implants, especially at the critical interface area between the implanted bone and the non-resected residual bone structure, before re-implantation in order to stimulate and enhance osteointegration of a bone implant.     

Osteoblast responses to orthopedic implant materials in vitro

Responses of neonatal rat calvarial osteoblasts to a variety of orthopedic implant materials were examined in vitro. Attachment, proliferation, and collagen synthesis of a well-characterized line of osteoblasts with 316L stainless steel, Ti-6Al-4V, Co-Cr-Mo, PMMA, hydroxyapatite, borosilicate glass, and tissue culture polystyrene were studied. Cell adhesion and growth were similar on nonapatitic materials. In contrast, attachment and growth of osteoblasts were significantly lower and slower, respectively, on hydroxyapatite. Collagen synthesis per cell and relative collagen synthesis, however, were comparable on all the materials tested.     

An in vitro model for the study of human implantation

Citation Holmberg JC, Haddad S, Wünsche V, Yang Y, Aldo PB, Gnainsky Y, Granot I, Dekel N, Mor G. An In vitro model for the study of human implantation. Am J Reprod Immunol 2012; 67: 169–178 Problem Implantation remains the rate‐limiting step for the success of in vitro fertilization. Appropriate models to study the molecular aspects of human implantation are necessary in order to improve fertility. Methods First trimester trophoblast cells are differentiated into blastocyst‐like spheroids (BLS) by culturing them in low attachment plates. Immortalized human endometrial stromal cells and epithelial cells (ECC‐1) were stably transfected with GFP or tdTomato. Co‐culture experiments were monitored using Volocity imaging analysis system. Results This method demonstrates attachment and invasion of BLS, formed by trophoblast cells, into stromal cells, but not to uterine epithelial cells. Conclusion We have developed an in vitro model of uterine implantation. The manipulation of this system allows for dual color monitoring of the cells over time. Additionally, specific compounds can be added to the culture media to test how this may affect implantation and invasion. This model is a helpful tool in understanding the complexity of human implantation.     

The effect of zoledronate-hydroxyapatite nanocomposites on osteoclasts and osteoblast-like cells in vitro

This study demonstrates that zoledronate containing hydroxyapatite nanocrystals (HA-ZOL) can be synthesized as a single crystalline phase up to a zoledronate content of about 7 wt% by direct synthesis in aqueous solution, at variance with what previously found for alendronate-hydroxyapatite nanocrystals (HA-AL). On increasing zoledronate incorporation, the length of the coherent crystalline domains and the crystal dimensions of hydroxyapatite decrease, whereas the specific surface area increases. Full profile fitting of the powder X-ray diffraction patterns does not indicate major structural modifications, but an increase of the hydroxyapatite unit cell, on increasing zoledronate content. These data, together with a structural similarity between hydroxyapatite and calcium zoledronate, suggest a preferential interaction between zoledronate and the hydroxyapatite faces parallel to the c-axis direction. Osteoblast-like MG-63 cells and human osteoclasts were cultured on HA-ZOL nanocrystals and as a comparison on HA-AL nanocrystals containing almost the same (about 7 wt%) bisphosphonate amount. The beneficial influence of bisphosphonates on osteoblast proliferation and differentiation is enhanced when the tests are performed in co-cultures. Similarly, the reduction of osteoclast proliferation and the increase of Caspase 3 production are dramatically enhanced in co-cultures, which highlight an even greater influence of HA-ZOL than HA-AL on osteoclast apoptosis.     

The titanium surface texture effects adherence and growth of human gingival keratinocytes and human maxillar osteoblast-like cells in vitro.

The adhesion, orientation and proliferation of human gingival epithelial cells and human maxillar osteoblast-like cells in primary and secondary culture were studied on glossy polished, sandblasted and plasma-sprayed titanium surfaces by scanning electron microscopy and in thin sections. The primary cultured explants of human gingival epithelial cells attached, spread and proliferated on all titanium surfaces with the greatest extension on the polished and the smallest extension on plasma-sprayed surfaces. In secondary suspension cultures of gingival keratinocytes, attachment spreading and growth was only observed on polished and plasma-sprayed surfaces, but not on sandblasted surfaces. Moreover, the attachment of these cells depended on the seeding concentration as well as on the coating with fetal calf serum. Cells on polished surfaces developed an extremely flat cell shape, but on sandblasted and plasma-sprayed surfaces a more cuboidal shape. In contrast human maxillar osteoblasts seeded as secondary suspension cultures attached very well to all three differently textured titanium surfaces and showed identical growth patterns independent of the titanium surface structure. These findings suggest that cell morphology, orientation, proliferation and adhesion of human gingival epithelial cells in primary or secondary culture are dependent on the texture of the titanium surface whereas no such differences were observed for maxillar osteoblast-like cells. In conclusion, the soft tissue integration and response is more influenced by the surface texture than the process of osseointegration.