Pierre Layrolle

Osteoblastic cell behaviour on different titanium implant surfaces.

The osseointegration of oral implants is related to the early interactions between osteoblastic cells and titanium surfaces. The behaviour of osteoblastic MC3T3-E1 cells was compared on four different titanium surfaces: mirror-polished (Smooth-Ti), alumina grit-blasted (Alumina-Ti) or biphasic calcium phosphate ceramic grit-blasted (BCP-Ti) and a commercially available implant surface (SLA). Scanning electron microscopy and profilometry showed distinct microtopographies. The BCP-Ti group had higher average surface roughness (Ra=2.5 microm) than the other grit-blasted groups. Hydrophilicity and surfaces energies were determined on the different substrates by dynamic contact angle measurements. The most hydrophilic surface was the Alumina-Ti discs, while SLA was the most hydrophobic. The titanium surfaces were all oxidized as TiO2 and polluted by carbon contaminants, as determined by X-ray photoelectron spectroscopy. Alumina-Ti samples also exhibited aluminium peaks as a result of the blasting. The BCP-Ti discs contained traces of calcium and phosphorus. MC3T3-E1 cells attached, spread and proliferated on the substrates. For both the SLA and BCP-Ti groups, the entire surface was covered with a layer of osteoblastic cells after 2 days. At high magnification, the cells exhibited cytoplasmic extensions and filopodia. Compared with plastic, cell viability was similar with the Smooth-Ti, slightly lower with the Alumina-Ti and superior with the SLA and BCP-Ti groups. Alkaline phosphatase activity increased with the culture time whatever the substrate. This study shows that BCP-blasting produces rough titanium implants without surface contaminants.

Macroporous Biphasic Calcium Phosphate Scaffold with High Permeability/Porosity Ratio

Macroporous biphasic calcium phosphate (BCP) with channel-shaped pores was produced by a novel dual-phase mixing method. The processing route includes mixing water-based BCP slurry and polymethylmethacrylate resin; shaping in a mold; and polymerization, drying, pyrolyzing, and sintering. After comparison with two other commercial macroporous BCP materials, which were produced along different routes, it was found that conventional parameters such as porosity and pore size cannot describe a macroporous structure precisely enough for the application as tissue-engineering scaffold. Instead, permeability can be seen as an intrinsic and quantitative parameter to describe the macroporous structure of various scaffolds, because it is independent of sample size and fluid used in the test. Another parameter, the permeability/porosity ratio, provides an indication of the percolative efficiency per unit porous volume of a scaffold. Structural characterizations and permeability studies of other macroporous scaffold materials were also performed, and it was found that permeability could reflect a combination of five important parameters for scaffold: (1) porosity, (2) pore size and distribution, (3) interconnectivity, (4) fenestration size and distribution, and (5) orientation of pores. Finally, the implications of relating permeability with biological performances are also discussed.

Biomimetic and electrolytic calcium phosphate coatings on titanium alloy: physicochemical characteristics and cell attachment.

Biomimetically deposited octacalcium phosphate (OCP) and carbonate apatite (BCA) as well as electrolytically deposited carbonate apatite (ECA) were considered as promising alternatives to conventional plasma spraying hydroxyapatite. This study compared their physicochemical characteristics and cell attachment behavior. The physicochemical characteristics included scanning electron microscopy observation, X-ray diffraction analysis, Fourier transform infrared spectroscopy analysis, surface roughness, coating thickness, dissolution test and scratch test. Cell attachment tests included morphology observation with stereomicroscopy and scanning electron microscopy as well as cell number count with DNA content assay. The OCP coating had 100% crystallinity and was about 40 microm thick, composed of large plate-like crystals of 30 microm, with the lowest surface roughness (R(a)=2.33 microm). The BCA coating had 60% crystallinity and was approximately 30 microm in thickness, composed of small crystals of 1-2 microm in size, with the highest surface roughness (R(a)=4.83 microm). The ECA coating had intermediate characteristics, with 78% crystallinity, 45 microm thickness, crystals of 5-6 microm and an average roughness of 3.87 microm. All coatings could be seen by eyes dissolving quickly and completely into acidic simulated body fluid (simulated physiological solutions-SPS, pH 3.0) but slowly and incompletely into neutral SPS (pH 7.3). It was suggested that the main factor determining coating dissolution in acidic SPS was the solubility isotherm, while some other factors including crystallinity and crystal size joined to determine coating dissolution in neutral SPS. In regard to adhesive strength, results of scratch test showed the critical load at the first crack of coating (L(c1)) was tightly related to crystal size as well as their arrangement, while the critical load at the total delamination of coating (L(c2)) was also related to the coating thickness. The ECA coating had the highest values. Owing to higher dissolution rate and globular appearance, BCA coating demonstrated the best goat bone marrow stromal cells attachment at 1 day or 3 days, followed by OCP and ECA coating.

Osteoblastic differentiation of human mesenchymal stem cells with platelet lysate.

Culture of expanded mesenchymal stem cells (MSCs) seeded on biomaterials may represent a clinical alternative to autologous bone graft in bone regeneration. Foetal bovine serum (FBS) is currently used for MSC expansion, despite risks of infectious disease transmission and immunological reaction due to its xenogenic origin. This study aimed to compare the osteogenic capacities of clinical-grade human MSCs cultured with FBS or allogenic human platelet lysate (PL). In vitro, MSCs cultured in PL both accelerate the expansion rate over serial passages and spontaneously induce osteoblastic gene expression such as alkaline phosphatase (ALP), bone sialoprotein (BSP), osteopontin (Op) and bone morphogenetic protein-2 (BMP-2). In vivo, ectopic bone formation is only observed on ceramics seeded with MSCs grown in PL medium implanted under the skin of immunodeficient mice for 7 weeks. In conclusion, allogenic human PL accelerates MSC proliferation and enhances MSC osteogenic differentiation.

Rapid prototyped porous titanium coated with calcium phosphate as a scaffold for bone tissue engineering.

High strength porous scaffolds and mesenchymal stem cells are required for bone tissue engineering applications. Porous titanium scaffolds (TiS) with a regular array of interconnected pores of 1000 microm in diameter and a porosity of 50% were produced using a rapid prototyping technique. A calcium phosphate (CaP) coating was applied to these titanium (Ti) scaffolds with an electrodeposition method. Raman spectroscopy and energy dispersive X-ray analysis showed that the coating consisted of carbonated hydroxyapatite. Cross-sectioned observations by scanning electron microscopy indicated that the coating evenly covered the entire structure with a thickness of approximately 25 microm. The bonding strength of the coating to the substrate was evaluated to be around 25 MPa. Rat bone marrow cells (RBMC) were seeded and cultured on the Ti scaffolds with or without coating. The Alamar Blue assay provided a low initial cell attachment (40%) and cell numbers were similar on both the uncoated and coated Ti scaffolds after 3 days. The Ti scaffolds were subsequently implanted subcutaneously for 4 weeks in syngenic rats. Histology revealed the presence of a mineralized collagen tissue in contact with the implants, but no bone formation. This study demonstrated that porous Ti scaffolds with high strength and defined geometry may be evenly coated with CaP layers and cultured mesenchymal stem cells for bone tissue engineering.

The human nose harbors a niche of olfactory ectomesenchymal stem cells displaying neurogenic and osteogenic properties.

We previously identified multipotent stem cells within the lamina propria of the human olfactory mucosa, located in the nasal cavity. We also demonstrated that this cell type differentiates into neural cells and improves locomotor behavior after transplantation in a rat model of Parkinsons disease. Yet, next to nothing is known about their specific stemness characteristics. We therefore devised a study aiming to compare olfactory lamina propria stem cells from 4 individuals to bone marrow mesenchymal stem cells from 4 age- and gender-matched individuals. Using pangenomic microarrays and immunostaining with 34 cell surface marker antibodies, we show here that olfactory stem cells are closely related to bone marrow stem cells. However, olfactory stem cells also exhibit singular traits. By means of techniques such as proliferation assay, cDNA microarrays, RT-PCR, in vitro and in vivo differentiation, we report that when compared to bone marrow stem cells, olfactory stem cells display (1) a high proliferation rate; (2) a propensity to differentiate into osseous cells; and (3) a disinclination to give rise to chondrocytes and adipocytes. Since peripheral olfactory stem cells originate from a neural crest-derived tissue and, as shown here, exhibit an increased expression of neural cell-related genes, we propose to name them olfactory ectomesenchymal stem cells (OE-MSC). Further studies are now required to corroborate the therapeutic potential of OE-MSCs in animal models of bone and brain diseases.

Bone Morphogenetic Protein 2 Incorporated into Biomimetic Coatings Retains Its Biological Activity

We have previously shown that proteins can be incorporated into the latticework of calcium phosphate layers when biomimetically coprecipitated with the inorganic components, upon the surfaces of titanium-alloy implants. In the present study, we wished to ascertain whether recombinant human bone morphogenetic protein 2 (rhBMP-2) thus incorporated retained its bioactivity as an osteoinductive agent. Titanium alloy implants were coated biomimetically with a layer of calcium phosphate in the presence of different concentrations of rhBMP-2 (0.1-10 microg/mL). rhBMP-2 was successfully incorporated into the crystal latticework, as revealed by protein blot staining. rhBMP-2 was taken up by the calcium phosphate coatings in a dose-dependent manner, as determined by ELISA. Rat bone marrow stromal cells were grown directly on these coatings for 8 days. Their osteogenicity was then assessed quantitatively by monitoring alkaline phosphatase activity. This parameter increased as a function of rhBMP-2 concentrations within the coating medium. rhBMP-2 incorporated into calcium phosphate coatings was more potent in stimulating the alkaline phosphatase activity of the adhering cell layer than was the freely suspended drug in stimulating that of cell layers grown on a plastic substratum. This system may be of osteoinductive value in orthopedic and dental implant surgery.

Correlating implant stability to bone structure

OBJECTIVES The aim of this study was to demonstrate a possible correlation between bone microarchitecture and primary implant stability. MATERIAL AND METHODS Twenty-two implants (Ankylos((R)) and Straumann((R))) were inserted into the maxillae and mandibles of human cadavers. Bone structure was determined by computed tomography in three specimens (male, age 53; female, 67; female, 80). A strict clinical protocol was used for implantation. Primary implant stability was measured by resonance frequency analysis (Osstell Mentor). The bone structure was analyzed by micro-computed tomography (CT). Bone histomorphometrical parameters were calculated and correlated to primary implant stability. RESULTS Implant stability quotients (ISQ) ranged from 50 to 70% depending on the specimens and sites. Histomorphometry indicated differences in the bone microstructures of the specimens. However, ISQ values were not related to trabecular bone histomorphometrical parameters. The sole correlation was found between ISQ values and cortical bone thickness. CONCLUSION This study confirms the relevance for primary stability of cortical thickness around implants. The thickness of cortical bone can be assessed using a standard clinical CT.

Bone regeneration: stem cell therapies and clinical studies in orthopaedics and traumatology.

•  Introduction •  Characterization of cells for bone regeneration in human beings •  State of the art in bone tissue engineering ‐  Biomaterials for scaffolding mesenchymal stem cells ‐  Future trends in bone tissue engineering •  Clinical targets for cell therapy in orthopaedics ‐  Current clinical problems and therapeutic approaches ‐  Potential clinical applications of cell based therapies for bone repair ‐  Data reported in clinical studies •  Selected paediatric bone disorders and cellular therapies ‐  Inborn errors of bone metabolism and cellular therapy ‐  Degenerative bone disorders in childhood •  Ethical aspects of EU clinical trials ‐  Ethics related to information and consent ‐  Approval of the studies ‐  Ethics related to privacy/data protection ‐  Ethics related to the risk‐benefit assessment ‐  Ethics related to protection of the health of persons involved in clinical trials ‐  Ethics related to transparency regarding research results •  Future directions and remarks

Enhanced osseointegration of titanium implants with nanostructured surfaces: an experimental study in rabbits.

Titanium and its alloys are commonly used for dental implants because of their good mechanical properties and biocompatibility. The surface properties of titanium implants are key factors for rapid and stable bone tissue integration. Micro-rough surfaces are commonly prepared by grit-blasting and acid-etching. However, proteins and cells interact with implant surfaces in the nanometer range. The aim of this study was to compare the osseointegration of machined (MA), standard alumina grit-blasted and acid-etched (MICRO) and nanostructured (NANO) implants in rabbit femurs. The MICRO surface exhibited typical random cavities with an average roughness of 1.5 μm, while the NANO surface consisted of a regular array of titanium oxide nanotubes 37±11 nm in diameter and 160 nm thick. The MA and NANO surfaces had a similar average roughness of 0.5 μm. The three groups of implants were inserted into the femoral condyles of New Zealand White rabbits. After 4 weeks, the pull-out test gave higher values for the NANO than for the other groups. Histology corroborated a direct apposition of bone tissue on to the NANO surface. Both the bone-to-implant contact and bone growth values were higher for the NANO than for the other implant surfaces. Overall, this study shows that the nanostructured surface improved the osseointegration of titanium implants and may be an alternative to conventional grit-blasted and acid-etched surface treatments.