Neural crest-derived cells in nasal conchae of adult mice contribute to bone regeneration.

Neural crest-derived cells (NCDCs), a class of adult stem cells not restricted to embryonic tissues, are attractive tissue regenerative therapy candidates because of their ease of isolation, self-renewing properties, and multipotency. Although adult NCDCs can undergo osteogenic differentiation in vitro, whether they induce bone formation in vivo remains unclear. Previously, our group reported findings showing high amounts of NCDCs scattered throughout nasal concha tissues of adult mice. In the present study, NCDCs in nasal conchae labeled with enhanced green fluorescent protein (EGFP) were collected from adult P0-Cre/CAG-CAT-EGFP double transgenic mice, then cultured in serum-free medium to increase the number. Subsequently, NCDCs were harvested and suspended in type I atelocollagen gel, then an atelocollagen sponge was used as a scaffold for the cell suspension. Atelocollagen scaffolds with NCDCs were placed on bone defects created in a mouse calvarial bone defect model. Over the ensuing 12 weeks, micro-CT and histological analysis findings showed that mice with scaffolds containing NCDCs had slightly greater bone formation as compared to those with a scaffold alone. Furthermore, Raman spectroscopy revealed spectral properties of bone in mice that received scaffolds with NCDCs similar to those of native calvarial bone. Bone regeneration is important not only for gaining bone mass but also chemical properties. These results are the first to show the validity of biomolecule-free adult nasal concha-derived NCDCs for bone regeneration, including the chemical properties of regenerated bone tissue.

Pentosidine correlates with nanomechanical properties of human jaw bone.

Initial intimate apposition between implant fixtures and host bone at the surgical site is a critical factor for osseointegration of dental implants. The advanced glycation end products accumulated in the jaw bone could lead to potential failure of a dental implant during the initial integration stage, because of the inferior bone mechanical property associated with the abnormal collagen cross-linking at the material level. Here, we demonstrate the lowered creep deformation resistance and reduced dimensional recovery of jaw bone in line with high levels of pentosidine accumulation in the bone matrix which likely correlate with the pentosidine level in blood plasma. Peripheral blood samples and cortical bone samples at the surgical site were obtained from patients scheduled for dental implants in the mandible. The pentosidine levels in blood plasma were assessed. Subsequently, the relative pentosidine levels and the mechanical properties of the jaw bone were quantified by Raman microspectroscopy and nanoindentation, respectively. The nanoindentation tests revealed less creep deformation resistance and reduced time-dependent dimensional recovery of bone samples with the increase in the relative pentosidine level in the bone matrix. Higher tan δ values at the various frequencies during the dynamic indentation tests also suggested that viscoelasticity is associated with the relative intensity of pentosidine in the jaw bone matrix. We found a positive correlation between the pentosidine levels in blood plasma and the bone matrix, which in turn reduced the mechanical property of the jaw bone at the material level. Increased creep and reduced dimensional recovery of the jaw bone may diminish the mechanical interlocking of dental implants during the initial integration stage. Given the likely correlation between the plasma pentosidine level and the mechanical properties of bone, measurement of the plasma pentosidine level could serve as a new index to assess jaw bone matrix quality in advance of implant surgery.

Nanomechanical characterization of time-dependent deformation/recovery on human dentin caused by radiation-induced glycation.

An increase in non-enzymatic collagen matrix cross-links, such as advanced glycation end-products (AGEs), is known to be a major complication in human mineralized tissues, often causing abnormal fractures. However, degradation of mechanical properties in relation to AGEs has not been fully elucidated at the material level. Here, we report nanoscale time-dependent deformation and dimensional recovery of human tooth dentin that has undergone glycation induced by x-ray irradiation. The reduction in enzymatic collagen cross-linking and the increased level of AGEs with concomitant growth of disordered collagen matrix diminished creep deformation recovery in the lower mineralized target region. However, the elevated AGEs level alone did not cause a reduction in time-dependent deformation and its recovery in the higher mineralized target region. In addition to the elevated AGEs level, the degradation of the mechanical properties of mineralized tissues should be assessed with care in respect to multiple parameters in the collagen matrix at the molecular level.

Nanoindentation time-dependent deformation/recovery suggestive of methylglyoxal induced glycation in calcified nodules.

Although empirical findings have indicated increase in bone fracture risk in type 2 diabetes patients, that has yet to be proven by results obtained at the material level. Here, we report evidence showing nanoscale time-dependent deformation/recovery of in vitro calcified nodules mimicking bone turnover in type 2 diabetes in respect to methylglyoxal (MG)-induced glycation. Nanoindentation test results revealed that calcified nodules cultured with MG did not show adequate dimensional recovery, despite a large creep rate during constant load indentation testing. This lesser recovery is likely based on the linear matrix polymerization network formed by advanced glycation end products (AGEs) as a secondary product of MG. Since elevated serum MG and abnormal bone turnover related to the amount of AGEs are observed in cases of type 2 diabetes, this time-dependent behavior may be one of the factors of the bone fracture mechanism at the material level in affected patients.

Antioxidant and osteogenic properties of anodically oxidized titanium.

Cells adhering onto implant surfaces are subjected to oxidative stress during wound healing processes. Although titanium and its alloys are among the most frequently used biomaterials in orthopedic and dental implants, titanium surfaces do not have antioxidant properties, and cells grown on these surfaces can show permanent oxidative stress. The present study assessed the antioxidant property and osteogenic properties of titanium samples with or without oxidation treatments. A thick rutile TiO2 film was observed on thermally oxidized titanium surfaces, while amorphous anatase TiO2 formed on anodically oxidized titanium surfaces prepared by discharging in 1 M Na2HPO4. A resistance to the depletion of reduced glutathione in adherent osteoblasts, which correlates with antioxidant behavior, occurred on anodically oxidized titanium. Enhanced osteogenic gene expressions and nano-biomechanical properties of mineralized tissue were achieved on anodically oxidized titanium, in comparison with thermally oxidized or untreated titanium. Thus, anodic oxidation by discharging in electrolyte is expected to be a useful surface modification for titanium implants.

Interfacial assembly of bioinspired nanostructures mediated by supersensitive crystals.

Laboratory-designed biocomposites structured by organic matrices impregnated with oriented biominerals have been significantly progressed by mimicking biological processes, although several problems associated with their formulation or antigenicity remain to be solved. Here, we describe a new strategy for the formulation of bioinspired nanostructures that involves spontaneous mediation by cooperative interactions between inorganic nanocrystals and host cells without the complex procedures required for laboratory-designed biocomposites. In the present study, osteoblastic cells were cultured on hydroxyapatite and beta-tricalcium phosphate nanocrystals prepared by discharging in electrolytes. Specifically, a high level of assembly of collagenous proteins associated with cell proliferation was achieved on nanoscale beta-tricalcium phosphate crystals by catalysis of polyphosphate chains produced during cell culture. Furthermore, a spatial structure that was practically composed of natural biocomposites found in bone and teeth was obtained on the nanocrystals due to increased cross-linking between inorganic molecules and biomolecules. Suggestions for the spontaneous formulation of bioinspired nanostructures in a living body mediated by inorganic biomaterials are also discussed.

Glow discharge plasma pretreatment enhances osteoclast differentiation and survival on titanium plates.

Despite the fact that several reports have demonstrated osteoclast activity on various bioactive ceramics, osteoclast functions on surface-modified titanium have not come under focus. This study aimed to examine whether the increasing surface energy of glow discharge plasma (GDP) involved in protein adhesion containing the RGD (Arg-Gly-Asp) sequence affects osteoclast responses on titanium plates. We examined osteoclast differentiation and survival rates on titanium plates with and without GDP. The amounts of osteoclasts on titanium plates were not increased by GDP after 1 week. However, osteoclast differentiation was greatly activated by GDP pretreatment, as tartrate-resistant acid phosphatase synthesis significantly increased on the titanium plates with GDP. Additionally, since the presence of osteoclasts was detected only on the titanium plates with GDP, even after 4h cultivation in a coculture test, the osteoclasts survival rate was increased by GDP pretreatment. As osteoclast responses were affected even on surface modified metallic materials, we concluded that novel approaches are needed not only for osteoclastic resorption on ceramic materials but also for osteoclast responses on surface-modified metallic materials.