Endogenus chondrocytes immobilized by G-CSF in nanoporous gels enable repair of critical-size osteochondral defects.

Injured articular cartilage is a leading cause for osteoarthritis. We recently discovered that endogenous stem/progenitor cells not only reside in the superficial zone of mouse articular cartilage, but also regenerated heterotopic bone and cartilage in vivo. However, whether critical-size osteochondral defects can be repaired by pure induced chemotatic cell homing of these endogenous stem/progenitor cells remains elusive. Here, we first found that cells in the superficial zone of articular cartilage surrounding surgically created 3 × 1 mm defects in explant culture of adult goat and rabbit knee joints migrated into defect-filled fibrin/hylaro1nate gel, and this migration was significantly more robust upon delivery of exogenous granulocyte-colony stimulating factor (G-CSF). Remarkably, G-CSF-recruited chondrogenic progenitor cells (CPCs) showed significantly stronger migration ability than donor-matched chondrocytes and osteoblasts. G-CSF-recruited CPCs robustly differentiated into chondrocytes, modestly into osteoblasts, and barely into adipocytes. In vivo, critical-size osteochondral defects were repaired by G-CSF-recruited endogenous cells postoperatively at 6 and 12 weeks in comparison to poor healing by gel-only group or defect-only group. ICRS and O'Driscoll scores of articular cartilage were significantly higher for both 6- and 12-week G-CSF samples than corresponding gel-only and defect-only groups. Thus, endogenous stem/progenitor cells may be activated by G-CSF, a Food and Drug Administration (FDA)-cleared bone-marrow stimulating factor, to repair osteochondral defects.

Alkaline activation of endogenous latent TGFß1 by an injectable hydrogel directs cell homing for in situ complex tissue regeneration.

Utilization of the body's regenerative potential for tissue repair is known as in situ tissue regeneration. However, the use of exogenous growth factors requires delicate control of the dose and delivery strategies and may be accompanied by safety, efficacy and cost concerns. In this study, we developed, for the first time, a biomaterial-based strategy to activate endogenous transforming growth factor beta 1 (TGFß1) under alkaline conditions for effective in situ tissue regeneration. We demonstrated that alkaline-activated TGFß1 from blood serum, bone marrow fluids and soaking solutions of meniscus and tooth dentin was capable of increasing cell recruitment and early differentiation, implying its broad practicability. Furthermore, we engineered an injectable hydrogel (MS-Gel) consisting of gelatin microspheres for loading strong alkaline substances and a modified gelatin matrix for hydrogel click crosslinking. In vitro models showed that alkaline MS-Gel controllably and sustainably activated endogenous TGFß1 from tooth dentin for robust bone marrow stem cell migration. More importantly, infusion of in vivo porcine prepared root canals with alkaline MS-Gel promoted significant pulp-dentin regeneration with neurovascular stroma and mineralized tissue by endogenous proliferative cells. Therefore, this work offers a new bench-to-beside translation strategy using biomaterial-activated endogenous biomolecules to achieve in situ tissue regeneration without the need for cell or protein delivery.

Parenchymal and stromal tissue regeneration of tooth organ by pivotal signals reinstated in decellularized matrix.

Cells are transplanted to regenerate an organs' parenchyma, but how transplanted parenchymal cells induce stromal regeneration is elusive. Despite the common use of a decellularized matrix, little is known as to the pivotal signals that must be restored for tissue or organ regeneration. We report that Alx3, a developmentally important gene, orchestrated adult parenchymal and stromal regeneration by directly transactivating Wnt3a and vascular endothelial growth factor. In contrast to the modest parenchyma formed by native adult progenitors, Alx3-restored cells in decellularized scaffolds not only produced vascularized stroma that involved vascular endothelial growth factor signalling, but also parenchymal dentin via the Wnt/ß-catenin pathway. In an orthotopic large-animal model following parenchyma and stroma ablation, Wnt3a-recruited endogenous cells regenerated neurovascular stroma and differentiated into parenchymal odontoblast-like cells that extended the processes into newly formed dentin with a structure-mechanical equivalency to native dentin. Thus, the Alx3-Wnt3a axis enables postnatal progenitors with a modest innate regenerative capacity to regenerate adult tissues. Depleted signals in the decellularized matrix may be reinstated by a developmentally pivotal gene or corresponding protein.

Preparation of well-distributed titania nanopillar arrays on Ti6Al4V surface by induction heating for enhancing osteogenic differentiation of stem cells.

Great effort has recently been devoted to the preparation of nanoscale surfaces on titanium-based implants to achieve clinically fast osteoinduction and osseointegration, which relies on the unique characteristics of the nanostructure. In this work, we used induction heating treatment (IHT) as a rapid oxidation method to fabricate a porous nanoscale oxide layer on the Ti6Al4V surface for better medical application. Well-distributed vertical nanopillars were yielded by IHT for 20-35 s on the alloy surface. The composition of the oxides contained rutile/anatase TiO2 and a small amount of Al2O3 between the TiO2 grain boundaries (GBs). This technology resulted in a reduction and subsequent increase of surface roughness of 26-32 nm when upregulating the heating time, followed by the successive enhancement of the thickness, wettability and adhesion strength of the oxidation layer to the matrix. The surface hardness also distinctly rose to 554 HV in the IHT-35 s group compared with the 350 HV of bare Ti6Al4V. The massive small-angle GBs in the bare alloy promoted the formation of nanosized oxide crystallites. The grain refinement and deformation texture reduction further improved the mechanical properties of the matrix after IHT. Moreover, in vitro experiments on a mesenchymal stem cell (BMSC) culture derived from human bone marrow for 1-7 days indicated that the nanoscale layers did not cause cytotoxicity, and facilitated cell differentiation in osteoblasts by enhancing the gene and osteogenesis-related protein expressions after 1-3 weeks of culturing. The increase of the IHT time slightly advanced the BMSC proliferation and differentiation, especially during long-term culture. Our findings provide strong evidence that IHT oxidation technology is a novel nanosurface modification technology, which is potentially promising for further clinical development.

Optimal internal fixation of anatomically shaped synthetic bone grafts for massive segmental defects of long bones.

BACKGROUND: Large segmental bone defects following tumor resection, high-energy civilian trauma, and military blast injuries present significant clinical challenges. Tissue engineering strategies using scaffolds are being considered as a treatment, but there is little research into optimal fixation of such scaffolds. METHODS: Twelve fresh-frozen paired cadaveric legs were utilized to simulate a critical sized intercalary defect in the tibia. Poly-ε-caprolactone and hydroxyapatite composite scaffolds 5 cm in length with a geometry representative of the mid-diaphysis of an adult human tibia were fabricated, inserted into a tibial mid-diaphyseal intercalary defect, and fixed with a 14-hole large fragment plate. Optimal screw fixation comparing non-locking and locking screws was tested in axial compression, bending, and torsion in a non-destructive manner. A cyclic torsional test to failure under torque control was then performed. FINDINGS: Biomechanical testing showed no significant difference for bending or axial stiffness with non-locking vs. locking fixation. Torsional stiffness was significantly higher (P=0.002) with the scaffold present for both non-locking and locking compared to the scaffold absent. In testing to failure, angular rotation was greater for the non-locking compared to locking constructs at each torque level up to 40 N-m (P<0.05). The locking constructs survived a significantly higher number of loading cycles before reaching clinical failure at 30 degrees of angular rotation (P<0.02). INTERPRETATION: The presence of the scaffold increased the torsional stiffness of the construct. Locking fixation resulted in a stronger construct with increased cycles to failure compared to non-locking fixation.

The self-assembly of a mini-fibril with axial periodicity from a designed collagen-mimetic triple helix.

In this work we describe the self-assembly of a collagen-like periodic mini-fibril from a recombinant triple helix. The triple helix, designated Col108, is expressed in Escherichia coli using an artificial gene and consists of a 378-residue triple helix domain organized into three pseudo-repeating sequence units. The peptide forms a stable triple helix with a melting temperature of 41 °C. Upon increases of pH and temperature, Col108 self-assembles in solution into smooth mini-fibrils with the cross-striated banding pattern typical of fibrillar collagens. The banding pattern is characterized by an axially repeating feature of ∼35 nm as observed by transmission electron microscopy and atomic force microscopy. Both the negatively stained and the positively stained transmission electron microscopy patterns of the Col108 mini-fibrils are consistent with a staggered arrangement of triple helices having a staggering value of 123 residues, a value closely connected to the size of one repeat sequence unit. A mechanism is proposed for the mini-fibril formation of Col108 in which the axial periodicity is instigated by the built-in sequence periodicity and stabilized by the optimized interactions between the triple helices in a 1-unit staggered arrangement. Lacking hydroxyproline residues and telopeptides, two factors implicated in the fibrillogenesis of native collagen, the Col108 mini-fibrils demonstrate that sequence features of the triple helical domain alone are sufficient to "code" for axially repeating periodicity of fibrils. To our knowledge, Col108 is the first designed triple helix to self-assemble into periodic fibrils and offers a unique opportunity to unravel the specific molecular interactions of collagen fibrillogenesis.

CA-074Me compound inhibits osteoclastogenesis via suppression of the NFATc1 and c-FOS signaling pathways.

The osteoclast is an integral cell of bone resorption. Since osteolytic disorders hinge on the function and dysfunction of the osteoclast, understanding osteoclast biology is fundamental to designing new therapies that curb osteolytic disorders. The identification and study of lysosomal proteases, such as cathepsins, have shed light on mechanisms of bone resorption. For example, Cathepsin K has already been identified as a collagen degradation protease produced by mature osteoclasts with high activity in the acidic osteoclast resorption pits. Delving into the mechanisms of cathepsins and other osteoclast related compounds provides new targets to explore in osteoclast biology. Through our anti-osteoclastogenic compound screening experiments we encountered a modified version of the Cathepsin B inhibitor CA-074: the cell membrane-permeable CA-074Me (L-3-trans-(Propylcarbamoyl) oxirane-2-carbonyl]-L-isoleucyl-L-proline Methyl Ester). Here we confirm that CA-074Me inhibits osteoclastogenesis in vivo and in vitro in a dose-dependent manner. However, Cathepsin B knockout mice exhibited unaltered osteoclastogenesis, suggesting a more complicated mechanism of action than Cathepsin B inhibition. We found that CA-074Me exerts its osteoclastogenic effect within 24 h of osteoclastogenesis stimulation by suppression of c-FOS and NFATc1 pathways.

Long Electron-Hole Separation of ZnO-CdS Core-Shell Quantum Dots.

The tunability of electronic and optical properties of semiconductor nanocrystal quantum dots (QDs) has been an important subject in nanotechnology. While control of the emission property of QDs in wavelength has been studied extensively, control of the emission lifetime of QDs has not been explored in depth. In this report, ZnO-CdS core-shell QDs were synthesized in a two-step process, in which we initially synthesized ZnO core particles, and then stepwise slow growth of CdS shells followed. The coating of a CdS shell on a ZnO core increased the exciton lifetime more than 100 times that of the core ZnO QD, and the lifetime was further extended as the thickness of shell increased. This long electron-hole recombination lifetime is due to a unique staggered band alignment between the ZnO core and CdS shell, so-called type II band alignment, where the carrier excitation holes and electrons are spatially separated at the core and shell, and the exciton lifetime becomes extremely sensitive to the thickness of the shell. Here, we demonstrated that the emission lifetime becomes controllable with the thickness of the shell in ZnO-CdS core-shell QDs. The longer excitonic lifetime of type II QDs could be beneficial in fluorescence-based sensors, medical imaging, solar cells photovoltaics, and lasers.

Crossbar assembly of antibody-functionalized peptide nanotubes via biomimetic molecular recognition.

Previously, a large scale assembly of nanowires in a parallel array configuration has been demonstrated, and one type of nanowire could interconnect two electrodes in the high-wire density. However, to assemble nanowires into practical logic-gate configurations in integrated circuits, we need more than the parallel assembly of nanowires. For example, when the assembling nanowires are monopolar semiconductors, logic gates such as AND, OR and NOR are to be assembled necessarily from two types of semiconducting nanowires, n-type and p-type, and some of these nanowires must cross perpendicularly to form a crossbar geometry for the logical operation. In this paper, the crossbar assembly of antibody-functionalized peptide nanotubes was demonstrated by a new biomimetic bottom-up technique. Molecular recognition between antigens and antibodies enabled two types of the antibody-functionalized bionanotubes to place them onto targeted locations on substrates, where their complementary antigens were patterned. When two rectangular pads of antigens, human IgG and mouse IgG, were patterned perpendicularly on an Au substrate by nanolithography and then the antihuman IgG nanotubes and the antimouse IgG nanotubes were incubated on this substrate in solution, these bionanotubes were attached onto corresponding locations to form the crossbar configuration.

Effect of electrode surface microstructure on electron transfer induced conformation changes in cytochrome c monitored by in situ UV and CD spectroelectrochemistry.

Electrochemical redox processes of bovine heart cytochrome c were investigated by in situ UV-vis and CD spectroelectrochemistry at bare glassy carbon electrode (GCE) and single-wall carbon nanotubes (SWNTs) modified glassy carbon electrode (SWNTs/GCE) using a long optical path thin layer cell. The spectra obtained at GCE and SWNTs/GCE reflect electrode surface microstructure-dependent changes in protein conformation during redox transition. Potential-dependent conformational distribution curves of cytochrome c obtained by analysis of in situ circular dichroism (CD) spectra using singular value decomposition least square (SVDLS) method show that SWNTs can retain conformation of cytochrome c. Some parameters of the electrochemical reduction process, i.e. the product of electron transfer coefficient and number of electrons (alpha n = 0.3), apparent formal potential (E0' = 0.04 V), were obtained by double logarithmic analysis and standard heterogeneous electron transfer rate constant k0 was obtained by electrochemistry and double logarithmic analysis, respectively.

In situ circular dichroic electrochemical study of bilirubin and bovine serum albumin complex.

The electrooxidation of bilirubin (BR) and bovine serum albumin (BSA) complexes was studied by in situ circular dichroism (CD) spectroelectrochemistry. The result showed that the mechanism of the whole electrooxidation process of this complex corresponded to electrochemical processes (EE mechanism) in aqueous solution. Some parameters of the process were obtained by double logarithm method, differential method and nonlinear regression method. In visible region, CD spectra of the two enantiomeric components of the complex and their fraction distribution against applied potentials were obtained by singular value decomposition least-square (SVDLS) method. Meanwhile, the distribution of the five components of secondary structure was also obtained by the same method in far-UV region. The peak potential gotten from EE mechanism corresponds to a turning point for the component transition, beyond which the whole reaction reaches a new equilibrium. Under applied positive potentials, the enantiomeric equilibrium between M and P form is broken and M form transfers to its enantiomer of P, while the fraction of alpha-helix increases and that improves the transition to P form.

The structural transition of DNA-Tris(1,10-phenanthroline) cobalt(III) complexes in ethanol-water solution.

The interaction of DNA with Tris(1,10-phenanthroline) cobalt(III) was studied by means of atomic force microscopy. Changes in the morphologies of DNA complex in the presence of ethanol may well indicate the crucial role of electrostatic force in causing DNA condensation. With the increase of the concentration of ethanol, electrostatic interaction is enhanced corresponding to a lower dielectric constant. Counterions condense along the sugar phosphate backbone of DNA when epsilon is lowered and the phosphate charge density can thus be neutralized to the level of DNA condensation. Electroanalytical measurement of DNA condensed with Co(phen)(3)(3+) in ethanol solution indicated that intercalating reaction remains existing. According to both the microscopic and spectroscopic results, it can be found that no secondary structure transition occurs upon DNA condensing. B-A conformation transition takes place at more than 60% ethanol solution.