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Mass spectrometry imaging (MSI) enables the spatial distributions of molecules possessing different mass‐to‐charge ratios to be mapped within complex environments revealing regional changes at the molecular level. Even at high mass resolving power, however, these images often reflect the summed distribution of multiple isomeric molecules, each potentially possessing a unique distribution coinciding with distinct biological function(s) and metabolic origin. Herein, this chemical ambiguity is addressed through an innovative combination of ozone‐induced dissociation reactions with MSI, enabling the differential imaging of isomeric lipid molecules directly from biological tissues. For the first time, we demonstrate both double bond‐ and sn‐positional isomeric lipids exhibit distinct spatial locations within tissue. This MSI approach enables researchers to unravel local lipid molecular complexity based on both exact elemental composition and isomeric structure directly from tissues.

Umecyanin (UMC) is a type 1 copper-containing protein which originates from horseradish roots and belongs to the stellacyanin subclass of the phytocyanins, a ubiquitous family of plant cupredoxins. The crystal structures of Cu(II) and Cu(I) UMC have been determined at 1.9 and 1.8 A, respectively. The protein has an overall fold similar to those of other phytocyanins. At the active site the cupric ion is coordinated by the N(delta1) atoms of His44 and His90, the S(gamma) of Cys85, and the O(epsilon)(1) of Gln95 in a distorted tetrahedral geometry. Both His ligands are solvent exposed and are surrounded by nonpolar and polar side chains on the protein surface. Thus, UMC does not possess a distinct hydrophobic patch close to the active site in contrast to almost all other cupredoxins. UMC has a large surface acidic patch situated approximately 10-30 A from the active site. The structure of Cu(I) UMC is the first determined for a reduced phytocyanin and demonstrates that the coordination environment of the cuprous ion is more trigonal pyramidal. This subtle change in geometry is primarily due to the Cu-N(delta1)(His44) and Cu-O(epsilon1)(Gln95) bond lengths increasing from 2.0 and 2.3 A in Cu(II) UMC to 2.2 and 2.5 A, respectively, in the reduced form, as a consequence of slight rotations of the His44 and Gln95 side chains. The limited structural changes upon redox interconversion at the active site of this stellacyanin are analogous to those observed in a typical type 1 copper site with an axial Met ligand and along with its surface features suggest a role for UMC in interprotein electron transfer.

Calcium silicate (CaSiO3, CS) ceramics have received significant attention for application in bone regeneration due to their excellent in vitro apatite-mineralization ability; however, how to prepare porous CS scaffolds with a controllable pore structure for bone tissue engineering still remains a challenge. Conventional methods could not efficiently control the pore structure and mechanical strength of CS scaffolds, resulting in unstable in vivo osteogenesis. The aim of this study is to set out to solve these problems by applying a modified 3D-printing method to prepare highly uniform CS scaffolds with controllable pore structure and improved mechanical strength. The in vivo osteogenesis of the prepared 3D-printed CS scaffolds was further investigated by implanting them in the femur defects of rats. The results show that the CS scaffolds prepared by the modified 3D-printing method have uniform scaffold morphology. The pore size and pore structure of CS scaffolds can be efficiently adjusted. The compressive strength of 3D-printed CS scaffolds is around 120 times that of conventional polyurethane templated CS scaffolds. 3D-Printed CS scaffolds possess excellent apatite-mineralization ability in simulated body fluids. Micro-CT analysis has shown that 3D-printed CS scaffolds play an important role in assisting the regeneration of bone defects in vivo. The healing level of bone defects implanted by 3D-printed CS scaffolds is obviously higher than that of 3D-printed β-tricalcium phosphate (β-TCP) scaffolds at both 4 and 8 weeks. Hematoxylin and eosin (H&E) staining shows that 3D-printed CS scaffolds induce higher quality of the newly formed bone than 3D-printed β-TCP scaffolds. Immunohistochemical analyses have further shown that stronger expression of human type I collagen (COL1) and alkaline phosphate (ALP) in the bone matrix occurs in the 3D-printed CS scaffolds than in the 3D-printed β-TCP scaffolds. Considering these important advantages, such as controllable structure architecture, significant improvement in mechanical strength, excellent in vivo osteogenesis and since there is no need for second-time sintering, it is indicated that the prepared 3D-printed CS scaffolds are a promising material for application in bone regeneration.

Mammalian cells cultured on 2D surfaces in microfluidic channels are increasingly used in drug development and biological research applications. These systems would have more biological or clinical relevance if the cells exhibit 3D phenotypes similar to the cells in vivo. We have developed a microfluidic channel based system that allows cells to be perfusion-cultured in 3D by supporting them with adequate 3D cell–cell and cell–matrix interactions. The maximal cell–cell interaction was achieved by perfusion-seeding cells through an array of micropillars; and 3D cell–matrix interactions were achieved by a polyelectrolyte complex coacervation process to form a thin layer of matrix conforming to the 3D cell shapes. Carcinoma cell lines (HepG2, MCF7), primary differentiated (hepatocytes) and primary progenitor cells (bone marrow mesenchymal stem cells) were perfusion-cultured for 72 hours to 1 week in the microfluidic channel, which preserved their 3D cyto-architecture and cell-specific functions or differentiation competence. This transparent 3D microfluidic channel-based cell culture system also allows direct optical monitoring of cellular events for a wide range of applications.

Although poly(alpha-hydroxy esters), especially the PLGA family of lactic acid/glycolic acid copolymers, have many properties which make them promising materials for tissue engineering, the inherent chemistry of surfaces made from these particular polymers is problematic. In vivo, they promote a strong foreign-body response as a result of nonspecific adsorption and denaturation of serum proteins, which generally results in the formation of a nonfunctional fibrous capsule. Surface modification post-production of the scaffolds is an often-utilized approach to solving this problem, conceptually allowing the formation of a scaffold with mechanical properties defined by the bulk material and molecular-level interactions defined by the modified surface properties. A promising concept is the so-called "blank slate": essentially a surface that is rendered resistant to nonspecific protein adsorption but can be readily activated to covalently bind bio-functional molecules such as extracellular matrix proteins, growth factors or polysaccharides. This study focuses on the use of the quartz crystal microbalance (QCM) to follow the layer-by-layer (LbL) electrostatic deposition of high molecular weight hyaluronic acid and chitosan onto PLGA surfaces rendered positively charged by aminolysis, to form a robust, protein-resistant coating. We further show that this surface may be further functionalized via the covalent attachment of collagen IV, which may then be used as a template for the self-assembly of basement membrane components from dilute Matrigel. The response of NIH-3T3 fibroblasts to these surfaces was also followed and shown to closely parallel the results observed in the QCM.

Collagen modification of scaffolds has been reported to promote matrix mineralization as an effective way to increase osseointegration of implants. The aim of this study was to investigate in vitro proliferation and differentiation of human alveolar osteoblasts (AOs) on medical-grade polycaprolactone–tricalcium phosphate (mPCL-TCP 80:20) scaffolds after collagen modification (mPCL-TCP-c) for 28 days. Collagen modification significantly increased the scaffold’s protein adsorption ability, and improved the initial seeding efficiency and cell attachment at day 1, compared with non-collagen-modified scaffolds. However, the total DNA content of both groups reached similar levels with no significant difference at 28 days’ culture. AOs were observed to spread along the collagen fibres and form extensive collagenous fibres with mineral nodules embedded, while multilayered cell sheets were formed in mPCL-TCP scaffolds. During culture, alkaline phosphatase (ALP) activity increased three- to five-fold in both groups, and collagen modification did not significantly affect either the metabolic rate or ALP activity kinetics of AOs. During osteogenic differentiation, similar gene expression of collagen type-I, osterix, osteopontin, and osteocalcin were detected in both groups. The mPCL-TCP group showed better organized mineralized tissue, but the mPCL-TCP-c showed more scattered and unorganized tissue. These results indicate that collagen modification improved the scaffold’s protein adsorption ability and encouraged initial cell attachment and distribution, but promoted fibrous-like tissue formation rather than mineralized tissue.