Vapor construction and modification of stem cell-laden multicomponent scaffolds for regenerative therapeutics.

Tissue engineering based on the combined use of isolated cells, scaffolds, and growth factors is widely used; however, the manufacture of cell-preloaded scaffolds faces challenges. Herein, we fabricated a multicomponent scaffold with multiple component accommodations, including bioactive molecules (BMs), such as fibroblast growth factor-2 (FGF-2) and l-ascorbic acid 2-phosphate (A2-P), and living cells of human adipose-derived stem cells (hASCs), within one scaffold construct. We report an innovative fabrication process based on vapor-phased construction using iced templates for vapor sublimation. Simultaneously, the vaporized water molecules were replaced by vapor deposition of poly-p-xylylene (PPX, USP Class VI, highly compatible polymer, FDA-approved records), forming a three-dimensional and porous scaffold matrix. More importantly, a multicomponent modification was achieved based on using nonvolatile solutes, including bioactive molecules of FGF-2 and A2-P, and living cells of hASCs, to prepare iced templates for sublimation. Additionally, the fabrication and construction resulted in a multicomponent scaffold product comprising the devised molecules, cells, and vapor-polymerized poly-p-xylylene as the scaffold matrix. The clean and dry fabrication process did not require catalysts, initiators or plasticizers, and potentially harmful solvents, and the scaffold products were produced in simple steps within hours of the processing time. Cell viability analysis showed a high survival rate (approximately 86.4%) for the accommodated hASCs in the fabricated scaffold product, and a surprising multilineage differentiation potential of hASCs was highly upregulated because of synergistic guidance by the same accommodated FGF-2 and A2-P components. Proliferation and self-renewal activities were also demonstrated with enhancement of the multicomponent scaffold product. Finally, in vivo calvarial defect studies further revealed that the constructed scaffolds provided blood vessels to grow into the bone defect areas with enhancement, and the induced conduction of osteoblast growth also promoted bone healing toward osseointegration. The reported scaffold construction technology represents a prospective tissue engineering scaffold product to enable accommodable and customizable versatility to control the distribution and composition of loading delicate BMs and living hASCs in one scaffold construct and demonstrates unlimited applications in tissue engineering repair and regenerative medicine applications.

Vapor-phased fabrication and modulation of cell-laden scaffolding materials.

Bottom-up approaches using building blocks of modules to fabricate scaffolds for tissue engineering applications have enabled the fabrication of structurally complex and multifunctional materials allowing for physical and chemical flexibility to better mimic the native extracellular matrix. Here we report a vapor-phased fabrication process for constructing three-dimensional modulated scaffold materials via simple steps based on controlling mass transport of vapor sublimation and deposition. We demonstrate the fabrication of scaffolds comprised of multiple biomolecules and living cells with built-in boundaries separating the distinct compartments containing defined biological configurations and functions. We show that the fabricated scaffolds have mass production potential. We demonstrate overall >80% cell viability of encapsulated cells and that modulated scaffolds exhibit enhanced cell proliferation, osteogenesis, and neurogenesis, which can be assembled into various geometric configurations. We perform cell co-culture experiments to show independent osteogenesis and angiogenesis activities from separate compartments in one scaffold construct.

Synergistically Enhanced Wound Healing of a Vapor-Constructed Porous Scaffold.

In this study, a porous, three-dimensional material of parylene (poly-p-xylylene) incorporating keratin was fabricated. As an FDA-approved material, parylene is highly stable and biocompatible. Keratin is an abundant natural material that can enhance cell adhesion and wound healing. A unique vapor deposition construction technique dispersed keratin homogenously in the parylene system. Instead of using a conventional template, a sublimating template was applied. This composite porous scaffold was investigated mechanically and biologically. In addition, human adipose stem cells were cultured on the scaffold. The synergistic functions, including biocompatibility, permeability, cell adhesion, and stem cell differentiation, were investigated. A mouse excisional wound-healing model further verified that using the porous composite scaffold with stem cells accelerated the wound healing process, supporting its in vivo efficacy. The positive results demonstrated that this material has the potential to serve as a wound repair platform.

Parylene-Based Porous Scaffold with Functionalized Encapsulation of Platelet-Rich Plasma and Living Stem Cells for Tissue Engineering Applications.

A scaffold was fabricated to synergistically encapsulate living human adipose-derived stem cells (hASCs) and platelet-rich plasma (PRP) based on a vapor-phase sublimation and deposition process. During the process, ice templates were prepared using sterile water as the solvent and were used to accommodate the sensitive living cells and PRP molecules. Under controlled processing conditions, the ice templates underwent vapor sublimation to evaporate water molecules, while at the same time, vapor-phase deposition of poly-p-xylylene (Parylene, USP Class VI highly biocompatible) occurred to replace the templates, and the final construction yielded a scaffold with Parylene as the matrix, with simultaneously encapsulated living hASCs and PRP molecules. Evaluation of the fabricated synergistic scaffold for the proliferation activities toward the encapsulated hASCs indicated significant augmentation of cell proliferation contributed by the PRP ingredients. In addition, osteogenic activity in the early stage by alkaline phosphatase expression and later stage with calcium mineralization indicated significant enhancement toward osteogenetic differentiation of the encapsulated hASCs, which were guided by the PRP molecules. By contrast, examinations of adipogenic activity by lipid droplet formation revealed an inhibition of adipogenesis with decreased intracellular lipid accumulation, and a statistically significant downregulation of adipogenic differentiation was postulated for the scaffold products when compared to the osteogenetic results and the control experiments. The reported fabrication method featured a clean and simple process to construct scaffolds that combined delicate living hASCs and PRP molecules inside the structure. The resultant synergistic scaffold and the selected commercially available hASCs and PRP are emerging as tissue engineering tools that provide multifunctionality for tissue repair and regeneration.

Nucleophilic Substitution of gem-Difluoroalkenes with TMSNu Promoted by Catalytic Amounts of Cs2CO3.

The efficient and practical nucleophilic cyanation and trifluoromethylation with appropriate trimethylsilyl nucleophiles were developed. Catalytic amounts of cheap and nontoxic Cs2CO3 were used to maintain a sufficiently high concentration of nucleophilic anion (CN- or CF3-) which could begin the catalytic cycle. The present methodologies provide diverse functionalized monofluoroalkenes bearing a cyano and trifluoromethyl group with excellent to moderate stereoselectivities.

Characterization of Mechanical Stability and Immunological Compatibility for Functionalized Modification Interfaces.

Surface modification layers are performed on the surfaces of biomaterials and have exhibited promise for decoupling original surface properties from bulk materials and enabling customized and advanced functional properties. The physical stability and the biological compatibility of these modified layers are equally important to ensure minimized delamination, debris, leaching of molecules, and other problems that are related to the failure of the modification layers and thus can provide a long-term success for the uses of these modified layers. A proven surface modification tool of the functionalized poly-para-xylylene (PPX) system was used as an example, and in addition to the demonstration of their chemical conjugation capabilities and the functional properties that have been well-documented, in the present report, we additionally devised the characterization protocols to examine stability properties, including thermostability and adhesive strength, as well as the biocompatibility, including cell viability and the immunological responses, for the modified PPX layers. The results suggested a durable coating stability for PPXs and firmly attached biomolecules under these stability and compatibility tests. The durable and stable modification layers accompanied by the native properties of the PPXs showed high cell viability against fibroblast cells and macrophages (MΦs), and the resulting immunological activities created by the MΦs exhibited excellent compatibility with non-activated immunological responses and no indication of inflammation.

Clickable and Photo-Erasable Surface Functionalities by Using Vapor-Deposited Polymer Coatings.

A prospective design for interface properties is enabled to perform precise functionalization, erasure capability for existing properties, reactivation of surface functionality to a second divergent property. A vapor-deposited, 2-nitro-5-(prop-2-yn-1-yloxy)methylbenzyl carbamate-functionalized poly-para-xylylene coating is synthesized in this study to realize such tasks by offering the accessibility of the azide/alkyne click reaction, an integrated photochemical decomposition/cleavage moiety, and the reactivation sites of amines behind the cleavage that allow the installation of a second surface function. With the benefits from the mild processing conditions used for the coatings and the rapid response of the photochemical reaction, the creation of sophisticated interface properties and localized chemical compositions was elegantly demonstrated with a hybrid functionality including a confined hydrophlic/hydrophobic wetting property and/or a cell adherent/repellent platform on such a coating surface.

Defined cell adhesion for silicon-based implant materials by using vapor-deposited functional coatings.

The field of implantable electronics relies on using silicon materials due to the merits of a well-established fabrication process and favorable properties; of particular interest is the surface modification of such materials. In the present study, we introduce a surface modification technique based on coatings of functionalized Parylene on silicon substrates, where the modified layers provide a defined cell adhesion capability for the resultant silicon materials/devices. Functionalization of Parylene was achieved during a one-step chemical vapor deposition (CVD) polymerization process, forming NHS ester-functionalized Parylene, and subsequent RGD attachment was enabled via a conjugation reaction between the NHS ester and amine groups. The modification procedures additionally provided a clean and gentle approach to avoid thermal excursions, intense irradiation, chemicals, or solvents that might damage delicate structures or sensitive molecules on the devices. The modification layers exhibited excellent mechanical strength on the substrate, meeting the high standards of the American Society for Testing and Materials (ASTM), and the resultant cell adherence property was verified by a centrifugation assay and the analysis of attached cell morphologies; the results collectively demonstrated robust and sustainable modification layers of the NHS ester-functionalized Parylene and confirmed that the cell-adherence property imparted by using this facile modification technique was effective. The modification technology is expected to benefit the design of prospective interface properties for silicon-based devices and related industrial products.

Synthesis of ß-Alkyl 2-Hydroxychalcones by Rhodium-Catalyzed Coupling of N-Phenoxyacetamides and Nonterminal Propargyl Alcohols.

An efficient rhodium-catalyzed coupling of N-phenoxyacetamides and nonterminal propargyl alcohols has been developed. A series of ß-Alkyl 2-hydroxychalcones bearing diverse functional groups were obtained with excellent regio- and stereoselectivity, and the desired chalcones could then be converted to triazole and chromene smoothly.

Vapor sublimation and deposition to build porous particles and composites.

The vapor deposition of polymers on regular stationary substrates is widely known to form uniform thin films. Here we report porous polymer particles with sizes controllable down to the nanometer scale can be produced using a fabrication process based on chemical vapor deposition (CVD) on a dynamic substrate, i.e., sublimating ice particles. The results indicate that the vapor deposition of a polymer is directed by the sublimation process; instead of forming a thin film polymer, the deposited polymers replicated the size and shape of the ice particle. Defined size and porosity of the polymer particles are controllable with respect to varying the processing time. Extendable applications are shown to install multiple functional sites on the particles in one step and to localize metals/oxides forming composite particles. In addition, one fabrication cycle requires approximately 60 min to complete, and potential scaling up the production of the porous particles is manageable.

Surface modification: activation and deactivation of osteogenic differentiation based on detachable growth factor protein.

An advanced material interface is modified by using a substrate-independent coating of detachable poly-para-xylylene, enabling dynamical control of the immobilization and detachment of biomolecules, and a previously installed biological function is deactivated or tuned with reduced activity. The induction of osteogenesis activity, and subsequent deactivation of such osteogenesis activity, is demonstrated.

Artificial intelligence in clinical medicine: Application and thinking / 第二军医大学学报

Artificial intelligence (AI) has become a national strategy in developed countries. AI has been a great success in many scenarios of medicine and health fields, including virtual assistant, medical imaging, drug mining, nutrition, hospital management, health management, mental illness, wearable devices, risk management, pathology and clinical diagnosis and treatment. Here, we reviewed and commented the recent progresses on the application of AI in the pathological diagnosis, ocular diseases, skin disorders, medical imaging, traditional Chinese medicine, electrocardiographic monitoring, medical robots, oncotherapy and translational researches, and summed up and prospected the problems of AI application in medicine and health fields. With the development of technology, AI will lead to a revolutionary progress in medical treatment.

Topologically Controlled Cell Differentiation Based on Vapor-Deposited Polymer Coatings.

In addition to the widely adopted method of controlling cell attachment for cell patterning, pattern formation via cell proliferation and differentiation is demonstrated using precisely defined interface chemistry and spatial topology. The interface platform is created using a maleimide-functionalized parylene coating (maleimide-PPX) that provides two routes for controlled conjugation accessibility, including the maleimide-thiol coupling reaction and the thiol-ene click reaction, with a high reaction specificity under mild conditions. The coating technology is a prime tool for the immobilization of sensitive molecules, such as growth factor proteins. Conjugation of fibroblast growth factor 2 (FGF-2) and bone morphogenetic protein (BMP-2) was performed on the coating surface by elegantly manipulating the reaction routes, and confining the conjugation reaction to selected areas was accomplished using microcontact printing (µCP) and/or UV irradiation photopatterning. The modified interface provides chemically and topologically defined signals that are recognized by cultured murine preosteoblast cells for proliferation (by FGF-2) and osteogenesis (by BMP-2) activities in specific locations. The reported technique additionally enabled synergistic pattern formation for both osteogenesis and proliferation activities on the same interface, which is difficult to perform using conventional cell attachment patterns. Because of the versatility of the coating, which can be applied to a wide range of materials and on curved and complex devices, the proposed technology is extendable to other prospective biomaterial designs and material interface modifications.

Stepwise and Programmable Cell Differentiation Pathways of Controlled Functional Biointerfaces.

An advanced control of biomaterial surfaces was created to enable the stepwise and switchable activities of the immobilized growth factor (GF) proteins for a programmed manipulation over cell differentiation pathways. The GF protein was immobilized on an advanced vapor-based coating of poly[(4-2-amide-2'-amine-dithiobisethyl-p-xylylene)-co-(p-xylylene)], and the equipped disulfide exchange mechanism of the coating enables the detachment and/or the displacement of the previously installed GF to reinstall a second GF protein. In this study, the controlled immobilization and displacement of the fibroblast growth factor (FGF-2) and bone morphogenetic protein (BMP-2) were demonstrated on cell culture substrates, and the resulting surfaces provided a programmable induction of cellular responses in proliferation and osteogenesis toward the cultured murine preosteoblasts (MC3T3-E1). A depreciated or stopped activity of the previously induced biological function, i.e., proliferation or osteogenesis, was found for MC3T3-E1 on the modified surface after the cleavage of the corresponding GF. In addition, with the approach to devise the displacement and reinstallation of FGF-2/BMP-2 proteins, a combined induction of proliferation and osteogenesis can be initiated in a timed latency to resolve programmable biological activities.

Controlling multi-function of biomaterials interfaces based on multiple and competing adsorption of functional proteins.

Multifunctional biomaterial surfaces can be created by controlling the competing adsorption of multiple proteins. To demonstrate this concept, bone morphogenetic protein 2 (BMP-2) and fibronectin were adsorbed to the hydrophobic surface of polychloro-para-xylylene. The resulting adsorption properties on the surface depended on the dimensional and steric characteristics of the selected protein molecule, the degree of denaturation of the adsorbed proteins, the associated adsorption of interphase water molecules within the protein layers, and the aggregation of proteins in a planar direction with respect to the adsorbent surface. Additionally, a defined surface composition was formed by the competing adsorption of multiple proteins, and this surface composition was directly linked to the composition of the protein mixture in the solution phase. Although the mechanism of this complex competing adsorption process is not fully understood, the adsorbed proteins were irreversibly adsorbed and were unaffected by the further adsorption of homologous or heterologous proteins. Moreover, synergistic biological activities, including cell osteogenesis and proliferation independently and specifically induced by BMP-2 or fibronectin, were observed on the modified surface, and these biological activities were positively correlated with the surface composition of the multiple adsorbed proteins. These results provide insights and important design parameters for prospective biomaterials and biointerfaces for (multi)functional modifications. The ability to control protein/interface properties will be beneficial for the processing of biomaterials for clinical applications and industrial products.

Vapor-based coatings for antibacterial and osteogenic functionalization and the immunological compatibility.

The immobilization of biofunctional molecules to biomaterial surfaces has enabled and expanded the versatility of currently available biomaterials to a wider range of applications. In addition, immobilized biomolecules offer modified surfaces that allow the use of smaller amounts of potentially harmful substances or prevent overdose, while the exhibited biological functions remain persistently effective. Surface concentrations of chlorhexidine (CHX) (1.40±0.08×10(-9)mol·cm(-2)) and bone morphogenetic protein 2 (BMP-2) (1.51±0.08×10(-11)mol·cm(-2)) immobilized molecules were determined in this study, and their specific biological functions in terms of antibacterial activity and osteogenesis potency, respectively, were demonstrated to be unambiguously effective. Immobilization exploits the use of vapor-based poly-p-xylylenes, which exhibit excellent biocompatibility and wide applicability for various substrate materials. This technique represents a practical and economical approach for the manufacture of certain industrial products. Furthermore, a minimal degree of macrophage activation was indicated on the modified surfaces via insignificant morphological changes and low levels of adverse inflammatory signals, including suppressed production of the pro-inflammatory cytokines IL-1ß and TNF-α as well as nitric oxide (NO). The results and the modification strategy illustrate a concept for designing prospective biomaterial surfaces such that the manipulation employed to elicit targeted biological responses does not compromise immunological compatibility.

Multifunctional and Continuous Gradients of Biointerfaces Based on Dual Reverse Click Reactions.

Chemical or biological gradients that are composed of multifunctional and/or multidirectional guidance cues are of fundamental importance for prospective biomaterials and biointerfaces. As a proof of concept, a general modification approach for generating multifunctional and continuous gradients was realized via two controlled and reversed click reactions, namely, thermo-activated thiol-yne and copper-free alkyne and azide click reactions. The cell adhesion property of fibroblasts was guided in a gradient with an enhancement, showing that the PEG molecule and RGD peptide were countercurrently immobilized to form such reversed gradients (with negating of the cell adhesion property). Using the gradient modification protocol to also create countercurrent distributions of FGF-2 and BMP-2 gradients, the demonstration of not only multifunctional but also gradient biointerfacial properties was resolved in time latencies on one surface by showing the manipulation in gradients toward proliferation and osteogenic differentiation for adipose-derived stem cells.

Fabrication of multipotent poly-para-xylylene particles in controlled nanoscopic dimensions.

In this study, poly-para-xylylene-based multifunctional nanoparticles (PPX-NPs) were fabricated. Based on the solubility characteristics determined for asymmetrically substituted poly-para-xylylenes in polar solvents, well-dispersed nanocolloids with a controllable size ranging from 50 to 800nm were produced in solution by the displacement of the solvent (water). These size ranges were found to have acceptable cellular compatibility through examinations of cultured 3T3 fibroblasts and adipose-derived stem cells treated with the PPX-NPs. In addition, these nanoscale PPX-NPs exhibited versatile bioconjugation properties in that a variety of available functional groups can be adopted from their counterpart, thin-film poly-para-xylylenes, during the production of these nanoparticles. For instance, bifunctional PPX-NPs with maleimide and benzoyl moieties were produced to enable immobilization via a maleimide-thiol reaction concurrent with a photochemical reaction. A cleavable PPX-NP was also produced with a thiol-exchangeable surface property. Additionally, by performing electrohydrodynamic jetting of parallel polymer solutions of selected poly-para-xylylenes, Janus-type or multicompartment PPX-NPs were created. The PPX-NPs can potentially be used for various biomedical applications such as combined diagnostics and drug delivery, multiplexing of detection, multiple-drug loading, and the targeted delivery of biomolecules or drugs.

Switching the Biointerface of Displaceable Poly-p-xylylene Coatings.

A new class of functionalized poly-p-xylyene coating has been synthesized to provide switchable and displaceable surface properties for biomaterials. The switchability is achieved through a mechanism for detaching/attaching biomolecules and/or a mechanism through which the programmed restoration of functions or their replacement by other functions can be carried out. This advanced version of poly-p-xylylene comprises an integrated disulfide moiety within the functional side group, and the switching phenomenon between the immobilized functional molecules is triggered by the redox thiol-disulfide interchange reaction. These dynamically well-defined molecules on the surfaces respond simultaneously to altered biological properties and controlled biointerfacial functions, for example, switching wettability or reversibly altered cell adhesion activity. Poly-p-xylylenes are a key player in controlling surface properties for many important applications, such as medical implants, biosensors, bioMEMS devices, and microfluidics. The introduction of this new facet of poly-p-xylylenes enables the dynamic mimicry of biological functions relevant to the design of new biomaterials.

Reduced protein expression of metastasis-related genes (nm23, KISS1, KAI1 and p53) in lymph node and liver metastases of gastric cancer.

PURPOSE: Metastasis remains an incurable common complication in patients with gastric cancer. A variety of theories have been proposed to explain the inefficiency of the metastatic process. To compare protein expression of metastasis-related genes (nm23, KISS1, KAI1 and p53) between primary tumours and metastatic tumours may be useful in illustrating these theories. METHODS: Metastasis-related tissue microarrays (including normal tissues, primary tumours, nodal metastases and liver metastases) were constructed. The protein expression of nm23, KISS1, KAI1 and p53 in lymph node and liver metastases from advanced gastric cancer specimens was mainly examined by immunohistochemical staining in relation to primary tumours. RESULTS: Immunohistochemical staining showed reduced protein expression of nm23, KISS1 and KAI1 in lymph node and liver metastases compared with primary tumours. Results for p53 were to the contrary. CONCLUSIONS: Our investigations revealed a tendency of reduced protein expression of metastasis suppressor genes nm23, KISS1 and KAI1 in gastric cancer with the progress of metastasis. This means that the progression theory is an important determinant of metastatic efficiency.