Guiding bone cell network formation in 3D via photosensitized two-photon ablation.

A long-standing challenge in skeletal tissue engineering is to reconstruct a three-dimensionally (3D) interconnected bone cell network in vitro that mimics the native bone microarchitecture. While conventional hydrogels are extensively used in studying bone cell behavior in vitro, current techniques lack the precision to manipulate the complex pericellular environment found in bone. The goal of this study is to guide single bone cells to form a 3D network in vitro via photosensitized two-photon ablation of microchannels in gelatin methacryloyl (GelMA) hydrogels. A water-soluble two-photon photosensitizer (P2CK) was added to soft GelMA hydrogels to enhance the ablation efficiency. Remarkably, adding 0.5 mM P2CK reduced the energy dosage threshold five-fold compared to untreated controls, enabling more cell-compatible ablation. By employing low-energy ablation (100 J/cm2) with a grid pattern of 1 µm wide and 30 µm deep microchannels, we induced dendritic outgrowth in human mesenchymal stem cells (hMSC). After 7 days, the cells successfully utilized the microchannels and formed a 3D network. Our findings reveal that cellular viability after low-energy ablation was comparable to unablated controls, whereas high-energy ablation (500 J/cm2) resulted in 42 % cell death. Low-energy grid ablation significantly promoted network formation and >40 µm long protrusion outgrowth. While the broad-spectrum matrix metalloproteinase inhibitor (GM6001) reduced cell spreading by inhibiting matrix degradation, cells invaded the microchannel grid with long protrusions. Collectively, these results emphasize the potential of photosensitized two-photon hydrogel ablation as a high-precision tool for laser-guided biofabrication of 3D cellular networks in vitro. STATEMENT OF SIGNIFICANCE: The inaccessible nature of osteocyte networks in bones renders fundamental research on skeletal biology a major challenge. This limit is partly due to the lack of high-resolution tools that can manipulate the pericellular environment in 3D cultures in vitro. To create bone-like cellular networks, we employ a two-photon laser in combination with a two-photon sensitizer to erode microchannels with low laser dosages into GelMA hydrogels. By providing a grid of microchannels, the cells self-organized into a 3D interconnected network within days. Laser-guided formation of 3D networks from single cells at micron-scale resolution is demonstrated for the first time. In future, we envisage in vitro generation of bone cell networks with user-dictated morphologies for both fundamental and translational bone research.

Hexakis-TIPS-Alkynylated Nonacenes: Persistent and Processible.

Four substituted nonacenes were prepared and characterized by UV-vis and EPR spectroscopy and X-ray crystallography. The compounds are the most stable and soluble nonacenes to date - due to six strategically placed triisopropylsilyl(TIPS)-ethynyl groups. They are stable for several weeks in the solid state. In dilute solution their half-life is 5-9 h. Crystal structure analyses of two nonacenes prove their structures. A nonacene derivative was tested in a solution-processed transistor and exhibits ambipolar charge transport (µe =0.007 cm2 /Vs; µh =0.023 cm2 /Vs).

Second Generation of Cata-Annulated Azaacene Bisimides: Towards Electron-Accepting Materials.

This work presents the 2nd generation of cata-annulated azaacene bisimides with increased electron affinities (up to -4.38 eV) compared to their consaguine conventional azaacenes. These compounds were synthesized via Buchwald-Hartwig coupling followed by oxidation with MnO2 . Crystal structure engineering through variation of the bisimide substituents furnished crystalline derivatives suitable for proof of concept organic field effect transistors with electron mobilities up to 2.2×10-4  cm2 (Vs)-1 . Moreover, we were able to characterize the charge carrying species, the radical anion, using electron paramagnetic resonance and absorption measurements.

Ion Mobility Measurements of Multianionic Metalloporphyrin Dimers: Structural Changes Induced by Countercation Exchange.

We present gas-phase structures of dimers of MnIII and FeIII meso-tetra(4-sulfonatophenyl)porphyrin multianions with various amounts of sodium and hydrogen counterions. The structural assignments are achieved by combining mass spectrometry, ion mobility measurements, quantum chemical calculations, and trajectory method collision cross section calculations. For a common charge state, we observe significant topological variations in the dimer structures of [(MTPPS)2+nX](6-n)- (M=MnIII, FeIII; X=H, Na; n = 1-3) induced by replacing hydrogen counterions by sodium. For sodium, the dimer structures are much more compact, a finding that can be rationalized by the stronger interactions of the sodium cations with the anionic sulfonic acid groups of the porphyrins as compared to hydrogen. Graphical Abstract ᅟ.

Photodissociation of Free Metalloporphyrin Dimer Multianions.

We have used action photofragmentation spectroscopy in the visible spectral range (410 to 650 nm) to investigate the optical properties of different monomeric and dimeric M(II)-meso-tetra-(4-sulfonatophenyl)-porphyrin (with M = Pd(II), Cu(II), Zn(II)) multianions isolated in the gas phase without solvent. In particular, we report the position of the Q-bands (S0 → S1 transitions) as a function of charge state, counterions, oligomerization, and dimer structure type. The results for the monomers (charge states = 4- and 3-, sodiated and protonated) are in good agreement with TDDFT calculations and condensed-phase spectra. For both homo and heterometallic dimers, photofragmentation spectra were recorded for two charge states, 5- and 3-, corresponding to coplanar and cofacial structure types, respectively. The fragmentation patterns observed for the dimers depend significantly on charge state, with fragmentation into monomers being dominant for the 5- species, while the 3- charge state predominantly fragments by SO2 loss. The monomer → dimer Q-band spectral shifts observed in the gas phase were compared with the optical properties of porphyrin aggregates in solution.

Structures of Metalloporphyrin-Oligomer Multianions: Cofacial versus Coplanar Motifs as Resolved by Ion Mobility Spectrometry.

We have combined ion mobility mass spectrometry with quantum chemical calculations to investigate the gas-phase structures of multiply negatively charged oligomers of meso-tetra(4-sulfonatophenyl)metalloporphyrins comprising the divalent metal centers ZnII, CuII, and PdII. Sets of candidate structures were obtained by geometry optimizations based on calculations at both the semiempirical PM7 and density functional theory (DFT) levels. The corresponding theoretical cross sections were calculated with the projection approximation and also with the trajectory method. By comparing these collision cross sections with the respective experimental values we were able to assign oligomer structures up to the tetramer. In most cases the cross sections of the lowest energy isomers predicted by theory were found to agree with the measurements to within the experimental uncertainty (2%). Specifically, we find that for a given oligomer size the structures are independent of the metal center but depend strongly on the charge state. Oligomers in low charge states with a correspondingly larger number of sodium counterions tend to form stacked, cofacial structures reminiscent of H-aggregate motifs observed in solution. By contrast, in higher charge states, the stack opens to form coplanar structures.

Q and Soret Band Photoexcitation of Isolated Palladium Porphyrin Tetraanions Leads to Delayed Emission of Nonthermal Electrons over Microsecond Time Scales.

We have used both action and photoelectron spectroscopy to study the response of isolated Pd(II) meso-tetra(4-sulfonatophenyl)porphyrin tetraanions ([PdTPPS](4-)) to electronic excitation over the 2.22-2.98 eV photon energy range. The action spectrum obtained by recording the wavelength-dependent intensity of charged decay products closely resembles the absorption spectrum of PdTPPS in aqueous solution (which shows pronounced Q and Soret absorption bands). The two main decay channels observed are sulfonate group loss and, predominantly, electron emission. To better understand the electron emission channel, we have also acquired photoelectron spectra at multiple detachment photon energies covering the range probed in action spectroscopy. Upon both Q and Soret band excitation, we find that electrons are emitted in three characteristic kinetic energy ranges. The corresponding detachment processes are identified as (delayed) tunneling emission from both excited singlet and triplet states (each of which is accessed by/after one-photon absorption) as well as resonant two-photon detachment. The first triplet state lifetime of isolated [PdTPPS](4-) is significantly longer than 10 µs, possibly on the 100 µs time scale. We estimate that more than 50% of the electron emission observed upon photoexcitation occurs by way of this triplet state.

The mixed gold-palladium polyoxo-noble-metalate [NaAu(III)4Pd(II)8O8(AsO4)8](11-).

The first fully inorganic, discrete gold-palladium-oxo complex [NaAu(III) 4 Pd(II) 8 O8 (AsO4 )8 ](11-) has been synthesized in aqueous medium. The combination of single-crystal XRD, elemental analysis, mass spectrometry, and DFT calculations allowed establishing the structure and composition of the novel polyanion, and UV/Vis studies suggest that it is stable in neutral aqueous media.