The effect of charge and albumin on cellular uptake of supramolecular polymer nanostructures.

Intracellular delivery of functional biomolecules by using supramolecular polymer nanostructures has gained significant interest. Here, various charged supramolecular ureido-pyrimidinone (UPy)-aggregates were designed and formulated via a simple "mix-and-match" method. The cellular internalization of these UPy-aggregates in the presence or absence of serum proteins by phagocytic and non-phagocytic cells, i.e., THP-1 derived macrophages and immortalized human kidney cells (HK-2 cells), was systematically investigated. In the presence of serum proteins the UPy-aggregates were taken up by both types of cells irrespective of the charge properties of the UPy-aggregates, and the UPy-aggregates co-localized with mitochondria of the cells. In the absence of serum proteins only cationic UPy-aggregates could be effectively internalized by THP-1 derived macrophages, and the internalized UPy-aggregates either co-localized with mitochondria or displayed as vesicular structures. While the cationic UPy-aggregates were hardly internalized by HK-2 cells and could only bind to the membrane of HK-2 cells. With adding and increasing the amount of serum albumin in the cell culture medium, the cationic UPy-aggregates were gradually taken up by HK-2 cells without anchoring on the cell membranes. It is proposed that the serum albumin regulates the cellular internalization of UPy-aggregates. These results provide fundamental insights for the fabrication of supramolecular polymer nanostructures for intracellular delivery of therapeutics.

Freezing-mediated formation of supraproteins using depletion forces.

Hypothesis Long-acting formulations such as microparticles, injectable depots and implantable devices can realize spatiotemporally controlled delivery of protein drugs to extend their therapeutic in vivo half-lives. To efficiently encapsulate the protein drugs into such drug delivery systems, (sub)micron-sized protein particles are needed. The formation of micronized supraproteins can be induced through the synergistic combination of attractive depletion forces and freezing. The size of the supraproteins can be fine-tuned from submicron to several microns by adjusting the ice crystallization rate through the freeze-quench depth, which is set by the target temperature. Methods Supraprotein micron structures were prepared from protein solutions under various conditions in the presence and absence of nonadsorbing polyethylene glycol. Scanning electron microscopy and dynamic light scattering were employed to determine the sizes of the supraproteins and real-time total internal reflection fluorescent microscopy was used to follow the supraprotein formation during freezing. The protein secondary structure was measured before and after micronization by circular dichroism. A phase diagram of a protein-polyethylene glycol mixture was theoretically predicted to investigate whether the depletion interaction can elucidate the phase behavior. Findings Micronized protein supraparticles could be prepared in a controlled manner by rapid freeze-drying of aqueous mixtures of bovine serum albumin, horseradish peroxidase and lysozyme mixed with polyethylene glycol. Upon freezing, the temperature quench initiates a phase separation process which is reminiscent of spinodal decomposition. This demixing is subsequently arrested during droplet phase separation to form protein-rich microstructures. The final size of the generated protein microparticles is determined by a competition between phase separation and cooling rate, which can be controlled by target temperature. The experimental phase diagram of the aqueous protein-polyethylene glycol dispersion aligns with predictions from depletion theory for charged colloids and nonadsorbing polymers.

Spleen-dedicated stiffness measurement performed well to rule out high-risk varices in HBV-related hepatocellular carcinoma: Screening for high-risk varices in HCC.

BACKGROUND: Esophagogastroduodenoscopy (EGD) is required to screen for high-risk varices (HRV) in patients with hepatocellular carcinoma (HCC), especially since overall survival rates have dramatically improved with new systemic therapies. AIM: To assess the Baveno VI and Baveno VII algorithms' ability to rule out HRV in hepatitis B virus (HBV)-related HCC METHODS: We prospectively enrolled consecutive patients with HBV related, compensated cirrhosis and newly diagnosed HCC who underwent liver stiffness measurement, spleen stiffness measurement (SSM) using a 100-Hz shear wave frequency, and EGD. RESULTS: From September 2021 to August 2023, we enrolled 219 patients with HCC, with 107 (48.9%) Barcelona Clinic Liver Cancer (BCLC) A, 28 (12.8%) BCLC B and 84 (38.3%) BCLC C, respectively. HRV prevalence was 28.8% (63/219). Baveno VI criteria safely (HRV missing rate, 3.2%) avoided 27.4% unnecessary EGDs, while the Baveno VII algorithm avoided 49.3% with HRV missing rate at 7.9% (5/63). The SSM ≤40 kPa avoided 47.5% of EGDs safely (HRV missing rate, 4.8%), significantly better than the Baveno VI criteria (p < 0.001) and comparable to the Baveno VII algorithm (p = 0.390). The SSM ≤40 kPa safely avoided EGDs in patient subgroups within Milan criteria, with portal vein tumour thrombosis or BCLC B/C or candidates for systemic therapy. CONCLUSIONS: We validated that the SSM ≤40 kPa using a 100-Hz probe could safely eliminate more unnecessary EGDs than the Baveno VI criteria in patients with HBV-related HCC. However, the efficacy of the Baveno VII algorithm in patients with HCC requires further investigation.

Iron Oxide Nanoparticles with Supramolecular Ureido-Pyrimidinone Coating for Antimicrobial Peptide Delivery.

Antimicrobial peptides (AMPs) can kill bacteria by disrupting their cytoplasmic membrane, which reduces the tendency of antibacterial resistance compared to conventional antibiotics. Their possible toxicity to human cells, however, limits their applicability. The combination of magnetically controlled drug delivery and supramolecular engineering can help to reduce the dosage of AMPs, control the delivery, and improve their cytocompatibility. Lasioglossin III (LL) is a natural AMP form bee venom that is highly antimicrobial. Here, superparamagnetic iron oxide nanoparticles (IONs) with a supramolecular ureido-pyrimidinone (UPy) coating were investigated as a drug carrier for LL for a controlled delivery to a specific target. Binding to IONs can improve the antimicrobial activity of the peptide. Different transmission electron microscopy (TEM) techniques showed that the particles have a crystalline iron oxide core with a UPy shell and UPy fibers. Cytocompatibility and internalization experiments were carried out with two different cell types, phagocytic and nonphagocytic cells. The drug carrier system showed good cytocompatibility (>70%) with human kidney cells (HK-2) and concentration-dependent toxicity to macrophagic cells (THP-1). The particles were internalized by both cell types, giving them the potential for effective delivery of AMPs into mammalian cells. By self-assembly, the UPy-coated nanoparticles can bind UPy-functionalized LL (UPy-LL) highly efficiently (99%), leading to a drug loading of 0.68 g g-1. The binding of UPy-LL on the supramolecular nanoparticle system increased its antimicrobial activity against E. coli (MIC 3.53 µM to 1.77 µM) and improved its cytocompatible dosage for HK-2 cells from 5.40 µM to 10.6 µM. The system showed higher cytotoxicity (5.4 µM) to the macrophages. The high drug loading, efficient binding, enhanced antimicrobial behavior, and reduced cytotoxicity makes ION@UPy-NH2 an interesting drug carrier for AMPs. The combination with superparamagnetic IONs allows potential magnetically controlled drug delivery and reduced drug amount of the system to address intracellular infections or improve cancer treatment.

Outstanding feasibility of spleen stiffness measurement by 100-Hz vibration-controlled transient elastography.

This novel spleen-dedicated FibroScan has high success rate and is easy to operate. The spleen stiffness is correlated with liver stiffness, which reflects the liver fibrosis stage. However, whether SSM is able to reflect the severity of liver disease warrants further observation.

Combined model with acoustic radiation force impulse to rule out high-risk varices in HBV-related cirrhosis with viral suppression.

AIMS: To prospectively evaluate the performance of spleen stiffness measurement (SSM) and liver stiffness measurement (LSM) via acoustic radiation force impulse (ARFI) imaging combined with platelet counts (PLT) in ruling out HRV in HBV-related cirrhotic patients with viral suppression. METHODS: Patients with cirrhosis enrolled between June 2020-March 2022 were divided into a derivation cohort and validation cohort. LSM and SSM ARFI-based, and esophagogastroduodenoscopy (EGD) were performed at enrollment. RESULTS: In the derivation cohort, overall, 236 HBV-related cirrhotic patients with maintained viral suppression were enrolled, and the prevalence of HRV was 19.5% (46/236). With the aim of identifying HRV, the most accurate LSM and SSM cut-offs were chosen of 1.46 m/s and 2.28 m/s, respectively. The combined model (LSM<1.46 m/s and PLT>150 × 109/L strategy combined with SSM ≤ 2.28 m/s) can spare 38.6% of EGDs and 4.3% of HRV cases were misclassified. In the validation cohort, we analysed 323 HBV-related cirrhotic patients with maintained viral suppression and validated the combined model can spare 33.4% (108/323) of EGD, and the HRV missed rate was 3.4%. CONCLUSIONS: A non-invasive prediction model combining LSM<1.46 m/s and PLT>150 × 109/L strategy with SSM ≤ 2.28 m/s exhibited excellent performance in ruling out HRV and avoided a significantly large number (38.6% vs 33.4%) of unnecessary EGDs in HBV-related cirrhotic patients with viral suppression.

Microfluidic-templated cell-laden microgels fabricated using phototriggered imine-crosslinking as injectable and adaptable granular gels for bone regeneration.

Injectable granular gels consisting of densely packed microgels serving as scaffolding biomaterial have recently shown great potential for applications in tissue regeneration, which allow administration via minimally invasive surgery, on-target cargo delivery, and high efficiency in nutrient/waste exchange. However, limitations such as insufficient mechanical strength, structural integrity, and uncontrollable differentiation of the encapsulated cells in the scaffolds hamper their further applications in the biomedical field. Herein, we developed a new class of granular gels via bottom-up assembly of cell-laden microgels via photo-triggered imine-crosslinking (PIC) chemistry based on the microfluidic technique. The particulate nature of the granular gels rendered them with shear-thinning and self-healing behavior, thereby functioning as an injectable and adaptable cellularized scaffold for bone tissue regeneration. Specifically, single cell-laden, monodisperse microgels composed of methacrylate- and o-nitrobenzene-functionalized hyaluronic acid and gelatin were prepared using a high-throughput microfluidic technique with a production rate up to 3.7 × 108 microgels/hr, wherein the PIC chemistry alleviated the oxygen inhibition on free-radical polymerization and facilitated enhanced fabrication accuracy, accelerated gelation rate, and improved network strength. Further in vitro and in vivo studies demonstrated that the microgels can serve as carriers to support the activity of the encapsulated mesenchymal stem cells; these cell-laden microgels can also be used as cellularized bone fillers to induce the regeneration of bone tissues as evidenced by the in vivo experiment using the rat femoral condyle defect model. In general, these results represent a significant step toward the precise fabrication of engineered tissue mimics with single-cell resolution and high cell-density and can potentially offer a powerful tool for the design and applications of a next generation of tissue engineering strategy. STATEMENT OF SIGNIFICANCE: Using microfluidic droplet-based technology, we hereby developed a new class of injectable and moldable granular gels via bottom-up assembly of cell-laden microgels as a versatile platform for tissue regeneration. Phototriggered imine-crosslinking chemistry was introduced for microgel cross-linkage, which allowed for the fabrication of microgels with improved matrix homogeneity, accelerated gelation process, and enhanced mechanical strength. We demonstrated that the microgel building blocks within the granular gels facilitated the proliferation and differentiation of the encapsulated mesenchymal stem cells, which can further serve as a cellularized scaffold for the treatment of bone defects.

Baveno VII algorithm outperformed other models in ruling out high-risk varices in individuals with HBV-related cirrhosis.

BACKGROUND & AIMS: The Baveno VII consensus recommends that spleen stiffness measurement (SSM) ≤40 kPa is safe for ruling out high-risk varices (HRVs) and avoiding endoscopic screening in patients who do not meet the Baveno VI criteria. This study aimed to validate the performance of the Baveno VII algorithm in individuals with HBV-related cirrhosis. METHODS: Consecutive individuals with HBV-related cirrhosis who underwent liver stiffness measurement (LSM) and SSM - using a 50 Hz shear wave frequency, spleen diameter measurement, and esophagogastroduodenoscopy (EGD) were prospectively enrolled from June 2020. A 100 Hz probe has been adopted for additional SSM assessment since July 2021. RESULTS: From June 2020 to January 2022, 996 patients were screened and 504 were enrolled for analysis. Among the 504 patients in whom SSM was assessed using a 50 Hz probe, the Baveno VII algorithm avoided more EGDs (56.7% vs. 39.1%, p <0.001) than Baveno VI criteria, with a comparable missed HRV rate (3.8% vs. 2.5%). Missed HRV rates were >5% for all other measures: 11.3% for LSM-longitudinal spleen diameter to platelet ratio score, 20.0% for platelet count/longitudinal spleen diameter ratio, and 8.8% for Rete Sicilia Selezione Terapia-hepatitis. SSM@100 Hz was assessed in 232 patients, and the Baveno VII algorithm with SSM@100 Hz spared more EGDs (75.4% vs. 59.5%, p <0.001) than that with SSM@50 Hz, both with a missed HRV rate of 3.0% (1/33). CONCLUSIONS: We validated the Baveno VII algorithm, demonstrating the excellent performance of SSM@50 Hz and SSM@100 Hz in ruling out HRV in individuals with HBV-related cirrhosis. Furthermore, the Baveno VII algorithm with SSM@100 Hz could safely rule out more EGDs than that with SSM@50 Hz. CLINICAL TRIAL NUMBER: NCT04890730. IMPACT AND IMPLICATIONS: The Baveno VII guideline proposed that for patients who do not meet the Baveno VI criteria, SSM ≤40 kPa could avoid further unnecessary endoscopic screening. The current study validated the Baveno VII algorithm using 50 Hz and 100 Hz probes, which both exhibited excellent performance in ruling out HRVs in individuals with HBV-related cirrhosis. Compared with the Baveno VII algorithm with SSM@50 Hz, SSM@100 Hz had a better capability to safely rule out unnecessary EGDs. Baveno VII algorithm will be a practical tool to triage individuals with cirrhosis in future clinical practice.

Tuning the affinity of amphiphilic guest molecules in a supramolecular polymer transient network.

Dynamicity plays a central role in biological systems such as in the cellular microenvironment. Here, the affinity and dynamics of different guest molecules in a transient supramolecular polymer hydrogel system, i.e. the host network, are investigated. The hydrogel system consists of bifunctional ureido-pyrimidinone (UPy) poly(ethylene glycol) polymers. A monofunctional complementary UPy guest is introduced, designed to interact with the host network based on UPy-UPy interactions. Furthermore, two other guest molecules are synthesized, being cholesterol and dodecyl (c12) guests; both designed to interact with the host network via hydrophobic interactions. At the nanoscale in solution, differences in morphology of the guest molecules were observed. The UPy-guest molecule formed fibers, and the cholesterol and c12 guests formed aggregates. Furthermore, cellular internalization of fluorescent guest molecules was studied. No cellular uptake of the UPy-cy5 guest was observed, whereas the cholesterol-cy5 guest showed membrane binding and cellular uptake. Also the c12-cy5 guest showed cellular uptake. Formulation of the guest molecules into the UPy hydrogel system was done to study the guest-host affinity. No changes in mechanical properties as measured with rheology were found upon guest-hydrogel formulation. Fluorescence recovery after photobleaching showed the diffusive properties of the cy5-functionalized guests throughout the host network. The c12 guest displayed a relatively fast mobility, the UPy guest displayed a decrease in mobility, and the cholesterol-guest remained relatively stable in the host network with little mobility. This demonstrates the tunable dynamic differences of affinity-based interaction between guest molecules and the host network. Interestingly, the cholesterol guest is internalized in cells and is robustly incorporated in the hydrogel network, while the UPy guest is not taken up by cells but shows an affinity to the hydrogel network. These results show the importance of guest-hydrogel affinity for future drug release. However, if modified with cholesterol these guests, or future drugs, will be taken up by cells; if modified with a UPy unit this does not occur. In this way both the drug-hydrogel interaction and the cell internalization behavior can be tuned. Regulating the host-guest dynamics in transient hydrogels opens the door to various drug delivery purposes and tissue engineering.

Monitoring local delivery of vancomycin from gelatin nanospheres in zebrafish larvae.

BACKGROUND: Infections such as biomaterial-associated infection and osteomyelitis are often associated with intracellular survival of bacteria (eg, Staphylococcus aureus). Treatment of these infections remains a major challenge due to the low intracellular efficacy of many antibiotics. Therefore, local delivery systems are urgently required to improve the therapeutic efficacy of antibiotics by enabling their intracellular delivery. PURPOSE: To assess the potential of gelatin nanospheres as carriers for local delivery of vancomycin into macrophages of zebrafish larvae in vivo and into THP-1-derived macrophages in vitro using fluorescence microscopy. MATERIALS AND METHODS: Fluorescently labeled gelatin nanospheres were prepared and injected into transgenic zebrafish larvae with fluorescent macrophages. Both the biodistribution of gelatin nanospheres in zebrafish larvae and the co-localization of vancomycin-loaded gelatin nanospheres with zebrafish macrophages in vivo and uptake by THP-1-derived macrophages in vitro were studied. In addition, the effect of treatment with vancomycin-loaded gelatin nanospheres on survival of S. aureus-infected zebrafish larvae was investigated. RESULTS: Internalization of vancomycin-loaded gelatin nanospheres by macrophages was observed qualitatively both in vivo and in vitro. Systemically delivered vancomycin, on the other hand, was hardly internalized by macrophages without the use of gelatin nanospheres. Treatment with a single dose of vancomycin-loaded gelatin nanospheres delayed the mortality of S. aureus-infected zebrafish larvae, indicating the improved therapeutic efficacy of vancomycin against (intracellular) S. aureus infection in vivo. CONCLUSION: The present study demonstrates that gelatin nanospheres can be used to facilitate local and intracellular delivery of vancomycin.

Nanostructured raspberry-like gelatin microspheres for local delivery of multiple biomolecules.

Multicompartment particles, which are particles composed of smaller building units, have gained considerable interest during the past decade to facilitate simultaneous and differential delivery of several biomolecules in various applications. Supercritical carbon dioxide (CO2) processing is an industrial technology widely used for large-scale synthesis and processing of materials. However, the application of this technology for production of multicompartment particles from colloidal particles has not yet been explored. Here, we report the formation of raspberry-like gelatin (RLG) microparticles composed of gelatin nanoparticles as colloidal building blocks through supercritical CO2 processing. We show that these RLG microparticles exhibit a high stability upon dispersion in aqueous media without requiring chemical cross-linking. We further demonstrate that these microparticles are cytocompatible and facilitate differential release of two different model compounds. The strategy presented here can be utilized as a cost-effective route for production of various types of multicompartment particles using colloidal particles with suitable interparticle interactions. STATEMENT OF SIGNIFICANCE: Multicompartment particles have gained considerable interest during the past decade to facilitate simultaneous and differential delivery of multiple biomolecules in various biomedical applications. Nevertheless, common methods employed for the production of such particles are often complex and only offer small-scale production. Here, we report the formation of raspberry-like gelatin (RLG) microparticles composed of gelatin nanoparticles as colloidal building blocks through supercritical CO2 processing. We show that these microparticles are cytocompatible and facilitate differential release of two model compounds with different molecular sizes, promising successful applications in various biomedical areas. Summarizing, this paper presents a novel strategy that can be utilized as a cost-effective route for production of various types of multicompartment particles using a wide range of colloidal building blocks.

Electrospun Nanofibrous Silk Fibroin Membranes Containing Gelatin Nanospheres for Controlled Delivery of Biomolecules.

Development of novel and effective drug delivery systems for controlled release of bioactive molecules is of critical importance in the field of regenerative medicine. Here, oppositely charged gelatin nanospheres are incorporated into silk fibroin nanofibers through a colloidal electrospinning technique. A novel fibrous nano-in-nano drug delivery system is fabricated without the use of any organic solvent. The distribution of fluorescently labeled gelatin A and B nanospheres inside the nanofibers can be fine-tuned by simple adjustment of the weight ratio between the nanospheres and the relative feeding rate of core and shell solutions containing nanospheres by using single and coaxial nozzle electrospinning, respectively. Incorporation of vancomycin-loaded gelatin B nanospheres into the silk fibroin nanofibrous membranes results in a more sustained release of vancomycin, compared to the gelatin nanospheres free membranes. In addition, these membranes exhibit excellent and prolonged antibacterial effects against Staphylococcus aureus. Moreover, these membranes support the attachment, spreading, and proliferation of periodontal ligament cells. These results suggest that the beneficial properties of gelatin nanospheres can be exploited to improve the biological functionality of electrospun nanofibrous silk fibroin membranes.

Electrophoretic Deposition of Chitosan Coatings Modified with Gelatin Nanospheres To Tune the Release of Antibiotics.

Orthopedic and dental implants are increasingly used in the medical field in view of their high success rates. Implant-associated infections, however, still occur and are difficult to treat. To combat these infections, the application of an active coating to the implant surface is advocated as an effective strategy to facilitate sustained release of antibacterial drugs from implant surfaces. Control over this release is, however, still a major challenge. To overcome this problem, we deposited composite coatings composed of a chitosan matrix containing gelatin nanospheres loaded with antibiotics onto stainless steel plates by means of the electrophoretic deposition technique. The gelatin nanospheres were distributed homogeneously throughout the coatings. The surface roughness and wettability of the coatings could be tuned by a simple adjustment of the weight ratio between the gelatin nanospheres and chitosan. Vancomycin and moxifloxacin were released in sustained and burst-type manners, respectively, while the coatings were highly cytocompatible. The antibacterial efficacy of the coatings containing different amounts of antibiotics was tested using a zone of inhibition test against Staphylococcus aureus, which showed that the coatings containing moxifloxacin exhibited an obvious inhibition zone. The coatings containing a high amount of vancomycin were able to kill bacteria in direct contact with the implant surface. These results suggest that the antibacterial capacity of metallic implants can be tuned by orthogonal control over the release of (multiple) antibiotics from electrophoretically deposited composite coatings, which offers a new strategy to prevent orthopedic implant-associated infections.

Antibacterial effects of electrospun chitosan/poly(ethylene oxide) nanofibrous membranes loaded with chlorhexidine and silver.

To prevent percutaneous device associated infections (PDAIs), we prepared electrospun chitosan/poly(ethylene oxide) (PEO) nanofibrous membrane containing silver nanoparticles as an implantable delivery vehicle for the dual release of chlorhexidine and silver ions. We observed that the silver nanoparticles were distributed homogeneously throughout the fibers, and a fast release of chlorhexidine in 2days and a sustained release of silver ions for up to 28days. The antibacterial efficacy of the membranes against Staphylococcus aureus showed that the membranes exhibited an obvious inhibition zone upon loading with either chlorhexidine (20µg or more per membrane) or AgNO3 (1 and 5wt% to polymer). Furthermore, long-term antibacterial effect up to 4days was verified using membranes containing 5wt% AgNO3. The results suggest that the membranes have strong potential to act as an active antibacterial dressing for local delivery of antibacterial agents to prevent PDAIs.

Increased acellular and cellular surface mineralization induced by nanogrooves in combination with a calcium-phosphate coating.

The current work evaluated the influence of nanoscale surface-topographies in combination with a calcium phosphate (CaP) coating on acellular and cellular surface mineralization. Four groups of substrates were produced, including smooth, grooved (940nm pitch, 430nm groove width, 185nm depth), smooth coated, and grooved coated. The substrates were characterized by scanning/transmission electron microscopy and atomic force microscopy. Osteoblast-like MC3T3 cells were cultured on the substrates for a period up to 35days under osteogenic conditions. Differentiation was observed by alkaline phosphatase assay and PCR of collagen I (COLI), osteopontin (OPN), osteocalcin (OC), bone-morphogenic protein 2 (BMP2), and bone sialoprotein (BSP). Mineralization was quantified by a calcium assay and Alizarin Red staining. In addition, acellular mineralization was determined after incubation of substrates in just cell culture medium without cells. Results showed that a reproducible nano-metric (∼50nm) CaP-layer could be applied on the substrates, without losing the integrity of the topographical features. While no relevant differences were found for cell viability, cells on smooth surfaces proliferated for a longer period than cells on grooved substrates. In addition, differentiation was affected by topographies, as indicated by an increased expression of OC, OPN and ALP activity. Deposition of a CaP coating significantly increased the acellular mineralization of smooth as well grooved substrate-surfaces. However, this mineralizing effect was strongly reduced in the presence of cells. In the cell seeded situation, mineralization was significantly increased by the substrate topography, while only a minor additive effect of the coating was observed. In conclusion, the model presented herein can be exploited for experimental evaluation of cell-surface interaction processes and optimization of bone-anchoring capability of implants. The model showed that substrates modified with CaP-coated coated nanogrooves display enhanced in vitro mineralization as compared to unmodified controls or substrates modified with either nanogrooves or CaP coatings. However, our results also indicated that acellular mineralization assays are not necessarily predictive for biological performance. STATEMENT OF SIGNIFICANCE: The manuscript describes the possibility to combine the mechanical properties of nanosized topographies with the biochemical properties of a calcium phosphate based coating for improvement of surface mineralization. Interestingly, our results demonstrate that further incubation of our surfaces in SBF type media allowed all surfaces to mineralize rapidly to a high extent. Moreover we prove that nanotexture be used to can stimulate and organize mineralization and that the combination surface of a CaP coating and a nanotexture has the potential to be effective as a bone-implant surface. Such experiments will be of considerable interest to those in the research community and industry, who are focusing on bio-mineralization processes and optimization of modern bone-implants.

Influence of the Molecular Weight and Charge of Antibiotics on Their Release Kinetics From Gelatin Nanospheres.

In this study, we investigated the fundamental relationship between the physicochemical characteristics of antibiotics and the kinetics of their release from gelatin nanospheres. We observed that antibiotics of high molecular weight (colistin and vancomycin) were released in a sustained manner from oppositely charged gelatin carriers for more than 14 d, as opposed to antibiotics of low molecular weight (gentamicin and moxifloxacin) which were released in a burst-like manner. The release kinetics of positively charged colistin strongly correlated with the rate of the enzymatic degradation of gelatin. To elucidate the differences among release kinetics of antibiotics, we explored the mechanism of interactions between antibiotics and gelatin nanospheres by monitoring the kinetics of release of antibiotics as a function of pH, ionic strength, and detergent concentrations. These studies revealed that the interactions between antibiotics and gelatin nanospheres were mainly dominated by (i) strong electrostatic forces for colistin; (ii) strong hydrophobic and electrostatic forces for vancomycin; (iii) weak electrostatic and hydrophobic forces for gentamicin; and (iv) weak hydrophobic forces for moxifloxacin. These results confirm that release of antibiotics from gelatin nanospheres strongly depends on the physicochemical characteristics of the antibiotics.

Sustained delivery of biomolecules from gelatin carriers for applications in bone regeneration.

Local delivery of therapeutic biomolecules to stimulate bone regeneration has matured considerably during the past decades, but control over the release of these biomolecules still remains a major challenge. To this end, suitable carriers that allow for tunable spatial and temporal delivery of biomolecules need to be developed. Gelatin is one of the most widely used natural polymers for the controlled and sustained delivery of biomolecules because of its biodegradability, biocompatibility, biosafety and cost-effectiveness. The current study reviews the applications of gelatin as carriers in form of bulk hydrogels, microspheres, nanospheres, colloidal gels and composites for the programmed delivery of commonly used biomolecules for applications in bone regeneration with a specific focus on the relationship between carrier properties and delivery characteristics.

Fast and continuous preparation of high polymerization degree cellulose nanofibrils and their three-dimensional macroporous scaffold fabrication.

C6-carboxy-cellulose with a carboxylate content of 0.8 mmol g(-1) was obtained by oxidation of once-dried cellulose, using the 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO)/NaClO/NaClO2 system at pH 6.8 and 60 °C for 16 h. This method, with the addition of reagents in the order TEMPO, NaClO and NaClO2, was 38 h faster than a previously published method. Individualized cellulose nanofibrils with a width of 3-5 nm and a length of several hundred nanometers were prepared by homogenizing the C6-carboxy-cellulose-water suspension. Macroporous cellulose nanofibril/poly(vinyl alcohol) scaffolds with interconnected large pores of 20-100 µm diameter and small pores of 2-10 µm diameter were fabricated. The cellulose nanofilaments formed nanofibrous structures on the surface of the PVA wall, which was similar to that of the collagen skeleton of the extracellular matrix. NIH/3T3 cells were cultured in the scaffolds for 4 weeks, SEM observation showed that the cells were anchored and clustered on the cellulose nanofilaments, forming spherical colonies. The extracellular matrix (ECM) was filled with mineralized particles.