Mechanical Properties, Drug Release, Biocompatibility, and Antibacterial Activities of Modified Emulsified Gelatin Microsphere Loaded with Gentamicin Composite Calcium Phosphate Bone Cement In Vitro.

Bone defects are commonly addressed with bone graft substitutes; however, surgical procedures, particularly for open and complex fractures, may pose a risk of infection. As such, a course of antibiotics combined with a drug carrier is often administered to mitigate potential exacerbations. This study involved the preparation and modification of emulsified (Em) crosslinking-gelatin (gel) microspheres (m-Em) to reduce their toxicity. The antibiotic gentamicin was impregnated into gel microspheres (m-EmG), which were incorporated into calcium phosphate bone cement (CPC). The study investigated the effects of m-EmG@CPC on antibacterial activity, mechanical properties, biocompatibility, and proliferation and mineralization of mouse progenitor osteoblasts (D1 cells). The average size of the gel microspheres ranged from 22.5 to 16.1 µm, with no significant difference between the groups (p > 0.05). Most of the oil content within the microspheres was transferred through modification, resulting in reduced cytotoxicity. Furthermore, antibiotic-impregnated m-EmG did not compromise the intrinsic properties of the microspheres and exhibited remarkably antibacterial effects. After combining with CPC (m-EmG@CPC), the microspheres did not significantly hinder the CPC reaction and produced the main product, hydroxyapatite (HA). However, the compressive strength of the largest microsphere content of 0.5 wt.% m-EmG in CPC decreased significantly from 59.8 MPa of CPC alone to 38.7 MPa of 0.5m-EmG@CPC (p < 0.05). The 0.5m-EmG@CPC composite was effective against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) in drug release and antibacterial tests. Compared with m-EmG alone, the 0.5m-EmG@CPC composite showed no toxicity to mouse fibroblast cells (L929). Additionally, the proliferation and mineralization of mouse osteoblastic osteoprogenitor cells (D1 cells) did not have a negative impact on the 0.5m-EmG@CPC composite over time in culture compared with CPC alone. Results suggest that the newly developed antibacterial 0.5m-EmG@CPC composite bone cement did not negatively affect the performance of osteoprogenitor cells and could be a new option for bone graft replacement in surgeries.

Hybrid-Aligned Fibers of Electrospun Gelatin with Antibiotic and Polycaprolactone Composite Membranes as an In Vitro Drug Delivery System to Assess the Potential Repair Capacity of Damaged Cornea.

The cornea lacks the ability to repair itself and must rely on transplantation to repair damaged tissue. Therefore, creating alternative therapies using dressing membranes based on tissue engineering concepts to repair corneal damage before failure has become a major research goal. Themost outstanding features that are important in reconstructing a damaged cornea are the mechanical strength and transparency of the membrane, which are the most important standard considerations. In addition, preventing infection is an important issue, especially in corneal endothelial healing processes. The purpose of this study was to produce aligned fibers via electrospinning technology using gelatin (Gel) composite polycaprolactone (PCL) as an optimal transport and antibiotic release membrane. The aim of the composite membrane is to achieve good tenacity, transparency, antibacterial properties, and in vitro biocompatibility. Results showed that the Gel and PCL composite membranes with the same electrospinning flow rate had the best transparency. The Gel impregnated with gentamicin antibiotic in composite membranes subsequently exhibited transparency and enhanced mechanical properties provided by PCL and could sustainably release the antibiotic for 48 h, achieving good antibacterial effects without causing cytotoxicity. This newly developed membrane has the advantage of preventing epidermal tissue infection during clinical operations and is expected to be used in the reconstruction of damaged cornea in the future.

Survival assessment of fractured porcelain-fused-to-metal crowns surface roughened by sandblasted and repaired by composite resin after in vitro thermal fatigue.

Background/purpose: Intraoral repair usually takes the convenience of the patient's daily life as the starting point, taking into account the bonding strength, operational feasibility, and safety. This study aimed to evaluate the survival of composite resin by simulating cavity fracture repair in porcelain-fused-to-metal (PFM) crowns and referring to the G.V. Black classification of caries as ceramic- and metal-site exposure. Materials and methods: Mechanical sandblast experimental and a nonsandblast control groups comprised 120 samples, and interfacial locking was enhanced through acid etching, bonding, and light-curing composite resin restoration. Classes of VI buccal (B), III mesial (M), and IV mesiobuccal (MB) types, were investigated. Load tests were performed on two sets, with one set at room temperature for 24 h and the other via thermal cycling at 5 °C/50 °C 720 times. Loading was gradually applied to the samples until a maximum of 450 N was reached. Results: Results showed that 24 h survival rates of B-, M - , and MB-repaired PFM crowns were 88%, 84%, and 88%, respectively. The repaired PFM survival rates for B, M, and MB were 52%, 44%, and 28%, respectively, after thermal cycling and loading tests. Multiple logistic regression and chi-square test (α = 0.05) showed that the regression results of factors affecting survival assessment were only significant between groups after thermal fatigue (P < 0.05). Survival rate of repairing metal-site in the MB model was significantly higher than that of ceramic-sites repairing in non-blasted samples. For the MB cavity model, sandblasting can significantly improve the survival rate of the repair of ceramic parts in the MB model (P < 0.05). Conclusion: Our results suggest that sandblasting can be further considered, especially for MB cavity fractures when ceramic-site restorations are required.

Development and In Vitro Analysis of Layer-by-Layer Assembled Membranes for Potential Wound Dressing: Electrospun Curcumin/Gelatin as Middle Layer and Gentamicin/Polyvinyl Alcohol as Outer Layers.

Nanofibrous membranes made of hydrogels have high specific surface areas and are suitable as drug carriers. Multilayer membranes fabricated by continuous electrospinning could delay drug release by increasing diffusion pathways, which is beneficial for long-term wound care. In this experiment, polyvinyl alcohol (PVA) and gelatin were used as membrane substrates, and a sandwich PVA/gelatin/PVA structure of layer-by-layer membranes was prepared by electrospinning under different drug loading concentrations and spinning times. The outer layers on both sides were citric-acid-crosslinked PVA membranes loaded with gentamicin as an electrospinning solution, and the middle layer was a curcumin-loaded gelatin membrane for the study of release behavior, antibacterial activity, and biocompatibility. According to the in vitro release results, the multilayer membrane could release curcumin slowly; the release amount was about 55% less than that of the single layer within 4 days. Most of the prepared membranes showed no significant degradation during immersion, and the phosphonate-buffered saline absorption rate of the multilayer membrane was about five to six times its weight. The results of the antibacterial test showed that the multilayer membrane loaded with gentamicin had a good inhibitory effect on Staphylococcus aureus and Escherichia coli. In addition, the layer-by-layer assembled membrane was non-cytotoxic but detrimental to cell attachment at all gentamicin-carrying concentrations. This feature could be used as a wound dressing to reduce secondary damage to the wound when changing the dressing. This multilayer wound dressing could be applied to wounds in the future to reduce the risk of bacterial infection and help wounds heal.

Synergistic effect of drug/antibiotic-impregnated micro/nanohybrid mesoporous bioactive glass/calcium phosphate composite bone cement on antibacterial and osteoconductive activities.

Calcium phosphate bone cements (CPC) can be used in minimally invasive surgery because of their injectability, and they can also be used to repair small and irregular bone defects. This study aimed to release the antibiotic gentamicin sulfate (Genta) to reduce tissue inflammation and prevent infection in the early stages of bone recovery. Subsequently, the sustained release of the bone-promoting drug ferulic acid (FA) mimicked the response of osteoprogenitor D1 cells interaction, thereby accelerating the healing process of the overall bone repair. Accordingly, the different particle properties of micro-nano hybrid mesoporous bioactive glass (MBG), namely, micro-sized MBG (mMBG) and nano-sized MBG (nMBG), were explored separately to generate different dose releases in MBG/CPC composite bone cement. Results show that nMBG had better sustained-release ability than mMBG when impregnated with the same dose. When 10 wt% of mMBG hybrid nMBG and composite CPC were used, the amount of MBG slightly shortened the working/setting time and lowered the strength but did not hinder the biocompatibility, injectability, anti-disintegration, and phase transformation of the composite bone cement. Furthermore, compared with 2.5wt%Genta@mMBG/7.5 wt% FA@nMBG/CPC, 5wt.%Genta@mMBG/5wt.%FA@nMBG/CPC exhibited better antibacterial activity, better compressive strength, stronger mineralization of osteoprogenitor cell, and similar 14-day slow-release trend of FA. The MBG/CPC composite bone cement developed can be used in clinical surgery to achieve the synergistic sustained release of antibacterial and osteoconductive activities.

Enhanced Cell Osteogenic Differentiation in Alendronate Acid and Flufenamic Acid Drug-Impregnated Nanoparticles of Mesoporous Bioactive Glass Composite Calcium Phosphate Bone Cement In Vitro.

This study aims to compare the anti-osteoporotic drugs alendronic acid (ALN) and flufenamic acid (FA) alone impregnate into nanoparticles of mesoporous bioactive glass (nMBG), which further composites calcium phosphate cement (CPC) and investigates their in vitro performance. The drug release, physicochemical properties, and biocompatibility of nMBG@CPC composite bone cement are tested, and the effect of the composites on improving the proliferation and differentiation efficiency of mouse precursor osteoblasts (D1 cells) is also investigated. Drug release shows that FA impregnates nMBG@CPC composite, a large amount of FA is released rapidly within 8 h, gradually reaching a stable release within 12 h, followed by a slow and sustained release within 14 days, and then reaches a plateau within 21 days. The release phenomenon confirms that the drug-impregnated nBMG@CPC composite bone cement effectively achieves slow drug delivery. The working time and setting time of each composite are within 4-10 min and 10-20 min, respectively, meeting the operational requirements of clinical applications. The addition of nMBG nanoparticles in the CPC matrix did not prevent the aggregation phenomenon under microstructural observation, thus resulting in a decrease in the strength of the nMBG@CPC composite. However, after 24 h of immersed reaction, the strength of each 5 wt.% nMBG impregnated with different concentrations of FA and ALN is still greater than 30 MPa, which is higher than the general trabecular bone strength. The drug-impregnated nMBG@CPC composites did not hinder the product formation and exhibit biocompatibility. Based on the proliferation and mineralization of D1 cells, the combination of nMBG with abundant FA and ALN in CPC is not conducive to the proliferation of D1 cells. However, when D1 cells are contact cultured for 21 days, alkaline phosphatase (ALP) enzyme activity shows higher ALP secretion from drug-impregnated nMBG@CPC composites than drug-free composites. Accordingly, this study confirms that nMBG can effectively impregnate the anti-osteoporosis drugs FA and ALN, and enhance the mineralization ability of osteoblasts. Furthermore, drug-impregnated nMBG applications can be used alone or in combination with CPC as a new option for osteoporotic bone-filling surgery.

Effect of Citric Acid on Swelling Resistance and Physicochemical Properties of Post-Crosslinked Electrospun Polyvinyl Alcohol Fibrous Membrane.

A series of electrospun polyvinyl alcohol (PVA) fiber membranes were crosslinked with citric acid (CA) at concentrations of 10, 20, and 30 wt.% (designated as CA10, CA20, and CA30). The effects of CA on the chemical structure, mechanical strength, swelling resistance, and cytotoxicity of the crosslinked PVA fibrous membranes were investigated. Infrared spectroscopy indicated the enhanced esterification of carboxyl and hydroxyl groups between CA and PVA. The modulus and strength of the electrospun PVA membrane increased due to the crosslinking between CA and PVA. The crosslinking of the PVA fiber matrix with CA increased the PVA binding point, thereby increasing the swelling resistance and modulus; however, the concentration of CA used was limited. Results showed that the water absorption of the PVA membranes decreased from 6.58 ± 0.04 g/g for CA10 to 3.56 ± 3.33 g/g for CA20 and 2.85 ± 0.40 g/g for CA30 with increasing CA. The water absorption remained unchanged after the membrane was soaked for a period of time, so no significant difference was found in the water absorption capacity of the same group after immersion from 1 h to 3 d. The tensile strength increased from 20.52 MPa of CA10 to 22.09 MPa of CA20. With an increased amount of CA used for crosslinking, the tensile strength and modulus of CA30 decreased to 11.48 and 13.94 MPa, respectively. Our study also showed that CA was not toxic to L929 cell viability when used for fiber crosslinking at less than 20 wt.% PVA, meaning it may be a good candidate as a support layer for guided tissue engineering.

In Vitro Characterizations of Post-Crosslinked Gelatin-Based Microspheres Modified by Phosphatidylcholine or Diammonium Phosphate as Antibiotic Delivery Vehicles.

Hydrogel-based microspheres prepared by emulsification have been widely used as drug carriers, but biocompatibility remains a challenging issue. In this study, gelatin was used as the water phase, paraffin oil was used as the oil phase, and Span 80 was used as the surfactant. Microspheres were prepared using a water-in-oil (W/O) emulsification. Diammonium phosphate (DAP) or phosphatidylcholine (PC) were further used to improve the biocompatibility of post-crosslinked gelatin microspheres. The biocompatibility of DAP-modified microspheres (0.5-10 wt.%) was better than that of PC (5 wt.%). The microspheres soaked in phosphate-buffered saline (PBS) lasted up to 26 days before fully degrading. Based on microscopic observation, the microspheres were all spherical and hollow inside. The particle size distribution ranged from 19 µm to 22 µm in diameter. The drug release analysis showed that the antibiotic gentamicin loaded on the microspheres was released in a large amount within 2 h of soaking in PBS. It was stabilized until the amount of microspheres integrated was significantly reduced after soaking for 16 days and then released again to form a two-stage drug release curve. In vitro experiments showed that DAP-modified microspheres at concentrations less than 5 wt.% had no cytotoxicity. Antibiotic-impregnated and DAP-modified microspheres had good antibacterial effects against Staphylococcus aureus and Escherichia coli, but these drug-impregnated groups hinder the biocompatibility of hydrogel microspheres. The developed drug carrier can be combined with other biomaterial matrices to form a composite for delivering drugs directly to the affected area in the future to achieve local therapeutic effects and improve the bioavailability of drugs.

In vitro evaluation of electrospun polyvinylidene fluoride hybrid nanoparticles as direct piezoelectric membranes for guided bone regeneration.

A polyvinylidene fluoride (PVDF) piezoelectric membrane containing carbon nanotubes (CNTs) and graphene oxide (GO) additives was prepared, with special emphasis on the piezoelectric activity of the aligned fibers. Fibroblast viability on membranes was measured to study cytotoxicity. Osteoprogenitor D1 cells were cultured, and mineralization of piezoelectric composite membranes was assessed by ultrasound stimulation. Results showed that the electrospun microstructures were anisotropically aligned fibers. As the GO content increased to 1.0 wt% (0.2 wt% interval), the ß phase in PVDF slightly increased but showed the opposite trend with the increase in CNT. Excessive addition of GO and CNT hindered the growth of the ß phase in PVDF. The direct piezoelectric activity and mechanical properties showed the same trend as the ß phase in PVDF. Moreover, GO/PVDF with the same nanoparticle content showed better performance than CNT/PVDF composites. In this study, a comparison of the generated piezoelectric specific voltage (unit: 10-3 Vg-1 cm-2, linear stretch, g33) with control PVDF only (0.55 ± 0.16) revealed that the two composites containing 0.8 wt% GO- and 0.2 wt% CNT- with 15 wt% PVDF exhibited excellent piezoelectric voltages, which were 3.37 ± 1.05 and 1.45 ± 0.07 (10-3 Vg-1 cm-2), respectively. In vitro cultures of these two groups in contact with D1 cells showed significantly higher alkaline phosphatase secretion than the PVDF only group within 1-10 days of cell culture. Further application of ultrasound stimulation showed that the piezoelectric membrane differentiated D1 cells earlier than without ultrasound and induced higher proliferation and mineralization. This developing piezoelectric effect is expected to generate voltage through activities to enhance microcurrent stimulation in vivo.

Injectability, Processability, Drug Loading, and Antibacterial Activity of Gentamicin-Impregnated Mesoporous Bioactive Glass Composite Calcium Phosphate Bone Cement In Vitro.

Calcium phosphate cement (CPC) is similar to bone in composition and has plasticity, while mesoporous bioactive glass (MBG) has the advantage of releasing Si, which can promote osteogenic properties and drug loading capacity. A sol-gel-prepared MBG micro-powder (mMBG) and further impregnated antibiotic gentamicin sulfate (Genta@mMBG: 2, 3, and 4 mg/mL) antibiotic were added to CPC at different weight ratios (5, 10, and 15 wt.%) to study CPC's potential clinical applications. Different ratios of mMBG/CPC composite bone cement showed good injectability and disintegration resistance, but with increasing mMBG addition, the working/setting time and compressive strength decreased. The maximum additive amount was 10 wt.% mMBG due to the working time of ~5 min, the setting time of ~10 min, and the compressive strength of ~51 MPa, indicating that it was more suitable for clinical surgical applications than the other groups. The 2Genta@mMBG group loaded with 2 mg/mL gentamicin had good antibacterial activity, and the 10 wt.% 2Genta@mMBG/CPC composite bone cement still had good antibacterial activity but reduced the initial release of Genta. 2Genta@mMBG was found to have slight cytotoxicity, so 2Genta@mMBG was composited into CPC to improve the biocompatibility and to endow CPC with more advantages for clinical application.

Morphological Changes, Antibacterial Activity, and Cytotoxicity Characterization of Hydrothermally Synthesized Metal Ions-Incorporated Nanoapatites for Biomedical Application.

The objective of this study was to prepare hydroxyapatite (HA) with potential antibacterial activity against gram-negative and gram-positive bacteria by incorporating different atomic ratios of Cu2+ (0.1-1.0%), Mg2+ (1.0-7.0%), and Zn2+ (1.0-7.0%) to theoretically replace Ca2+ ions during the hydrothermal synthesis of grown precipitated HA nanorods. This study highlights the role of comparing different metal ions on synthetic nanoapatite in regulating the antibacterial properties and toxicity. The comparisons between infrared spectra and between diffractograms have confirmed that metal ions do not affect the formation of HA phases. The results show that after doped Cu2+, Mg2+, and Zn2+ ions replace Ca2+, the ionic radius is almost the same, but significantly smaller than that of the original Ca2+ ions, and the substitution effect causes the lattice distance to change, resulting in crystal structure distortion and reducing crystallinity. The reduction in the length of the nanopatites after the incorporation of Cu2+, Mg2+, and Zn2+ ions confirmed that the metal ions were mainly substituted during the growth of the rod-shape nanoapatite Ca2+ distributed along the longitudinal site. The antibacterial results show that nanoapatite containing Cu2+ (0.1%), Mg2+ (3%), and Zn2+ (5-7%) has obvious and higher antibacterial activity against gram-positive bacteria Staphylococcus aureus within 2 days. The antibacterial effect against the gram-negative bacillus Escherichia coli is not as pronounced as against Staphylococcus aureus. The antibacterial effect of Cu2+ substituted Ca2+ with an atomic ratio of 0.1~1.0% is even better than that of Mg2+- and Zn2+- doped with 1~7% groups. In terms of cytotoxicity, nanoapatites with Cu2+ (~0.2%) exhibit cytotoxicity, whereas Mg2+- (1-5%) and Zn2+- (~1%) doped nanoapatites are biocompatible at low concentrations but become cytotoxic as ionic concentration increases. The results show that the hydrothermally synthesized nanoapatite combined with Cu2+ (0.2%), Mg2+ (3%), and Zn2+ (3%) exhibits low toxicity and high antibacterial activity, which provides a good prospect for bypassing antibiotics for future biomedical applications.

Advances of Hydroxyapatite Hybrid Organic Composite Used as Drug or Protein Carriers for Biomedical Applications: A Review.

Hydroxyapatite (HA), especially in the form of HA nanoparticles (HANPs), has excellent bioactivity, biodegradability, and osteoconductivity and therefore has been widely used as a template or additives for drug delivery in clinical applications, such as dentistry and orthopedic repair. Due to the atomically anisotropic distribution on the preferred growth of HA crystals, especially the nanoscale rod-/whisker-like morphology, HA can generally be a good candidate for carrying a variety of substances. HA is biocompatible and suitable for medical applications, but most drugs carried by HANPs have an initial burst release. In the adsorption mechanism of HA as a carrier, specific surface area, pore size, and porosity are important factors that mainly affect the adsorption and release amounts. At present, many studies have developed HA as a drug carrier with targeted effect, porous structure, and high porosity. This review mainly discusses the influence of HA structures as a carrier on the adsorption and release of active molecules. It then focuses on the benefits and effects of different types of polymer-HA composites to re-examine the proteins/drugs carry and release behavior and related potential clinical applications. This literature survey can be divided into three main parts: 1. interaction and adsorption mechanism of HA and drugs; 2. advantages and application fields of HA/organic composites; 3. loading and drug release behavior of multifunctional HA composites in different environments. This work also presents the latest development and future prospects of HA as a drug carrier.