Bone repair and regenerative biomaterials: Towards recapitulating the microenvironment

© 2019 by the authors. Biomaterials and tissue engineering scaffolds play a central role to repair bone defects. Although ceramic derivatives have been historically used to repair bone, hybrid materials have emerged as viable alternatives. The rationale for hybrid bone biomaterials is to recapitulate the native bone composition to which these materials are intended to replace. In addition to the mechanical and dimensional stability, bone repair scaffolds are needed to provide suitable microenvironments for cells. Therefore, scaffolds serve more than a mere structural template suggesting a need for better and interactive biomaterials. In this review article, we aim to provide a summary of the current materials used in bone tissue engineering. Due to the ever-increasing scientific publications on this topic, this review cannot be exhaustive; however, we attempted to provide readers with the latest advance without being redundant. Furthermore, every attempt is made to ensure that seminal works and significant research findings are included, with minimal bias. After a concise review of crystalline calcium phosphates and non-crystalline bioactive glasses, the remaining sections of the manuscript are focused on organic-inorganic hybrid materials

生分解性有機-無機複合球状粒子の創製とその生物学的性質に関する研究

Antibiotic resistance in bacteria is a serious problem that requires researchers to engineer new strategies to tackle this growing threat. The limited intracellular bioavailability of antibiotics decreases the efficacy of the treatments and, as a consequence promotes bacterial resistance towards antibiotics. Therefore, the development and improvement of drug controlled release systems is vital to create new approaches to deliver in the most effective manner the drugs or other bioactive compounds to the desired location. Especially if the targeted site is the gastrointestinal track, where the environmental conditions are harsh for biomolecules to maintain its stability and function. Polymeric microspheres are attractive due to their biodegradability and ability to encapsulate drugs or bioactive agents, therefore increasing their bioavailability. To address these poor bioavailability or unsustained drug release challenges, chitosan microspheres are adequate as drug delivery carriers for the gastrointestinal track due to mucoadhesive properties, which allows the drug dosage to be retained in the gastrointestinal track for extended periods of time, in addition to the presence of reactive sites in chitosan which allow the interaction with biomolecules to be carried to the targeted site. Spherical particles were produced using chitosan and γ-glycidoxypropyltrimethoxysilane (GPTMS) as an organic-inorganic hybrid compound resorting to two different methods. The first method consisted in a microfluidic approach using chitosan–GPTMS–β-glycerophosphate (chitosan–GPTMS–β-GP) to produce microspheres with uniform size and spherical shape around 650 μm and 285 μm. Whereas, in the second method beads with diameter around 2 mm with micropores were synthetized by dropping the hybrid precursor sol into liquid nitrogen followed by a freeze drying process. The physicochemical characterization of the microspheres from the microfluidic system was performed in which the formation of siloxane (Si-O-Si) networks was confirmed in the chitosan polymeric matrix, as well as the spheres stability in solutions. The degradation of microspheres with different GPTMS molar ratios was evaluated under simulated gastric fluids (SGF) and neutral conditions. The microspheres incubated at pH 7.4 had the lowest weight loss (27%–32%), whereas those incubated at pH 1.7 and pH 5.4 showed greater weight losses of 43–59% and 69–77%, respectively. The inhibition of the degradation at low pH was dependent on the siloxane network formed in the chitosan matrix. Additionally, GPTMS was released with the chitosan chains via hydrolysis of the chitosan molecules. Pelargonidin is a natural antioxidant which was incorporated in the microspheres and the releasing behavior was observed under SGF conditions and simulated time of digestion cycle in humans. The release profile observed leads to believe that these microspheres are promising for gastrointestinal drug delivery applications due to its resistance to low pH conditions present in the upper gastrointestinal track, in addition to the controlled and sustained release rate of pelargonidin and its ability to retain it in the matrix even after 57 h. Cerium compounds have been described to possess antibacterial activity, and new strategies of treating pathogenic bacteria are needed due to the rapid increase of bacterial resistance towards common antibiotics. The bacterial behavior of Escherichia coli and Staphylococcus aureus was observed with the chitosan–GPTMS–β-GP spheres and hydrogels containing cerium(III) chloride (CeCl3), and no antibacterial effect was observed due to the immediate interaction between β-GP and cerium, via the complex formation of cerium with the amino groups of chitosan, making it inaccessible to the bacteria. Furthermore, the bacterial viability increased for both gram-negative and gram-positive strains on hydrogels with and without cerium. To achieve antibacterial applications using the chitosan-siloxane hybrid, beads without β-GP were prepared by dropping the chitosan-GPTMS precursor sols into liquid nitrogen. The beads were synthetized with and without cerium. The bacterial activity was greatly reduced with the highest tested amounts of cerium against both gram-negative (Escherichia coli) and gram-positive (Staphylococcus aureus) strains. This microporous beads have the potential to be applied for soft tissue defect fillers materials with antibacterial properties to reduce or eradicate in situ bacterial infection.九州工業大学博士学位論文 学位記番号：生工博甲第312号 学位授与年月日：平成30年3月23日1. GENERAL INTRODUCTION|2. SYNTHESIS OF CHITOSAN-SILOXANE HYBRID MICROSPHERES USING A MICROFLUIDIC APPROACH AND RELEASE OF PELARGONIDIN IN GASTROINTESTINAL SIMULATED CONDITIONS|3. BACTERIAL BEHAVIOR ON CHITOSAN-SILOXANE HYBRID MICROSPHERES AND HYDROGELS CONTAINING CERIUM|4. BACTERIAL BEHAVIOR WITH CHITOSAN-SILOXANE HYBRID SPHERICAL BEADS CONTAINING CERIUM九州工業大学平成29年

Polymer Crosslinking: a new Strategy to Enhance Mechanical Properties and Structural Stability of Bioactive Glasses

The organic-inorganic hybrids fabricated by the sol-gel method are intrinsically bioactive materials with extensive applications in bone tissue engineering. The brittleness and limited water uptake capacity of these monoliths, however, restrict their applications for engineering the soft tissues and their interfaces with bone. To address these challenges, we developed a unique method in which polymer crosslinking was used to cease the over-condensation of a bioactive glass component and eradicate the formation of brittle structure. In this study, an organosilane-functionalized gelatin methacrylate was covalently bonded to a bioactive glass during the sol-gel process, and the condensation of silica networks was controlled by polymer-crosslinking. The physicochemical properties and mechanical strength of these hybrid hydrogels were then tuned by the incorporation of secondary crosslinking agents such as poly(ethylene glycol diacrylate). The resulting elastic hydrogels displayed tuneable compressive modulus in the range of 42 kPa to 530 kPa. The swelling behaviours of these hybrids and their structural integrities were also favourable for tissue engineering applications. Moreover, these hybrid hydrogels kept their structures for more than 28 days in simulated body fluid. The bioactivity of the constructs due to the presence of silica networks were confirmed by detecting nearly 2-fold increase in the alkaline phosphatase activity of the cultured bone progenitor cells on these hybrid hydrogels within 28 days of in vitro culture. Within the same period, in vivo studies on mice subcutaneous model showed that the hybrid hydrogels were highly biocompatible and well-tolerated. In summary, the bioactivity of the constructs, their tuneable physicochemical properties, the outstanding biocompatibility, and biodegradability of the hybrid hydrogels showed the high potential of the developed technique for fabrication of constructs for a variety of soft and hard tissue regeneration

Polymer Crosslinking: a new Strategy to Enhance Mechanical Properties and Structural Stability of Bioactive Glasses

The organic-inorganic hybrids fabricated by the sol-gel method are intrinsically bioactive materials with extensive applications in bone tissue engineering. The brittleness and limited water uptake capacity of these monoliths, however, restrict their applications for engineering the soft tissues and their interfaces with bone. To address these challenges, we developed a unique method in which polymer crosslinking was used to cease the over-condensation of a bioactive glass component and eradicate the formation of brittle structure. In this study, an organosilane-functionalized gelatin methacrylate was covalently bonded to a bioactive glass during the sol-gel process, and the condensation of silica networks was controlled by polymer-crosslinking. The physicochemical properties and mechanical strength of these hybrid hydrogels were then tuned by the incorporation of secondary crosslinking agents such as poly(ethylene glycol diacrylate). The resulting elastic hydrogels displayed tuneable compressive modulus in the range of 42 kPa to 530 kPa. The swelling behaviours of these hybrids and their structural integrities were also favourable for tissue engineering applications. Moreover, these hybrid hydrogels kept their structures for more than 28 days in simulated body fluid. The bioactivity of the constructs due to the presence of silica networks were confirmed by detecting nearly 2-fold increase in the alkaline phosphatase activity of the cultured bone progenitor cells on these hybrid hydrogels within 28 days of in vitro culture. Within the same period, in vivo studies on mice subcutaneous model showed that the hybrid hydrogels were highly biocompatible and well-tolerated. In summary, the bioactivity of the constructs, their tuneable physicochemical properties, the outstanding biocompatibility, and biodegradability of the hybrid hydrogels showed the high potential of the developed technique for fabrication of constructs for a variety of soft and hard tissue regeneration

The effect of magnesium on bioactivity, rheology and biology behaviors of injectable bioactive glass-gelatin-3-glycidyloxypropyl trimethoxysilane nanocomposite-paste for small bone defects repair

Injectable bioactive glass-based pastes represent promising biomaterials to fill small bone defects thus improving and speed up the self-healing process. Accordingly, injectable nanocomposite pastes based on bioactive glass-gelatin-3-glycidyloxypropyl trimethoxysilane (GPTMS) were here synthesized via two different glasses 64SiO2. 27CaO. 4MgO. 5P2O5 (mol.%) and 64SiO2.31CaO. 5P2O5 (mol.%). In particular, the effects of MgO on bioactivity, rheology, injectability, disintegration resistance, compressive strength and cellular behaviors were investigated. The results showed that the disintegration resistance and compressive strength of the composite were improved by the replacement of MgO; thus, leading to an increase in the amount of storage modulus (G′) from 26800 to 43400 Pa, equal to an increase in the viscosity of the paste from 136 × 103 to 219 × 103 Pa s. Since the release rate of ions became more controllable, the formation of calcite was decreased after immersion of the Mg bearing samples in the SBF solution. Specimens’ cytocompatibility was firstly verified towards human osteoblasts by metabolic assay as well as visually confirmed by the fluorescent live/dead staining; finally, the ability of human fibroblasts to penetrate within the pores of 3D composites was verified by a migration assay simulating the devices repopulation upon injection in the injured site

Characterization and degradation study of chitosan-siloxane hybrid microspheres synthesized using a microfluidic approach

Chitosan microspheres can address challenges associated with poor bioavailability or unsustained drug release when used as drug delivery systems thanks to their mucoadhesiveness, which allows the drug dosage to be retained in the gastrointestinal track for extended periods. Chitosan-3-glycidoxypropyltrimethoxysilane-β-glycerophosphate (chitosan-GPTMS-β-GP) hybrid microspheres were synthetized through sol-gel processing using a microfluidic approach. Microspheres with uniform spherical shapes and sizes of approximately 650 μm were obtained. The microstructures of the microspheres consisted of four different siloxane structures. The degradation behaviors of the hybrid microspheres were examined under acidic pH conditions mimicking those found in the gastrointestinal track. Microspheres with different GPTMS molar ratios were incubated under several pH conditions for 2 weeks. The microspheres incubated at pH 7.4 extended the lowest weight loss (27%–32%), whereas those incubated at pH 1.7 and pH 5.4 showed greater weight losses of 43–59% and 69–77%, respectively. The inhibition of the degradation at low pH was dependent on the siloxane network in the chitosan matrix. Phosphate was mostly released in early stages, and the released amount of silicon was dependent on the composition. GPTMS was released with a chitosan chain via the hydrolysis of a chitosan molecule. The pelargonidin was incorporated in the microspheres and the slow releasing was observed at acidic condition. The resistance of these hybrid microspheres to low-pH conditions for longer than a full digestion cycle is promising for gastrointestinal drug delivery applications

Covalently Crosslinked Organic/Inorganic Hybrid Biomaterials for Bone Tissue Engineering Applications

Scaffolds are key components for bone tissue engineering and regeneration. They guide new bone formation by mimicking bone extracellular matrix for cell recruitment and proliferation. Ideally, scaffolds for bone tissue engineering need to be osteoconductive, osteoinductive, porous, degradable and mechanically competent. As a single material can not provide all these requirements, composites of several biomaterials are viable solutions to combine various properties. However, conventional composites fail to fulfil these requirements due to their distinct phases at the microscopic level. Organic/inorganic (O/I) class II hybrid biomaterials, where the organic and inorganic phases are chemically crosslinked on a molecular scale, hence the phases are homogenously dispersed, are the ideal choices for bone tissue engineering. In this research, polycaprolactone/borophosphosilicate glass (PCL/BPSG) and poly(vinylpyrrolidone-co-triethoxyvinylsilane)/bioactive glass (Poly(VP-co-TEVS)/BG) class II hybrid biomaterials were successfully prepared via a sol-gel process. PCL was functionalized with 3-glycidoxypropyl trimethoxysilane at both ends prior to hybrid syntheses. Trimethoxysilane-functionalized PCL was then polycondensed with the glass precursors via non-aqueous sol gel reactions to form covalently bonded O/I network with -C-Si-O-Si- bonds. The resultant amorphous and transparent hybrid materials exhibited apatite depositions when incubated with simulated body fluid. The ultimate compressive stress, modulus and toughness of these hybrids were significantly greater compared with their conventional composites counterparts, attributed to the covalent bonding between the O/I phases. In addition, these hybrids exhibited more controlled degradation and subsequent ion release without showing any abrupt features. Pre-osteoblast cells seeded on the hybrid biomaterials displayed enhanced spreading, focal adhesion formation, and cell number, indicating cytocompatibility. PCL/BPSG hybrid scaffolds were prepared by a solvent-free casting and particulate leaching methods to obtain consistent pore size distribution, controllable porosity and pore interconnectivity. Significant number of cell infiltration and adhesion into the scaffolds were observed in cell culture conditions. Bone-associated gene expression by induced pluripotent stem cells on these scaffolds revealed that the hybrid scaffolds had an upregulating effect on gene expressions for alkaline phosphatase, osteopontin and osteocalcin

Radiopaque Crystalline, Non-Crystalline and Nanostructured Bioceramics

Radiopacity is sometimes an essential characteristic of biomaterials that can help clinicians perform follow-ups during pre- and post-interventional radiological imaging. Due to their chemical composition and structure, most bioceramics are inherently radiopaque but can still be doped/mixed with radiopacifiers to increase their visualization during or after medical procedures. The radiopacifiers are frequently heavy elements of the periodic table, such as Bi, Zr, Sr, Ba, Ta, Zn, Y, etc., or their relevant compounds that can confer enhanced radiopacity. Radiopaque bioceramics are also intriguing additives for biopolymers and hybrids, which are extensively researched and developed nowadays for various biomedical setups. The present work aims to provide an overview of radiopaque bioceramics, specifically crystalline, non-crystalline (glassy), and nanostructured bioceramics designed for applications in orthopedics, dentistry, and cancer therapy. Furthermore, the modification of the chemical, physical, and biological properties of parent ceramics/biopolymers due to the addition of radiopacifiers is critically discussed. We also point out future research lacunas in this exciting field that bioceramists can explore further