Synthesis and characterization of hyaluronic acid–poly(ethylene glycol) hydrogels via Michael addition: An injectable biomaterial for cartilage repair

Injectable hydrogels based on hyaluronic acid (HA) and poly(ethylene glycol) (PEG) were designed as biodegradable matrices for cartilage tissue engineering. Solutions of HA conjugates containing thiol functional groups (HA-SH) and PEG vinylsulfone (PEG-VS) macromers were cross-linked via Michael addition to form a three-dimensional network under physiological conditions. Gelation times varied from 14 min to less than 1 min, depending on the molecular weights of HA-SH and PEG-VS, degree of substitution (DS) of HA-SH and total polymer concentration. When the polymer concentration was increased from 2% to 6% (w/v) in the presence of 100 U ml^?1 hyaluronidase the degradation time increased from 3 to 15 days. Hydrogels with a homogeneous distribution of cells were obtained when chondrocytes were mixed with the precursor solutions. Culturing cell–hydrogel constructs prepared from HA185k-SH with a DS of 28 and cross-linked with PEG5k-4VS for 3 weeks in vitro revealed that the cells were viable and that cell division took place. Gel–cell matrices degraded in approximately 3 weeks, as shown by a significant decrease in dry gel mass. At day 21 glycosaminoglycans and collagen type II were found to have accumulated in hydrogels. These results indicate that these injectable hydrogels have a high potential for cartilage tissue engineering.     

Solvent-dependent properties of electrospun fibrous composites for bone tissue regeneration

Biodegradable polymer–ceramic composite scaffolds have gained importance in recent years in the field of orthopedic biomaterials and tissue engineering scaffolds for improving the rate of degradation and limited mechanical properties of bioactive ceramics. This study sought to create composites using the electrospinning process to achieve fibrous scaffolds with uniform fiber morphologies and uniform ceramic dispersions. Composites consisting of 20% hydroxyapatite/80% β-tricalcium phosphate (20/80 HA/TCP) and poly (ε-caprolactone) (PCL) were fabricated. The 20/80 HA/TCP composition was chosen as the ceramic component because of previous reports of greater bone tissue formation in comparison with HA or TCP alone. For electrospinning, PCL was dissolved in either methylene chloride (Composite–MC) or a combination of methylene chloride (80%) and dimethylformamide (20%) (Composite–MC + DMF). Composite–MC mats contained a bimodal distribution of fiber diameters with nanofibers between larger, micron-sized fibers with an average pore size of 79.6 ± 67 μm, whereas Composite–MC + DMF fibers had uniform fiber diameters with an average pore size of 7.0 ± 4.2 μm. Elemental mapping determined that the ceramic was distributed throughout the mat and inside the fiber for both composites. However, physical characterization using differential scanning calorimetry (DSC) and mechanical testing revealed that the ceramic in the mats produced with MC + DMF were more uniformly dispersed than the ceramic in the mats produced with MC alone. Maximum tensile stress and strain were significantly higher for Composite–MC + DMF mats compared with Composite–MC mats and were comparable with the mechanical properties of mats of PCL alone. For both composites, there was molecular interaction between the PCL and the ceramic, as demonstrated by a maximum increase of ～10 °C in the glass transition values with the addition of the ceramic, as confirmed by Fourier transform infrared analysis. In addition, the crystallization behavior of the composites suggested that the ceramic was acting as a nucleating agent. Cell viability studies using human mesenchymal stem cells (MSC) showed that both composite scaffolds supported cell growth. However, cell numbers at early time points in culture were significantly higher on mats produced from MC + DMF compared with mats prepared with MC alone. Further examination revealed that cells were able to infiltrate the pores of the Composite–MC mats, but remained on the outer surface of the Composite–MC + DMF and unfilled PCL mats during the culture period. The results of this study demonstrate that the solvent or solvent combination used in preparing the electrospun composite mats plays a critical role in determining its properties, which may, in turn, affect cell behavior.     

Molecular dynamics simulations on the interaction between polymers and hydroxyapatite with and without coupling agents

Molecular dynamics (MD) simulations were employed to study hydroxyapatite/biopolymer interface interactions in composites for biomedical applications. The study analyzed the binding energies between hydroxyapatite (HA) and three polymers: polyethylene (PE), polyamide (PA) and polylactic acid (PLA). The interactions of polymers on HA crystallographic planes (0 0 1), (1 0 0) and (1 1 0) were simulated. The effects of the silane coupling agent (A174) on interfacial binding energies were also examined. The results show that HA (1 1 0) has the highest binding energy with these polymers because of its higher planar atom density than that of HA (0 0 1) and (1 0 0). The binding energies of PA/HA and PLA/HA are much higher than that of PE/HA, which might be attributed to large number of polar groups in PA and PLA chains. The silane coupling agent A174 increases the binding energy between PE and HA, but not for the PA/HA and PLA/HA systems. The MD results can be used to guide the design of polymer/HA composites and to select proper coupling agents.     

Composite fibrous biomaterials for tissue engineering obtained using a supercritical CO2 antisolvent process

Several techniques have been proposed for producing porous structures or scaffolds for tissue engineering but, as yet, with no optimal solution. With regard to this topic, this paper focuses on the preparation of biocompatible nanometric filler–polymer composites organized in a network of fibers. Titanium dioxide (TiO₂) or hydroxyapatite (HAP) nanopowders as the guest particles and poly(lactic acid) (L-PLA) or the blend poly(methylmethacrylate)/poly(ε-caprolactone) (PMMA/PCL) as the polymer carrier were selected as model systems for this purpose. A supercritical antisolvent technique was used to produce the composites. In the process developed, the non-soluble particulate filler was suspended in a polymer solution, and both components were sprayed simultaneously into supercritical carbon dioxide (scCO₂). Using this technique, polymeric matrices were loaded with ～10–20 wt.% of inorganic phase distributed throughout the composite. Two different hybrid materials were prepared: a PMMA/PCL + TiO₂ system where either fibers or microparticles were prepared by varying the molecular weight of the used PMMA; and fibers in the case of L-PLA + HAP system. After further post-processing in a three-dimensional network, these nanofibers can potentially be used as scaffolds for tissue engineering.     

Study of nanoscale structures in hydrated biomaterials using small-angle neutron scattering

Distribution of water in three classes of biomedically relevant and degradable polymers was investigated using small-angle neutron scattering. In semicrystalline polymers, such as poly(lactic acid) and poly(glycolic acid), water was found to diffuse preferentially into the non-crystalline regions. In amorphous polymers, such as poly(d,l-lactic acid) and poly(lactic-co-glycolic acid), the scattering after 7 days of incubation was attributed to water in microvoids that form following the hydrolytic degradation of the polymer. In amorphous copolymers containing hydrophobic segments (desaminotyrosyl-tyrosine ethyl ester) and hydrophilic blocks (poly(ethylene glycol) (PEG)), a sequence of distinct regimes of hydration were observed: homogeneous distribution (～10 Å length scales) at <13 wt.% PEG (～1 water per EG), clusters of hydrated domains (～50 Å radius) separated at 24 wt.% PEG (1–2 water per EG), uniformly distributed hydrated domains at 41 wt.% PEG (～4 water per EG) and phase inversion at >50 wt.% PEG (>6 water per EG). Increasing the PEG content increased the number of these domains with only a small decrease in distance between the domains. These discrete domains appeared to coalesce to form submicron droplets at ～60 °C, above the melting temperature of crystalline PEG. The significance of such observations on the evolution of micrometer-size channels that form during hydrolytic erosion is discussed.     

Synthesis,characterization and surface modification of low moduli poly(ether carbonate urethane)ureas for soft tissue engineering

Flexible scaffolds are of great interest in engineering functional and mechano-active soft tissues as such scaffolds might allow mechanical stimuli to transfer effectively from the scaffolds to cells during tissue development. Towards this end, we have developed a family of flexible poly(ether carbonate urethane)ureas (PECUUs) with a triblock copolymer poly(trimethylene carbonate)–poly(ethylene oxide)–poly(trimethylene carbonate) (PTMC–PEO–PTMC) or pentablock copolymers PTMC–PEO–PPO–PEO–PTMC (PPO, polypropylene oxide) as soft segments, linked by 1,4-diisocyanatobutane and putrescine. All of the PECUUs had low glass transition temperatures (<?46 °C). The PTMC–PEO–PTMC-containing PECUUs had low tensile strength and breaking strain. Replacing PEO with the similar length PEO–PPO–PEO resulted in highly flexible and soft PECUUs possessing breaking strains of 362–711%, tensile strengths of 8–18 MPa and moduli of 5.5–7.4 MPa at room temperature in air. Under aqueous conditions at 37 °C, these polymers remained flexible while their moduli were decreased to 3.4–4.0 MPa. PECUUs based on PTMC–PEO–PPO–PEO–PTMC were thermosensitive as the water content at 37 °C was lower than that at 4 °C. PECUU using PTMC–PEO–PTMC as a soft segment showed 30% weight loss over 6 weeks in PBS at 37 °C, while that using PTMC–PEO–PPO–PEO–PTMC as a soft segment had weight loss <6%. Degradation products were found to lack cytotoxicity. The mechanical stresses and moduli of PECUUs based on PTMC–PEO–PPO–PEO–PTMC were unchanged during the degradation. To enhance cell adhesion, PECUUs were surface modified with Arg-Gly-Asp-Ser (RGDS). Smooth muscle cell adhesion was 114% of tissue culture polystyrene for unmodified PECUU and >180% for RGDS-modified PECUUs, with cell viability on both surfaces increasing during culture. These low moduli polyurethanes may find applications in engineering cardiovascular or other soft tissues.     

Semi-interpenetrating networks of hyaluronic acid in degradable PEG hydrogels for cartilage tissue engineering

Hydrolytically biodegradable poly(ethylene glycol) (PEG) hydrogels offer a promising platform for chondrocyte encapsulation and tuning degradation for cartilage tissue engineering, but offer no bioactive cues to encapsulated cells. This study tests the hypothesis that a semi-interpenetrating network of entrapped hyaluronic acid (HA), a bioactive molecule that binds cell surface receptors on chondrocytes, and crosslinked degradable PEG improves matrix synthesis by encapsulated chondrocytes. Degradation was achieved by incorporating oligo (lactic acid) segments into the crosslinks. The effects of HA molecular weight (MW) (2.9 × 10⁴ and 2 × 10⁶ Da) and concentration (0.5 and 5 mg g⁻¹) were investigated. Bovine chondrocytes were encapsulated in semi-interpenetrating networks and cultured for 4 weeks. A steady release of HA was observed over the course of the study with 90% released by 4 weeks. Incorporation of HA led to significantly higher cell numbers throughout the culture period. After 8 days, HA increased collagen content per cell, increased aggrecan-positive cells, while decreasing the deposition of hypertrophic collagen X, but these effects were not sustained long term. Measuring total sulfated glycosaminoglycan (sGAG) and collagen content within the constructs and released to the culture medium after 4 weeks revealed that total matrix synthesis was elevated by high concentrations of HA, indicating that HA stimulated matrix production although this matrix was not retained within the hydrogels. Matrix-degrading enzymes were elevated in the low-, but not the high-MW HA. Overall, incorporating high-MW HA into degrading hydrogels increased chondrocyte number and sGAG and collagen production, warranting further investigations to improve retention of newly synthesized matrix molecules.     

Poly(ethylene glycol) analogs grafted with low molecular weight poly(ethylene imine) as non-viral gene vectors

A novel class of non-viral gene vectors consisting of low molecular weight poly(ethylene imine) (PEI) (molecular weight 800 Da) grafted onto degradable linear poly(ethylene glycol) (PEG) analogs was synthesized. First, a Michael addition reaction between poly(ethylene glycol) diacrylates (PEGDA) (molecular weight 258 Da) and d,l-dithiothreitol (DTT) was carried out to generate a linear polymer (PEG–DTT) having a terminal thiol, methacrylate and pendant hydroxyl functional groups. Five PEG–DTT analogs were synthesized by varying the molar ratio of diacrylates to thiols from 1.2:1 to 1:1.2. Then PEI (800 Da) was grafted onto the main chain of the PEG–DTTs using 1,1′-carbonyldiimidazole as the linker. The above reaction gave rise to a new class of non-viral gene vectors, (PEG–DTT)–g-PEI copolymers, which can effectively complex DNA to form nanoparticles. The molecular weights and structures of the copolymers were characterized by gel permeation chromatography, ¹H nuclear magnetic resonance and Fourier transform infrared spectroscopy. The size of the nanoparticles was <200 nm and the surface charge of the nanoparticles, expressed as the zeta potential, was between +20 and +40 mV. Cytotoxicity assays showed that the copolymers exhibited much lower cytotoxicities than high molecular weight PEI (25 kDa). Transfection was performed in cultured HeLa, HepG2, MCF-7 and COS-7 cells. The copolymers showed higher transfection efficiencies than PEI (25 kDa) tested in four cell lines. The presence of serum (up to 30%) had no inhibitory effect on the transfection efficiency. These results indicate that this new class of non-viral gene vectors may be a promising gene carrier that is worth further investigation.     

Peptide-grafted poly(ethylene glycol) hydrogels support dynamic adhesion of endothelial progenitor cells

This study investigated the dynamic adhesion of endothelial progenitor cells (EPCs) to peptide-grafted poly(ethylene glycol) diacrylate (PEGDA) hydrogels and determined the relative ability of RGDS, REDV and YIGSRG peptides to reduce the velocity of EPC rolling. Circulating EPCs are key mediators of endothelium repair and have been shown to accelerate re-endothelialization, which is important in reducing the incidence of restenosis following stent placement and occlusion of small diameter vascular grafts. However, to exploit these capabilities for tissue engineering applications, more knowledge is needed about EPC binding to the vascular wall under shear and, in particular, whether the incorporation of peptide ligands into biomaterials can support the process of EPC rolling or maintain EPC adhesion. This study specifically examined one type of EPCs endothelial colony forming cells (ECFCs), based on their ability to be expanded in culture and differentiate into mature endothelial cells. The amount of grafted PEG–peptide was shown to be dependent on the concentration of PEG–peptide grafting solution photopolymerized onto the hydrogel surface. The ECFC strength of adhesion on PEG–RDGS grafted hydrogels exceeded 350 dyn cm^?2 for 85% of adherent cells. PEG–RGDS grafted hydrogels supported ECFC rolling, whereas ECFC velocity on the negative control PEG–RGES grafted hydrogels and on the “blank slate” PEGDA hydrogels was substantially higher than the cutoff velocity for cell rolling. The ECFC rolling velocity on PEG–RDGS grafted hydrogels depended on the shear rate; as shear rate was increased from 20 s^?1 to 120 s^?1, ECFC rolling velocity increased from 103 ± 3 μm s^?1 to 741 ± 28 μm s^?1. REDV and YIGSRG, which are known to preferentially support endothelial cell adhesion, also supported ECFC rolling. Interestingly, the rolling velocity of ECFCs on PEG–REDV grafted hydrogels was significantly lower than on PEG–YIGSRG or on PEG–RGDS grafted hydrogels. Understanding the dynamic adhesion of ECFCs to peptide-grafted hydrogels is the first step towards understanding the similarities and differences of EPCs from mature endothelial cells and improving the ability to sequester EPCs to biomaterial surfaces in order to promote intravascular re-endothelialization.     

Injectable,rapid gelling and highly flexible hydrogel composites as growth factor and cell carriers

A family of injectable, rapid gelling and highly flexible hydrogel composites capable of releasing insulin-like growth factor (IGF-1) and delivering mesenchymal stromal cell (MSC) were developed. Hydrogel composites were fabricated from Type I collagen, chondroitin sulfate (CS) and a thermosensitive and degradable hydrogel copolymer based on N-isopropylacrylamide, acrylic acid, N-acryloxysuccinimide and a macromer poly(trimethylene carbonate)-hydroxyethyl methacrylate. The hydrogel copolymer was gellable at body temperature before degradation and soluble at body temperature after degradation. Hydrogel composites exhibited LCSTs around room temperature. They could easily be injected through a 26-gauge needle at 4 °C, and were capable of gelling within 6 s at 37 °C to form highly flexible gels with moduli matching those of the rat and human myocardium. The hydrogel composites showed good oxygen permeability; the oxygen pressure within the hydrogel composites was similar to that in the air. The effects of collagen and CS contents on LCST, gelation time, injectability, mechanical properties and degradation properties were investigated. IGF-1 was loaded into the hydrogel composites for enhanced cell survival/growth. The released IGF-1 remained bioactive during a 2-week release period. Small fraction of CS in the hydrogel composites significantly decreased IGF-1 release rate. The release kinetics appeared to be controlled mainly by hydrogel composite water content, degradation and interaction with IGF-1. Human MSC adhesion on the hydrogel composites was comparable to that on the tissue culture plate. MSCs were encapsulated in the hydrogel composites and were found to grow inside during a 7-day culture period. IGF-1 loading significantly accelerated MSC growth. RT-PCR analysis demonstrated that MSCs maintained their multipotent differentiation potential in hydrogel composites with and without IGF-1. These injectable and rapid gelling hydrogel composites demonstrated attractive properties for serving as growth factor and cell carriers for cardiovascular tissue engineering applications.     

Therapeutic foam scaffolds incorporating biopolymer-shelled mesoporous nanospheres with growth factors

A novel therapeutic scaffolding system of engineered nanocarriers within a foam matrix for the long-term and sequential delivery of growth factors is reported. Mesoporous silica nanospheres were first functionalized to have an enlarged mesopore size (12.2 nm) and aminated surface, which was then shelled by a biopolymer, poly(lactic acid) (PLA) or poly(ethylene glycol) (PEG), via electrospraying. The hybrid nanocarrier was subsequently combined with collagen to produce foam scaffolds. Bovine serum albumin (BSA), used as a model protein, was effectively loaded within the enlarged nanospheres. The biopolymer shell substantially prolonged the release period of BSA (2–3 weeks from shelled nanospheres vs. within 1 week from bare nanospheres), and the release rate was highly dependent on the shell composition (PEG > PLA). Collagen foam scaffolding of the shelled nanocarrier further slowed down the protein release, while enabling the incorporation of a rapidly releasing protein, which is effective for sequential protein delivery. Acidic fibroblast growth factor (aFGF), loaded onto the shelled-nanocarrier scaffolds, was released over a month at a highly sustainable rate, profiling a release pattern similar to that of BSA. The biological activity of the aFGF was evidenced by the significant proliferation of osteoblastic precursor cells in the aFGF-releasing scaffolds. Furthermore, the aFGF-delivering scaffolds implanted in rat subcutaneous tissue for 2 weeks showed a substantially enhanced invasion of fibroblasts with a homogeneous population. Taken together, it is concluded that the biopolymer encapsulation of mesoporous nanospheres effectively prolongs the release of growth factors over weeks to a month, providing a nanocarrier platform for a long-term growth factor delivery. Moreover, the foam scaffolding of the nanocarrier system is a potential therapeutic three-dimensional matrix for cell culture and tissue engineering.     

Modulating rheological and degradation properties of temperature-responsive gelling systems composed of blends of PCLA–PEG–PCLA triblock copolymers and their fully hexanoyl-capped derivatives

In this study, the ability to modulate rheological and degradation properties of temperature-responsive gelling systems composed of aqueous blends of poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide) (PCLA–PEG–PCLA) triblock copolymers (i.e. uncapped) and their fully capped derivatives was investigated. Uncapped and capped PCLA–PEG–PCLA triblock copolymers, abbreviated as degree of modification 0 and 2 (DM0 and DM2, respectively), were composed of identical PCLA and PEG blocks but different end groups: namely hydroxyl and hexanoyl end groups. DM0 was synthesized by ring opening polymerization of l-lactide and ε-caprolactone in toluene using PEG as initiator and tin(II) 2-ethylhexanoate as the catalyst. A portion of DM0 was subsequently reacted with an excess of hexanoyl chloride in solution to yield DM2. The cloud point and phase behaviour of DM0 and DM2 in buffer as well as that of their blends were determined by light scattering in a diluted state and by vial tilting and rheological measurements in a concentrated state. Degradation/dissolution properties of temperature-responsive gelling systems were studied in vitro at pH 7.4 and 37 °C. The cloud points of DM0/DM2 blends were ratio-dependent and could be tailored from 15 to 40 °C for blends containing 15 to 100 wt.% DM0. Vial tilting and rheological experiments showed that, with solid contents between 20 and 30 wt.%, DM0/DM2 blends (15/85 to 25/75 w/w) had a sol-to-gel transition temperature at 10–20 °C, whereas blends with less than 15 wt.% DM0 formed gels below 4 °C and the ones with more than 25 wt.% DM0 did not show a sol-to-gel transition up to 50 °C. Complete degradation of temperature-responsive gelling systems took ～100 days, independent of the DM0 fraction and the initial solid content. Analysis of residual gels in time by GPC and ¹H-NMR showed no chemical polymer degradation, but indicated gel degradation by dissolution. Preferential dissolution of lactoyl-rich polymers induced enrichment of the residual gels in caproyl-rich polymers. To the best of our knowledge, degradation of temperature-responsive gelling systems by dissolution has not been reported or hypothesized as being the consequence of acylation of polymers. In conclusion, blending of PCLA–PEG–PCLA triblock polymers composed of identical backbones but different end groups provides for a straightforward preparation of temperature-responsive gelling systems with well-characterized rheological properties and potential in drug delivery. Furthermore, acylation of triblock copolymers may allow for the design of bioerodible systems with control over degradation by polymer dissolution.     

Interfacial optimization of fiber-reinforced hydrogel composites for soft fibrous tissue applications

Meniscal tears are the most common orthopedic injuries to the human body, yet the current treatment of choice is a partial meniscectomy, which is known to lead to joint degeneration and osteoarthritis. As a result, there is a significant clinical need to develop materials capable of restoring function to the meniscus following an injury. Fiber-reinforced hydrogel composites are particularly suited for replicating the mechanical function of native fibrous tissues due to their ability to mimic the native anisotropic property distribution present. A critical issue with these materials, however, is the potential for the fiber–matrix interfacial properties to severely limit composite performance. In this work, the interfacial properties of an ultra-high-molecular-weight polyethylene (UHMWPE) fiber-reinforced poly(vinyl alcohol) (PVA) hydrogel are studied. A novel chemical grafting technique, confirmed using X-ray photoelectron spectroscopy, is used to improve UHMWPE–PVA interfacial adhesion. Interfacial shear strength is quantified using fiber pull-out tests. Results indicate significantly improved fiber–hydrogel interfacial adhesion after chemical grafting, where chemically grafted samples have an interfacial shear strength of 256.4 ± 64.3 kPa compared to 11.5 ± 2.9 kPa for untreated samples. Additionally, scanning electron microscopy of fiber surfaces after fiber pull-out reveal cohesive failure within the hydrogel matrix for treated fiber samples, indicating that the UHMWPE–PVA interface has been successfully optimized. Lastly, inter-fiber spacing is observed to have a significant effect on interfacial adhesion. Fibers spaced further apart have significantly higher interfacial shear strengths, which is critical to consider when optimizing composite design. The results in this study are applicable in developing similar chemical grafting techniques and optimizing fiber–matrix interfacial properties for other hydrogel-based composite systems.     

Preparation and Drug-Delivery Potential of Metronidazole-Loaded PELA Tri-block Co-polymeric Electrospun Membranes

The aim of this study was to investigate the potential of poly(ethylene glycol-co-lactide) (PELA tri-block with a segmental sequence of PLA–PEG–PLA) electrospun membranes as drug-delivery vehicles using metronidazole as a model drug. PELA membranes with smooth surfaces and no bead defects were electrospun from polymer solutions containing 20% (w/v) PELA in 8:2 N,N-dimethyl formamide (DMF)/acetone. The morphology of the drug-loaded electrospun membranes was influenced by electrospinning parameters such as the flow rate and voltages during preparation. Metronidazole could be released from the electrospun membranes and was characterized by an initial burst effect. Higher voltages led to faster release rates, while an increase in the flow rate decreased the drug release. The incorporation of metronidazole into the electrospun membranes decreased their surface hydrophilicity. The amount of drug released from the electrospun membranes was effective in inhibiting microbial growth. Cell adhesion on the PELA membranes with or without drug was less than that on the homo-polymeric PDLLA membranes. Proliferation of L929 mouse fibroblasts on the PELA membranes was observed. This study confirms the potential of metronidazole-loaded PELA biodegradable electrospun membranes for optimizing the clinical therapy of post-surgical adhesions and infections.     

Poly(lactide)–vitamin E derivative/montmorillonite nanoparticle formulations for the oral delivery of Docetaxel

Four systems of nanoparticles of biodegradable polymers were developed in this research for oral delivery of anticancer drugs with Docetaxel used as a model drug, which include the poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs), the poly(lactide)–vitamin E TPGS nanoparticles (PLA–TPGS NPs), the poly(lactic-co-glycolic acid)–montmorillonite nanoparticles (PLGA/MMT NPs) and the poly(lactide)–vitamin E TPGS/montmorillonite nanoparticles (PLA–TPGS/MMT NPs). Vitamin E TPGS stands for d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), which is a water-soluble derivative of natural vitamin E formed by esterification of vitamin E succinate with polyethylene glycol (PEG) 1000. The design was made to take advantages of TPGS in nanoparticle technology such as high emulsification effects and high drug encapsulation efficiency, and those in drug formulation such as high cellular adhesion and adsorption. MMT of similar effects is also a detoxifier, which may cure some side effects caused by the formulated drug. The drug-loaded NPs were prepared by a modified solvent extraction/evaporation method and then characterized for their MMT content, size and size distribution, surface charge and morphology, physical status and encapsulation efficiency of the drug in the NPs, and in vitro drug release profile. Cellular uptake of the coumarin 6-loaded NPs was investigated. In vitro cancer cell viability experiment showed that judged by IC₅₀, the PLA–TPGS/MMT NP formulation was found 2.89, 3.98, 2.12-fold more effective and the PLA–TPGS NP formulation could be 1.774, 2.58, 1.58-fold more effective than the Taxotere^® after 24, 48, 72 h treatment, respectively. In vivo PK experiment with SD rats showed that oral administration of the PLA–TPGS/MMT NP formulation and the PLA–TPGS NP formulation could achieve 26.4 and 20.6 times longer half-life respectively than i.v. administration of Taxotere^® at the same 10 mg/kg dose. One dose oral administration of the NP formulations could realize almost 3 week sustained chemotherapy in comparison of 22 h of i.v. administration of Taxotere^®. The oral bioavailability can be enhanced from 3.59% for Taxotere^® to 78% for the PLA–TPGS/MMT NP formulation and 91% for the PLA–TPGS NP formulation respectively. Oral chemotherapy by nanoparticles of biodegradable polymers is feasible.     

Novel nanostructured biodegradable polymer matrices fabricated by phase separation techniques for tissue regeneration

Biomimetic nanostructures have a wide range of applications. In particular, biodegradable polymer nanostructures that mimic the subtleties of extracellular matrix may provide favorable cell interactions. In this study, a co-solvent system was developed to configure a thermodynamically metastable biodegradable polymer solution, from which novel nanostructured matrices subsequently formed via wet phase separation (quaternary) or a combination with thermally induced phase separation. Three-dimensional (3D) nanostructured porous matrices were further fabricated by combination with particle-leaching (100–300 μm glucose). The new co-solvent system may generate matrices with reproducible nanostructures from a variety of biodegradable polymers such as poly(d,l-lactide) (PLA), poly(ε-caprolactone) (PCL) and PCL-based polyurethane. In vitro cell culture experiments were performed with mouse pre-osteoblasts (MC3T3-E1) and human bone marrow-derived mesenchymal stem cells (hBM-MSC) to evaluate the osteoinductive potential of PLA nanostructures. The results showed that nanofibrous (<100 nm) membranes promoted the bone-related marker gene expression and matrix mineralization of MC3T3-E1 at 14 days. Nanofibrous 3D matrices seeded with hBM-MSC without osteogenic induction supplements demonstrated a 2.5-fold increase in bone matrix deposition vs. the conventional microporous matrices after 14 and 21 days. Antimicrobial nanofibers were further obtained by plasma-assisted coating of chitosan on PLA nanofibers. This study reveals a platform for fabricating novel biodegradable nanofibrous architecture, with potential in tissue regeneration.     

Injectable chitosan hyaluronic acid hydrogels for cartilage tissue engineering

Injectable cartilaginous constructs that can form gels in tissue defects have many advantages in tissue engineering applications. In this study we created an injectable hydrogel consisting of methacrylated glycol chitosan (MeGC) and hyaluronic acid (HA) by photocrosslinking with a riboflavin photoinitiator under visible light. A minimum irradiation time of 40 s was required to produce stable gels for cell encapsulation with 87–90% encapsulated chondrocyte viability. Although increasing the irradiation time from 40 to 600 s significantly enhanced the compressive modulus of the hydrogels up to 11 or 17 kPa for MeGC or MeGC/HA, respectively, these conditions reduced the encapsulated cell viability to 60–65%. The majority of chondrocytes encapsulated in MeGC hydrogels after 300 s irradiation maintained a rounded shape with a high cell viability of ～80–87% over a 21 day culture period. The incorporation of HA in MeGC hydrogels increased the proliferation and deposition of cartilaginous extracellular matrix by encapsulated chondrocytes. These findings demonstrate that MeGC/HA composite hydrogels have the potential for cartilage repair.     

In vitro degradation and cytotoxicity of Mg/Ca composites produced by powder metallurgy

Mg/Ca (1 wt.%, 5 wt.%, 10 wt.% Ca) composites were prepared from pure magnesium and calcium powders using the powder metallurgy method, aiming to enlarge the addition of Ca content without the formation of Mg₂Ca. The microstructures, mechanical properties and cytotoxicities of Mg/Ca composite samples were investigated. The corrosion of Mg/Ca composites in Dulbecco’s modified Eagle’s medium (DMEM) for various immersion intervals was studied by electrochemical impedance spectroscopy measurements and environmental scanning electron microscope, with the concentrations of released Mg and Ca ions in DMEM for various immersion time intervals being measured. It was shown that the main constitutional phases were Mg and Ca, which were uniformly distributed in the Mg matrix. The ultimate tensile strength (UTS) and elongation of experimental composites decreased with increasing Ca content, and the UTS of Mg/1Ca composite was comparable with that of as-extruded Mg–1Ca alloy. The corrosion potential increased with increasing Ca content, whereas the current density and the impedance decreased. It was found that the protective surface film formed quickly at the initial immersion stage. With increasing immersion time, the surface film became compact, and the corrosion rate of Mg/Ca composites slowed down. The surface film consisted mainly of CaCO₃, MgCO₃·3H₂O, HA and Mg(OH)₂ after 72 h immersion in DMEM. Mg/1Ca and Mg/5Ca composite extracts had no significant toxicity (p > 0.05) to L-929 cells, whereas Mg/10Ca composite extract induced ～40% reduced cell viability.     

Hollow hydroxyapatite microspheres: A novel bioactive and osteoconductive carrier for controlled release of bone morphogenetic protein-2 in bone regeneration

The regeneration of large bone defects is a common and significant clinical problem. Limitations associated with existing treatments such as autologous bone grafts and allografts have increased the need for synthetic bone graft substitutes. The objective of this study was to evaluate the capacity of novel hollow hydroxyapatite (HA) microspheres to serve as a carrier for controlled release of bone morphogenetic-2 (BMP2) in bone regeneration. Hollow HA microspheres (106–150 μm) with a high surface area (>100 m² g^?1) and a mesoporous shell wall (pore size 10–20 nm) were created using a glass conversion technique. The release of BMP2 from the microspheres into a medium composed of diluted fetal bovine serum in vitro was slow, but it occurred continuously for over 2 weeks. When implanted in rat calvarial defects for 3 or 6 weeks, the microspheres loaded with BMP2 (1 μg per defect) showed a significantly better capacity to regenerate bone than those without BMP2. The amount of new bone in the defects implanted with the BMP2-loaded microspheres was 40% and 43%, respectively, at 3 and 6 weeks, compared to 13% and 17%, respectively, for the microspheres without BMP2. Coating the BMP2-loaded microspheres with a biodegradable polymer, poly(lactic-co-glycolic acid), reduced the amount of BMP2 released in vitro and, above a certain coating thickness, significantly reduced bone regeneration in vivo. The results indicate that these hollow HA microspheres could provide a bioactive and osteoconductive carrier for growth factors in bone regeneration.     

Tungsten disulfide nanotubes reinforced biodegradable polymers for bone tissue engineering

In this study, we have investigated the efficacy of inorganic nanotubes as reinforcing agents to improve the mechanical properties of poly(propylene fumarate) (PPF) composites as a function of nanomaterial loading concentration (0.01–0.2 wt.%). Tungsten disulfide nanotubes (WSNTs) were used as reinforcing agents in the experimental group. Single- and multi-walled carbon nanotubes (SWCNTs and MWCNTs) were used as positive controls, and crosslinked PPF composites were used as the baseline control. Mechanical testing (compression and three-point bending) shows a significant enhancement (up to 28–190%) in the mechanical properties (compressive modulus, compressive yield strength, flexural modulus and flexural yield strength) of WSNT-reinforced PPF nanocomposites compared to the baseline control. In comparison to the positive controls, significant improvements in the mechanical properties of WSNT nanocomposites were also observed at various concentrations. In general, the inorganic nanotubes (WSNTs) showed mechanical reinforcement better than (up to 127%) or equivalent to that of carbon nanotubes (SWCNTs and MWCNTs). Sol fraction analysis showed significant increases in the crosslinking density of PPF in the presence of WSNTs (0.01–0.2 wt.%). Transmission electron microscopy (TEM) analysis on thin sections of crosslinked nanocomposites showed the presence of WSNTs as individual nanotubes in the PPF matrix, whereas SWCNTs and MWCNTs existed as micron-sized aggregates. The trend in the surface area of nanostructures obtained by Brunauer–Emmett–Teller (BET) surface area analysis was SWCNTs > MWCNTs > WSNTs. The BET surface area analysis, TEM analysis and sol fraction analysis results taken together suggest that chemical composition (inorganic vs. carbon nanomaterials), the presence of functional groups (such as sulfide and oxysulfide) and individual dispersion of the nanomaterials in the polymer matrix (absence of aggregation of the reinforcing agent) are the key parameters affecting the mechanical properties of nanostructure-reinforced PPF composites and the reason for the observed increases in the mechanical properties compared to the baseline and positive controls.