Acrylonitrile Butadiene Styrene/Thermoplastic Polyurethane Blends for Material Extrusion Three-Dimensional Printing: Effects of Blend Composition on Printability and Properties.

Blend filaments of acrylonitrile butadiene styrene (ABS) and thermoplastic polyurethane (TPU) were prepared at different weight ratios, i.e., 100:0, 70:30, 50:50, 30:70, and 0:100, for FDM printing; the prepared filaments, with an average diameter of 2.77 ± 0.19 mm, were encoded as A100, A70T30, A50T50, A30T70, and T100, respectively. The properties and printability of the filaments were thoroughly investigated. The blend composition, as well as the printing parameters, were optimized to obtain the FDM-printed objects with a well-defined surface structure and minimized warpages. The glass transition temperatures of ABS and TPU in the blends were not much altered from those of the parent filaments, whereas the thermal degradation characteristics of the blend filaments still fell between those of the neat filaments. The fractured surfaces of the filaments, observed by SEM, appeared smoother when higher amounts of TPU integrated; the smoothest surface of the ABS-based filament was found in A30T70, indicating the well-compatible blend characteristic. This was also confirmed by its rheological behavior examined by a parallel plate rheometer at 225 °C. Not only was the printability of the filaments improved, but also the warpages of the 3D-printed specimens were decreased when increasing amount of TPU was incorporated into the filaments. Among the printed objects, the A30T70 specimen exhibited the evenest surface morphology with the lowest surface roughness value of 32.9 ± 13.2 nm and the most uniform and consistent linear printing structure when being fabricated at the nozzle temperature of 225 °C and the printing bed temperature of 60 °C. However, the incorporation of TPU into the filaments markedly cut down both strength and modulus values of the fabricated materials up to about half but assisted the printed articles to absorb more energy, demonstrating that this polymer served as a good and effective toughener for ABS.

Dual-Functional Drug Delivery System for Bisphosphonate-Related Osteonecrosis Prevention and Its Bioinspired Releasing Model and In Vitro Assessment.

Clindamycin (CDM)/geranylgeraniol (GGOH)-loaded plasma-treated mesoporous silica nanoparticles/carboxymethyl chitosan composite hydrogels (CHG60 and CHG120) were developed for the prevention of medication-related osteonecrosis of the jaw associated with bisphosphonates (MRONJ-B). The pore structure and performances of CHGs, e.g., drug release profiles and kinetics, antibacterial activity, zoledronic acid (ZA)-induced cytotoxicity reversal activity, and acute cytotoxicity, were evaluated. The bioinspired platform mimicking in vivo fibrin matrices was also proposed for the in vitro/in vivo correlation. CHG120 was further encapsulated in the human-derived fibrin, generating FCHG120. The SEM and µCT images revealed the interconnected porous structures of CHG120 in both pure and fibrin-surrounding hydrogels with %porosity of 75 and 36%, respectively, indicating the presence of fibrin inside the hydrogel pores, besides its peripheral region, which was evidenced by confocal microscopy. The co-presence of GGOH moderately decelerated the overall releases of CDM from CHGs in the studied releasing fluids, i.e., phosphate buffer saline-based fluid (PBB) and simulated interstitial fluid (SIF). The whole-lifetime release patterns of CDM, fitted by the Ritger-Peppas equation, appeared nondifferentiable, divided into two releasing stages, i.e., rapid and steady releasing stages, whereas the biphasic drug release patterns of GGOH were observed with Phase I and II releases fitted by the Higuchi and Ritger-Peppas equations, respectively. Notably, the burst releases of both drugs were subsided with lengthier durations (up to 10-12 days) in SIF, compared with those in PBB, enabling CHGs to elicit satisfactory antibacterial and ZA cytotoxicity reversal activities for MRONJ-B prevention. The fibrin network in FCHG120 further reduced and sustained the drug releases for at least 14 days, lengthening bactericidal and ZA cytotoxicity reversal activities of FCHG and decreasing in vitro and in ovo acute drug toxicity. This highlighted the significance of fibrin matrices as appropriate in vivo-like platforms to evaluate the performance of an implant.

Biodegradable Dual-Function Nanocomposite Hydrogels for Prevention of Bisphosphonate-Related Osteonecrosis of the Jaw.

This study presents the development of composite hydrogels, comprising a biodegradable polymer (carboxymethyl chitosan (CMCS or CM)) and a mixture of plasma-treated mesoporous silica nanoparticles (PMCM-41 or PM) and amine-functionalized mesoporous silica nanoparticles (NMCM-41 or NM), coloaded with a hydrophilic antibiotic (clindamycin hydrochloride (CDM or C)) and a poorly water-soluble compound (geranylgeraniol (GGOH or G)) for prevention of bisphosphonate-related osteonecrosis of the jaw (BRONJ). The CG-loaded hydrogel stabilities were better maintained when CDM-preloaded PMCM-41 and NMCM-41 were initially used and governed by weight ratios of CDM-loaded PMCM-41 to NMCM-41 and CDM quantity utilized. 5PM240C-1NM-CM demonstrated the best CDM-loaded hydrogel for GGOH postloading. The scanning electron microscopy (SEM) and X-ray microcomputer-tomography (µCT) images of 5PM240C-1NM-CM revealed a porous structure with homogeneously distributed nanoparticles. Two GGOH-loaded 5PM240C-1NM-CM hydrogels were generated after GGOH loadings. Their biphasic drug release profiles were fitted by Ritger-Peppas and Hixson-Crowell models. The copresence of GGOH could hinder CDM releases, while GGOH was released with a slower rate. The hydrogels prolonged the CDM and GGOH releases up to 9 days. They possessed antibacterial activities against Streptococcus sanguinis for up to 14 days and satisfactorily provided good cytoprotection against zoledronic acid for osteoclastic and osteoblastic progenitors, thus preserving a pool of viable progenitor cells that had the capacity to differentiate into mature osteoclasts and osteoblasts in vitro, suggesting their potential local application for prevention of BRONJ.

Chondrogenic Differentiation of Human Mesenchymal Stem Cells and Macrophage Polarization on 3D-Printed Poly(ε-caprolactone)/Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Blended Scaffolds with Different Secondary Porous Structures.

This study was aimed to evaluate the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) and polarization of THP-1-derived macrophages cultured on poly(ε-caprolactone) (PC)/poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PH) blended scaffolds with dual primary (PP) and secondary (SP) pores, which were fabricated via a 3D printing technique, i.e., fused deposition modeling, followed by a salt-leaching process at 50 °C for varied times, i.e., 15, 30, and 60 min. Sodium chloride (SC), a porogen, was initially incorporated in the blend at varied weight percentages, i.e., 0, 25, and 50%, whereas 1 M NaOH solution and deionized water were used as salt-leaching agents. To elucidate the surface properties of the developed scaffolds, directly governed by the amount of the salt originally mixed and the salt-leaching efficiency, several characterization techniques, e.g., scanning electron microscopy, X-ray microcomputed tomography, mercury intrusion porosimetry, atomic force microscopy, and contact angle measurement, were used. Meanwhile, the salt-leaching efficiency was determined by means of weight loss measurement and thermogravimetric analysis. It was found that the alkaline solution could satisfactorily leach out the salt particles in 60 min with a mild etching of the polymer framework. The most immensely and homogeneously pitted filament surface was observed in the NaOH-treated scaffold initially integrated with 50% salt, i.e., 60B_PC/PH/50SC; the SP structure was mostly open and interconnected. The size of most of micropores was about 0.14 µm. With its suitable microsurface roughness and hydrophilicity, 60B_PC/PH/50SC could properly support the initial attachment and lamellipodia formation of hMSCs, which was favorable for chondrogenesis. Consequently, a significantly increased ratio of glycosaminoglycans/deoxyribonucleic acid and a superior expression of the COL2A1 gene were detected when cells were grown on this material. Although 60B_PC/PH/50SC induced the macrophages to secrete a slightly high level of IL-1ß during the first few days of culture, the polarized M1 cells could return to a nearly normal stage at Day7, suggesting no unfavorable chronic inflammation caused by the material.

Antibacterial and osteogenic activities of clindamycin-releasing mesoporous silica/carboxymethyl chitosan composite hydrogels.

Conventional treatment of jaw bone infection is often ineffective at controlling bacterial infection and enhancing bone regeneration. Biodegradable composite hydrogels comprised of carboxymethyl chitosan (CMCS) and clindamycin (CDM)-loaded mesoporous silica nanoparticles (MCM-41), possessing dual antibacterial activity and osteogenic potency, were developed in the present study. CDM was successfully loaded into both untreated and plasma-treated MCM-41 nanoparticles, denoted as (p)-MCM-41, followed by the incorporation of each of CDM-loaded (p)-MCM-41 into CMCS. The resulting CDM-loaded composite hydrogels, (p)-MCM-41-CDM-CMCS, demonstrated slow degradation rates (about 70% remaining weight after 14-day immersion), while the CDM-free composite hydrogel entirely disintegrated after 4-day immersion. The plasma treatment was found to improve drug loading capacity and slow down initial drug burst effect. The prolonged releases of CDM from both (p)-MCM-41-CDM-CMCS retained their antibacterial effect against Streptococcus sanguinis for at least 14 days in vitro. In vitro assessment of osteogenic activity showed that the CDM-incorporated composite hydrogel was cytocompatible to human mesenchymal stem cells (hMSCs) and induced hMSC mineralization via p38-dependent upregulated alkaline phosphatase activity. In conclusion, novel (p)-MCM-41-CDM-CMCS hydrogels with combined controlled release of CDM and osteogenic potency were successfully developed for the first time, suggesting their potential clinical benefit for treatment of intraoral bone infection.

Hyaluronic acid-coated gold nanorods enhancing BMP-2 peptide delivery for chondrogenesis.

Bone morphogenic protein-2 (BMP-2) knuckle epitope peptide has been recently discovered and known to activate chondrogenesis. However, the applications of this soluble peptide remain very limited due to rapid diffusion resulting in poor cellular uptake into target cells. We herein designed nanoparticles made from hyaluronic acid functionalized gold nanorods (GNRs) to conjugate with thiolated BMP-2 knuckle epitope peptide via a two-step reaction. Hyaluronic acid was modified to have thiol functional groups to replace the cetyl trimethylammonium bromide ligands on the surface of GNRs. The thiolated peptides were subsequently reacted with hyaluronic acid on the surface on GNRs via a maleimide-hydrazide crosslinker. The conjugation was confirmed by the change of surface charge of GNRs and the plasmon shift. A colorimetric peptide assay suggested more than 69% of the thiolated peptides were conjugated with the hyaluronic acid coated gold nanorods. Moreover, in vitro cell viability showed that BMP-2 conjugated hyaluronic acid functionalized gold nanorods (B2HGR) were cytocompatible and did not cause cytotoxicity to fibroblast cells. The B2HGRs also significantly promote cellular uptake of the BMP-2 peptides in both human mesenchymal stem cells and porcine chondrocytes due to multivalent ligand binding to the BMP receptors on the cell surface resulting in receptor-mediated endocytosis. The enhanced cellular uptake was clearly observed under a confocal microscope resulting in the significant activation of type II collagen gene expression and glucosaminoglycan secretion in those cells. Furthermore, our delivery system is a proof-of-concept of using scaffolds in combination with nanodelivery platform to enhance cartilaginous repair. The peptide loading capacity and the release is not limited by the scaffolds. Therefore, our delivery platform has potential applications for cartilage regeneration in a preclinical and clinical setting in the future.

Chondrogenic phenotype in responses to poly(ɛ-caprolactone) scaffolds catalyzed by bioenzymes: effects of surface topography and chemistry.

Biodegradable poly(ε-caprolactone) (PCL) has been increasingly investigated as a promising scaffolding material for articular cartilage tissue repair. However, its use can be limited due to its surface hydrophobicity and topography. In this study, 3D porous PCL scaffolds fabricated by a fused deposition modeling (FDM) machine were enzymatically hydrolyzed using two different biocatalysts, namely Novozyme®435 and Amano lipase PS, at varied treatment conditions in a pH 8.0 phosphate buffer solution. The improved surface topography and chemistry of the PCL scaffolds were anticipated to ultimately boost the growth of porcine articular chondrocytes and promote the chondrogenic phenotype during cell culture. Alterations in surface roughness, wettability, and chemistry of the PCL scaffolds after enzymatic treatment were thoroughly investigated using several techniques, e.g., SEM, AFM, contact angle and surface energy measurement, and XPS. With increasing enzyme content, incubation time, and incubation temperature, the surfaces of the PCL scaffolds became rougher and more hydrophilic. In addition, Novozyme®435 was found to have a higher enzyme activity than Amano lipase PS when both were used in the same enzymatic treatment condition. Interestingly, the enzymatic degradation process rarely induced the deterioration of compressive strength of the bulk porous PCL material and slightly reduced the molecular weight of the material at the filament surface. After 28 days of culture, both porous PCL scaffolds catalyzed by Novozyme®435 and Amano lipase PS could facilitate the chondrocytes to not only proliferate properly, but also function more effectively, compared with the non-modified porous PCL scaffold. Furthermore, the enzymatic treatments with 50 mg of Novozyme®435 at 25 °C from 10 min to 60 min were evidently proven to provide the optimally enhanced surface roughness and hydrophilicity most significantly favorable for induction of chondrogenic phenotype, indicated by the greatest expression level of cartilage-specific gene and the largest production of total glycosaminoglycans.

Bisphosphonate activation of crystallized bioglass scaffolds for enhanced bone formation.

The interplay between bone formation by osteoblasts and bone resorption by osteoclasts has a critical effect on bone remodelling processes, and resultant bone quality. Bone scaffolds combined with anti-resorptive bisphosphonate drugs are a promising approach to achieving bone regeneration. Here, we have examined the synergistic effects of the bisphosphonate alendronate (ALD) coated onto calcium phosphate (CaP) modified, sintered bioactive glass 45S5 (BG) scaffolds, on osteoblast stimulation and osteoclast inhibition. After BG pre-treatment with ALD (10-8 M) for 5 days, human MG-63 osteoblasts displayed increased cellular proliferation and significantly enhanced alkaline phosphatase activity (ALP), in comparison with a non-ALD control BG. In contrast, human THP-1-derived osteoclasts cultured with 10-8 M ALD pretreated BG scaffolds showed a significant decrease in tartrate-resistant acid phosphatase (TRAcP) activity, and morphological changes indicative of functional inhibition, including reduced cell size and disruption of the osteoclast sealing zone (F-actin rings). These findings indicate that ALD-coated BG scaffolds promote osteoblast activity and inhibit osteoclast function to enhance bone formation.

Effect of pre-treatment of crystallized bioactive glass with cell culture media on structure, degradability, and biocompatibility.

The silicate glass 45S5 Bioglass® (BG) is a potential scaffold material for bone regeneration because of its excellent bioactivity, biocompatibility and ability to form a strong bond with bone tissues, via the formation of an apatite layer on its surface. The evaluation of in vitro bioactivity in physiological body fluids, whilst challenging, can offer some insights for developing the bone-bonding ability of these glasses in vivo. In this study, we investigated the influence of three different cell culture and tissue fluid-like solutions on the dissolution and calcium-phosphate (CaP) based re-precipitation behaviour at the glass-liquid interface. We also examined pre-treatment of BG with these biological solutions, and how its influence on bone-forming MG-63 osteoblastic cell proliferation, viability and adhesion. The biological solutions used in this comparative study were: commercial cell culture medium (DMEM), a DMEM solution without organic components (DML) and a simulated body fluid (SBF), incorporating TRIS-buffer. Incubation of BG in these solutions over 28 days resulted in differences in weight loss, solution pH and ion release, and the development of CaP-based surface layers. XRD and FT-IR analyses showed clear differences in the characteristics of the CaP-based coating layers formed by the different solutions. The interfacial reactivity between the glass and the solutions depended on the composition and properties of the solutions. The formation of the CaP layer occurred more rapidly in SBF due to the presence of TRIS-buffer, which also significantly accelerated glass dissolution, further reducing the BG mass in SBF. MG-63 osteoblasts proliferated and spread more rapidly across the surfaces of all pre-conditioned BG, compared to fresh BG. The experimental results of this work help clarify differences between in vitro bioactivity of BG observed in cell culture solutions and in vivo BG bioactivity.

Porous 45S5 Bioglass®-based scaffolds using stereolithography: Effect of partial pre-sintering on structural and mechanical properties of scaffolds.

Scaffolds made from 45S5 Bioglass® ceramic (BG) show clinical potential in bone regeneration due to their excellent bioactivity and ability to bond to natural bone tissue. However, porous BG scaffolds are limited by their mechanical integrity and by the substantial volume contractions occurring upon sintering. This study examines stereolithographic (SLA) methods to fabricate mechanically robust and porous Bioglass®-based ceramic scaffolds, with regular and interconnected pore networks and using various computer-aided design architectures. It was found that a diamond-like (DM) architecture gave scaffolds the most controllable results without any observable closed porosity in the fired scaffolds. When the pore dimensions of the DM scaffolds of the same porosity (~60vol%) were decreased from 700 to 400µm, the compressive strength values increased from 3.5 to 6.7MPa. In addition, smaller dimensional shrinkage could be obtained by employing partially pre-sintered bioglass, compared to standard 45S5 Bioglass®. Scaffolds derived from pre-sintered bioglass also showed marginally improved compressive strength.

Enhancement of chondrocyte proliferation, distribution, and functions within polycaprolactone scaffolds by surface treatments.

Enhancement of porcine chondrocyte growth, distribution and functions within polycaprolactone (PCL) scaffolds was attempted using alkaline hydrolysis and oxygen plasma treatment. The hydrolysis of PCL was performed either before or after scaffold fabrication in the preparations of pre-hydrolyzed PCL (pre-HPCL) or post-HPCL scaffolds, respectively. The PCL, pre-HPCL, and post-HPCL scaffolds were subsequently plasma-treated to yield plasma-treated PCL, plasma-treated pre-HPCL, and plasma-treated post-HPCL scaffolds, respectively. All scaffolds were comparatively characterized, in terms of surface morphology, hydrophilicity, and atomic composition using scanning electron microscopy, contact angle measurement and X-ray photoelectron spectroscopy, respectively. The interactions of chondrocytes with individual scaffolds were assessed, in terms of cartilage-gene expression and cartilaginous matrix production using reverse transcription polymerase chain reaction analysis and glycosaminoglycans (GAGs) assay, respectively. The cell infiltration and cartilaginous matrix distribution were investigated by histological and immunofluorescence analysis. The results revealed that the plasma treatment exhibited a more prominent effect on the enhancement of surface roughness and hydrophilicity of the scaffolds than the alkaline hydrolysis. The scaffolds subjected to both surface treatments stimulated the cells to secret more GAGs and type II collagen. The sequence of hydrolysis of PCL also evidently played a crucial role in the hydrophilicity of the materials and the cartilage-gene expression and cartilaginous matrix production of the cultured chondrocytes. The hydrolysis of PCL prior to the fabrication, followed by the oxygen plasma treatment of the resulting fabricated scaffold, yielded plasma-treated pre-HPCL scaffold with homogeneous hydrophilic characteristics all over the material. Consequently, the cells could proliferate well, infiltrate most deeply and ultimately produce the highest amounts of the cartilage-specific substances throughout this scaffold.

Bone tissue engineering scaffolding: computer-aided scaffolding techniques.

Tissue engineering is essentially a technique for imitating nature. Natural tissues consist of three components: cells, signalling systems (e.g. growth factors) and extracellular matrix (ECM). The ECM forms a scaffold for its cells. Hence, the engineered tissue construct is an artificial scaffold populated with living cells and signalling molecules. A huge effort has been invested in bone tissue engineering, in which a highly porous scaffold plays a critical role in guiding bone and vascular tissue growth and regeneration in three dimensions. In the last two decades, numerous scaffolding techniques have been developed to fabricate highly interconnective, porous scaffolds for bone tissue engineering applications. This review provides an update on the progress of foaming technology of biomaterials, with a special attention being focused on computer-aided manufacturing (Andrade et al. 2002) techniques. This article starts with a brief introduction of tissue engineering (Bone tissue engineering and scaffolds) and scaffolding materials (Biomaterials used in bone tissue engineering). After a brief reviews on conventional scaffolding techniques (Conventional scaffolding techniques), a number of CAM techniques are reviewed in great detail. For each technique, the structure and mechanical integrity of fabricated scaffolds are discussed in detail. Finally, the advantaged and disadvantage of these techniques are compared (Comparison of scaffolding techniques) and summarised (Summary).

Fibroblast interaction with carboxymethylchitosan-based hydrogels.

The interaction between L929 cells and carboxymethylchitosan (CM-chitosan)-based hydrogels, hydrogels from pure CM-chitosan and its blends, was examined in this study. Cytotoxicity of all materials was also assessed. The cellular morphology and behavior on the surfaces of the hydrogels were observed by scanning electron microscopy (SEM). The effects of various parameters, e.g., type and content of blended polymers, surface structure of hydrogels, and steaming condition used for the preparation of the hydrogels, on the cell-material response were investigated. The results of the cytotoxicity test revealed that all hydrogels were non-cytotoxic. The SEM micrographs demonstrated that the cells proliferated and spread onto a porous CM-chitosan sample. Better cell spreading was found on a flat surface of a CM-chitosan film. Rounded cells were observed when poly(vinyl alcohol) (PVA) was incorporated into CM-chitosan. Fewer cells were found when the content of PVA increased. Spherical clusters of the aggregated cells existed in the blends with ultra high viscosity carboxymethylcellulose (CM-cellulose). In contrast, with the use of low viscosity CM-cellulose, the cells appeared more spreading. The attached cells on the CM-chitosan film steamed at the highest temperature and longest period appeared to spread the most among all tested steaming conditions.

Preparation of acrylic grafted chitin for wound dressing application.

Chitin grafted with poly(acrylic acid) (chitin-PAA) was prepared with the aim of obtaining a hydrogel characteristic for wound dressing application. The chitin-PAA films were synthesized at various acrylic acid feed contents to investigate its effect on water sorption ability. Acrylic acid (AA) was first linked to chitin, acting as the active grafting sites on the chain that was further polymerized to form a network structure. The evidences of grafting were found from FTIR and solid state 13C NMR spectra. The TGA results exhibited the high degradation temperature of the grafted product suggesting the formation of a network structure. The degree of swelling (DS) of chitin-PAA films was found in the range of 30-60 times of their original weights depending upon the monomer feed content. The chitin-PAA film with 1:4 weight ratio of chitin:AA, possessed optimal physical properties. The cytocompatibility of the film was investigated with a cell line of L929 mouse fibroblasts. The morphology and behavior of the cells on the chitin-PAA film were determined after different time periods of culture up to 14 days. The L929 cells proliferated and attached well onto the film. These results suggested that the 1:4 chitin-PAA has a potential to be used as a wound dressing.