Scaffold geometry and computational fluid dynamics simulation supporting osteogenic differentiation in dynamic culture.

Geometry of porous scaffolds is critical to the success of cell adhesion, proliferation, and differentiation in bone tissue engineering. In this study, the effect of scaffold geometry on osteogenic differentiation of MC3T3-E1 pre-osteoblasts in a perfusion bioreactor was investigated. Three geometries of oligolactide-HA scaffolds, named Woodpile, LC-1000, and LC-1400, were fabricated with uniform pore size distribution and interconnectivity using stereolithography (SL) technique, and tested to evaluate for the most suitable scaffold geometry. Compressive tests revealed sufficiently high strength of all scaffolds to support new bone formation. The LC-1400 scaffold showed the highest cell proliferation in accordance with the highest level of osteoblast-specific gene expression after 21 days of dynamic culture in a perfusion bioreactor; however, it deposited less amount of calcium than the LC-1000 scaffold. Computational fluid dynamics (CFD) simulation was employed to predict and explain the effect of flow behavior on cell response under dynamic culture. The findings concluded that appropriate flow shear stress enhanced cell differentiation and mineralization in the scaffold, with the LC-1000 scaffold performing best due to its optimal balance between permeability and flow-induced shear stress.

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.

Development of chondrocyte-laden alginate hydrogels with modulated microstructure and properties for cartilage regeneration.

Alginate hydrogel is an attractive biomaterial for cell microencapsulation. The microarchitecture of hydrogels can regulate cellular functions. This study aims to investigate the applicability of sodium citrate buffer (SCB) as a culture medium supplement for modulating the microstructure of alginate microbeads to provide a favorable microenvironment for chondrogenic induction. The chondrocyte-laden microbeads, with and without TGF-ß3 incorporation, were produced through an encapsulator. The obtained small-sized microbeads (~300 µm) were exposed to a treatment medium containing SCB, composed of varied concentrations of sodium citrate (1.10-1.57 mM), sodium chloride (3.00-4.29 mM), and ethylenediaminetetraacetic acid (0.60-0.86 mM) to partially degrade their crosslinked structure for 3 days, followed by culture in a normal medium until day 21. Scanning electron microscope micrographs demonstrated a loose hydrogel network with an enhanced pore size in the SCB-treated microbeads. Increasing the concentration of SCB in the treatment medium reduced the calcium content of the microbeads via a Na+ /Ca2+ exchange process and improved the water absorption of the microbeads, resulting in a higher swelling ratio. All the tested SCB concentrations were non-cytotoxic. Increases in aggrecan and type II collagen gene expression and their corresponding extracellular matrix accumulation, glycosaminoglycans, and type II collagen were vividly detected in the TGF-ß3-containing microbeads with increasing SCB concentrations in the treatment medium. Our findings highlighted that the combination of SCB treatment and TGF-ß3 incorporation in the chondrocyte-laden microbeads is a promising strategy for enhancing cartilage regeneration, which may contribute to a versatile application in cell delivery and tissue engineering.

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.

Role of laminin and cognate receptors in cholangiocarcinoma cell migration.

Extensive desmoplasia in cholangiocarcinoma (CCA) is associated with tumor aggressiveness, indicating a need for further understanding of CCA cell-matrix interaction. This study demonstrated laminin as the most potent attractant for CCA cell migration and the vast elevation of its receptor integrin ß4 (ITGB4) in CCA cell lines. Besides, their high expressions in CCA tissues were correlated with lymphatic invasion and the presence of ITGB4 was also associated with short survival time. ITGB4 silencing revealed it as the receptor for laminin-induced HuCCA-1 migration, but KKU-213 utilized 37/67-kDa laminin receptor (LAMR) instead. These findings highlight the role of ITGB4 and LAMR in transducing laminin induction of CCA cell migration and the potential of ITGB4 as diagnostic and prognostic biomarkers for CCA.

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.

PCL/PHBV blended three dimensional scaffolds fabricated by fused deposition modeling and responses of chondrocytes to the scaffolds.

In this study, poly(ε-caprolactone)/poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PCL/PHBV) blended porous scaffolds were fabricated by fused deposition modeling (FDM). PCL/PHBV filaments, initially prepared at different weight ratios, that is, 100/0, 75/25, 50/50, and 25/75, were fabricated by the lay-down pattern of 0/90/45/135° to obtain scaffolds with dimension of 6.0 × 6.0 × 2.5 mm3 and average filament diameters and channel sizes in the ranges of 370-390 µm and 190-210 µm, respectively. To enhance the surface hydrophilicity of the materials, the scaffolds were subsequently subjected to a low pressure oxygen plasma treatment. The untreated and plasma-treated scaffolds were comparatively characterized, in terms of surface properties, mechanical strength, and biological properties. From SEM, AFM, water contact angle, and XPS results, the surface roughness, wettability, and hydrophilicity of the blended scaffolds were found to be enhanced after plasma treatment, while the compressive strength of the scaffolds was scarcely changed. It was, however, found to increase with an increasing content of PHBV incorporated. The porcine chondrocytes exhibited higher proliferative capacity and chondrogenic potential when being cultured on the scaffolds with greater PHBV contents, especially when they were plasma-treated. The PCL/PHBV scaffolds were proven to possess good physical, mechanical, and biological properties that could be appropriately used in articular cartilage regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1141-1150, 2017.

Mechanical and biological properties of photocurable oligolactide-HA composites investigated under accelerated degradation.

The major concern related to biodegradable bone substitute materials is the loss of mechanical strength which can be undesirable when occurring too quickly before new bone formation. In this study, the multifunctional lactide oligomers having 2, 3, and 4 arms end capped with methacrylate groups were synthesized with the aim of improving the degradation properties. Their composites with hydroxyapatite (HA) were photopolymerized and subjected to accelerated degradation at 60 °C. The results showed that increasing number of arms significantly improved thermal and mechanical properties as well as biocompatibility of the composites. All composites although varying in number of arms had similar levels of bone-specific gene expression and calcification indicating their equal bioactivity in supporting bone formation. The high HA content in the composites was proposed to be responsible for enhanced osteoblast response, and this tended to suppress the effects of polymeric structure.

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.