Dang Quang Svend Le

Surface-modified functionalized polycaprolactone scaffolds for bone repair: In vitro and in vivo experiments

A porcine calvaria defect study was carried out to investigate the bone repair potential of three-dimensional (3D)-printed poly-ε-caprolactone (PCL) scaffolds embedded with nanoporous PCL. A microscopic grid network was created by rapid prototyping making a 3D-fused deposition model (FDM-PCL). Afterward, the FDM-PCL scaffolds were infused with a mixture of PCL, water, and 1,4-dioxane and underwent a thermal-induced phase separation (TIPS) followed by lyophilization. The TIPS process lead to a nanoporous structure shielded by the printed microstructure (NSP-PCL). Sixteen Landrace pigs were divided into two groups with 8 and 12 weeks follow-up, respectively. A total of six nonpenetrating holes were drilled in the calvaria of each animal. The size of the cylindrical defects was h 10 mm and Ø 10 mm. The defects were distributed randomly using following groups: (a) NSP-PCL scaffold, (b) FDM-PCL scaffold, (c) autograft, (d) empty defect, (a1) NSP-PCL scaffold + autologous mononuclear cells, and (a2) NSP-PCL scaffold + bone morphogenetic protein 2. Bone volume to total volume was analyzed using microcomputed tomography (µCT) and histomorphometry. The µCT and histological data showed significantly less bone formation in the NSP-PCL scaffolds in all three variations after both 8 and 12 weeks compared to all other groups. The positive autograft control had significantly higher new bone formation compared to all other groups except the FDM-PCL when analyzed using histomorphometry. The NSP-PCL scaffolds were heavily infiltrated with foreign body giant cells suggesting an inflammatory response and perhaps active resorption of the scaffold material. The unmodified FDM-PCL scaffold showed good osteoconductivity and osseointegration after both 8 and 12 weeks.

Enhanced efficacy of chemotherapy for breast cancer stem cells by simultaneous suppression of multidrug resistance and antiapoptotic cellular defense.

UNLABELLED While chemotherapy is universally recognized as a frontline treatment strategy for breast cancer, it is not always successful; among the leading causes of treatment failure is existing and/or acquired multidrug resistance. Cancer stem cells (CSCs), which constitute a minority of the cells of a tumor, are acknowledged to be responsible for increased resistance to chemo-drugs through a combination of increased expression of ATP-binding cassette transporters (ABC transporters), an increased anti-apoptotic defense, and/or the ability for extensive DNA repair like normal stem cells. Consequently, more effective therapy, especially targeted to CSCs, is urgently required. We studied the characteristics of 231-CSCs (CD44+/CD24-) sorted from human MDA-MB-231 breast cancer cells and demonstrated that 231-CSCs exhibited enhanced capacities for proliferation, migration, tumorigenesis and chemotherapy resistance. To address these multifunctional facets of CSCs, we devised a non-ionic surfactant-based vesicle (niosome) co-delivery system to simultaneously deliver siRNAs, targeted to both the ABC transporter (ABCG2) and the anti-apoptosis defense gene (BCL2), and doxorubicin (DOX) to CSCs. The rationale is to sensitize CSCs to DOX by down regulating the drug-resistance gene ABCG2 and simultaneously induce apoptosis by lowering BCL2 expression. The co-delivery system (CDS) successfully delivered siRNAs and DOX to the cytoplasm and nuclei, respectively, and resulted in a down-regulation of ABCG2- and BCL2 mRNAs in CSCs by 60% and 65%, respectively, compared to the control. A corresponding decrease in protein expression was observed using Western blotting. The IC50 of DOX in CSCs concurrently decreased significantly. Our result established CDS as a promising multi-drug delivery platform for cancer treatment. STATEMENT OF SIGNIFICANCE Cancer stem cells (CSCs) are acknowledged to be responsible for increased resistance to chemo-drugs through a combination of increased expression of ABC transporters, an increased anti-apoptotic defense, and/or the ability for extensive DNA repair like normal stem cells. Consequently, effective therapy, especially to CSCs, is urgently required. In current study, we studied the characteristics of 231-CSCs sorted from human MDA-MB-231 breast cancer cells and found that 231-CSCs possessed enhanced proliferation, migration, tumorigenesis, and DOX resistance. We employed a non-ionic surfactant-based vesicle (niosome) delivery system to simultaneously deliver siRNAs targeted to multi-drug resistance genes, and DOX to kill 231-CSCs. The CDS showed an enhanced therapeutic effect by resensitizing 231-CSCs to DOX and may constitute a promising candidate for cancer chemotherapy.

Engineered three-dimensional nanofibrous multi-lamellar structure for annulus fibrosus repair

Repairing annulus fibrosus (AF) defects is one of the most challenging topics in intervertebral disc disease treatment research. The highly oriented native structure offers mechanical functionality to the spine, however manufacturing scaffolds with such a structure still presents a challenge for tissue engineering. Here, a three-dimensional (3D) multi-lamellar scaffold with hierarchically aligned nano- and micro-fibers for AF tissue engineering was successfully developed. Aligned polycaprolactone (PCL) nano-fiber sheets, which were fabricated by electrospinning, were inserted into fused-deposit-modeling (FDM) micro-fibers to build a layer-by-layer structure, with the thickness of each layer being 0.7 mm and the angle of fiber alignment in each adjacent layer being 60°. Human mesenchymal stem cells (hMSCs) were used for in vitro compatibility studies. The architecture of the scaffold was characterized by scanning electron microscopy (SEM). Uniaxial tensile testing showed closed mechanical properties of the scaffold to native AF tissue. The XTT cell viability and DNA quantification of the cells on the multi-lamellar scaffold were found to be significantly higher than the FDM scaffolds without nano-fibers. Confocal microscopy demonstrated that the cells spread evenly on the surface of the electrospun sheet and oriented along the nano-fiber direction. This 3D multi-lamellar scaffold has the advantages of stability from the FDM micro-fibers, and unique characteristics from the aligned electrospun nano-fibers, such as mimicking the extracellular matrix (ECM), and an ultrahigh surface area for improved hMSC attachment, proliferation and contact guidance of cell morphology. The newly designed scaffold mimics the native structure of AF and has a great potential as a substrate for the regeneration of AF.

Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model

Introduction: The osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs) was compared with that of dental pulp-derived stromal cells (DPSCs) in vitro and in a pig calvaria critical-size bone defect model. Methods: BMSCs and DPSCs were extracted from the tibia bone marrow and the molar teeth of each pig, respectively. BMSCs and DPSCs were cultured in monolayer and on a three-dimensional (3D) polycaprolactone (PCL) – hyaluronic acid – tricalcium phosphate (HT-PCL) scaffold. Population doubling (PD), alkaline phosphatase (ALP) activity, and calcium deposition were measured in monolayer. In the 3D culture ALP activity, DNA content, and calcium deposition were evaluated. Six non-penetrating critical-size defects were made in each calvarium of 14 pigs. Three paired sub-studies were conducted: (1) empty defects vs. HT-PCL scaffolds; (2) PCL scaffolds vs. HT-PCL scaffolds; and (3) autologous BMSCs on HT-PCL scaffolds vs. autologous DPSCs on HT-PCL scaffolds. The observation time was five weeks. Bone volume fractions (BV/TV) were assessed with micro-computed tomography (μCT) and histomorphometry. Results and discussion: The results from the in vitro study revealed a higher ALP activity and calcium deposition of the DPSC cultures compared with BMSC cultures. Significantly more bone was present in the HT-PCL group than in both the pure PCL scaffold group and the empty defect group in vivo. DPSCs generated more bone than BMSCs when seeded on HT-PCL. In conclusion, DPSCs exhibited a higher osteogenic potential compared with BMSCs both in vitro and in vivo, making it a potential cell source for future bone tissue engineering.

Cyanoacrylate medical glue application in intervertebral disc annulus defect repair: Mechanical and biocompatible evaluation

In an attempt to find an ideal closure method during annulus defect repair, we evaluate the use of medical glue by mechanical and biocompatible test. Cyanoacrylate medical glue was applied together with a multilayer microfiber/nanofiber polycaprolactone scaffold and suture in annulus repair. Continuous axial loading and fatigue mechanical test was performed. Furthermore, the in vitro response of mesenchymal stem cell (MSC) to the glue was evaluated by cell viability assay. The in vivo response of annulus tissue to the glue and scaffold was also studied in porcine lumbar spine; histological sections were evaluated after 3 months. Cyanoacrylate glue significantly improved the closure effect in the experimental group with failure load 2825.7 ± 941.6 N, compared to 774.1 ± 281.3 N in the control group without glue application (p < 0.01). The experimental group also withstood the fatigue test. No toxic effect was observed by in vitro cell culture and in vivo implantation. On the basis of this initial evaluation, the use of cyanoacrylate medical glue improves closure effect with no toxicity in annulus defect repair. This method of annulus repair merits further effectiveness study in vivo.

In vivo drug release behavior and osseointegration of a doxorubicin-loaded tissue-engineered scaffold

Bone tissue-engineered scaffolds with therapeutic effects must meet the basic requirements as to support bone healing at the defect side and to release an effect drug within the therapeutic window. Here, a rapid prototyped PCL scaffold embedded with a chitosan/nanoclay/β-tricalcium phosphate composite (DESCLAYMR) loaded with the chemotherapeutic drug doxorubicin (DESCLAYMR_DOX) is proposed as a potential multifunctional medical application for patients who undergo bone tumor resection. We showed the DESCLAYMR_DOX scaffold released DOX locally in a sustained manner in mice without significantly increasing the plasma DOX concentrations. The evaluation of osseointegration in a porcine study showed increased mineralized bone formation, unmineralized collagen fibers and significantly higher alpha Smooth Muscle Actin (α-SMA) positive areas relative to the total investigated area (TA) in defects treated solely with the DESCLAYMR scaffold than in the DESCLAYMR_DOX; and alkaline phosphatase activity, α-SMA/TA and bone formation were higher in the DESCLAYMR loaded with 100 μg per scaffold DOX (DOX_low) than with 400 μg per scaffold DOX (DOX_high). Our results suggest that the DESCLAYMR_DOX can be a viable candidate as a multifunctional medical application by delivering the chemotherapeutic agent to target remaining tumor cells and facilitate bone formation.

Biomechanical evaluation of annulus fibrosus repair with scaffold and soft anchors in an ex vivo porcine model

Introduction: Altered biomechanical properties, due to intervertebral disc (IVD) degeneration and missing nucleus fibrosus, could be thought as one of the reasons for the back pain many herniation patients experience after surgery. It has been suggested to repair annulus fibrosus (AF) to restore stability and allow nucleus pulposus (NP) replacement and furthermore prevent reherniation. The aim of this study was to evaluate a new method for closing a defect in AF for use in herniation surgery. Methods: Our repair method combines a polycaprolactone (PCL) scaffold plugging herniation and soft anchors to secure the plug. Ex vivo biomechanical testing was carried out in nine porcine lumbar motion segments. Flexion–extension, lateral bending and rotation were repeated three times: first in healthy specimens, second with a full thickness circular defect applied, and third time with the specimens repaired. Finally push out tests were performed to check whether the plug would remain in. Results: Tests showed that applying a defect to the AF increases the range of motion (ROM), neutral zone (NZ) and neutral zone stiffness (NZS). In flexion/extension it was found significant for ROM, NZ, and NZS. For lateral bending and rotation a significant increase in ROM occurred. After AF repair ROM, NZ and NZS were normalized. All plugs remained in the AF during push out test up until 4000 N, but NP was squeezed out through the pores of the scaffold. Discussion: A defect in the AF changes the biomechanical properties in the motion segment, changes that point to instability. Repairing the defect with a PCL plug and soft anchors brought the biomechanical behavior back to native state. This concept is promising and might be a viable way to repair the IVD after surgery.

Surgical Repair of Annulus Defect with Biomimetic Multilamellar Nano/microfibrous Scaffold in a Porcine Model.

Annulus defect is associated with reherniation and disc degeneration after discectomy; currently there is no effective treatment that addresses this problem. The annulus is a hierarchical lamellar structure, where each lamella consists of aligned collagen fibres, which are parallel and tilted at 30° to the spinal axis. In this study, a biomimetic biodegradable scaffold consisting of multilamellar nano/microfibres, sharing nanotopography and microporosity similar to the native lamellar structure, was assessed in a porcine model, aided by sealing with fascia and medical glue and subsequent suture fixation. After 6‐ and 12‐week observation, we found that this treatment restored nucleus volume and slowed down disc degeneration, as indicated by magnetic resonance imaging of T1/T2‐weighted, T2‐mapping, T1‐ρ imaging. Histological analysis showed aligned collagen fibres organized in the scaffold and integrated with surrounding native annulus tissue. The autologous bone marrow concentrate‐seeded scaffolds showed slightly earlier collagen fibre formation at 6 weeks. This novel treatment could efficiently close the annulus defect with newly formed, organized and integrated collagen fibres in a porcine model. Copyright

Calcium–MicroRNA Complex-Functionalized Nanotubular Implant Surface for Highly Efficient Transfection and Enhanced Osteogenesis of Mesenchymal Stem Cells

Controlling mesenchymal stem cell (MSC) differentiation by RNA interference (RNAi) is a promising approach for next-generation regenerative medicine. However, efficient delivery of RNAi therapeutics is still a limiting factor. In this study, we have developed a simple, biocompatible, and highly effective delivery method of small RNA therapeutics into human MSCs (hMSCs) from an implant surface by calcium ions. First, we demonstrated that simple Ca/siRNA targeting green fluorescent protein (GFP) nanocomplexes were able to efficiently silence GFP in GFP-expressing hMSCs with adequate Ca2+ concentration (>5 mM). In addition, a single transfection could obtain a long-lasting silencing effect for more than 2 weeks. All three of the main endocytosis pathways (clathrin- and caveolin-mediated endocytosis and macropinocytosis) were involved in the internalization of the Ca/siRNA complexes by MSCs, and macropinocytosis plays the most dominant role. Furthermore, the Ca/siRNA complexes could be efficiently loaded onto the titanium implant surface when pretreated with anodization to create a nanotube (NT) layer. Because of the hydrophilic property of the NT surface, the Ca/siRNA was quickly loaded (less than 4 h) with high efficiency (nearly 100%), forming an even amorphous coating. The Ca/siRNA-coated NT surface showed an initial burst release of 80% of the siRNA complexes over 2 h, which is adequate to achieve robust gene silencing of attached hMSCs. To demonstrate the therapeutic potential of our Ca/siRNA coating technology, Ca/antimiR-138 complexes were loaded on to the NT surface, which strongly enhanced the osteogenic differentiation of hMSCs. In conclusion, our findings suggest that Ca2+ is an effective and biocompatible carrier to deliver small RNA therapeutics into hMSCs, both in solution and from functionalized surfaces, which provides a novel approach to control the MSC differentiation and tissue regeneration.