Jens Vinge Nygaard

Mechanical stresses in carotid plaques using MRI-based fluid-structure interaction models

Risk assessment in patients with carotid atherosclerosis relies on the degree of luminal stenosis. Incorporating morphological information on plaque composition obtained noninvasively through the use of magnetic resonance imaging (MRI) could include other variables besides the degree of stenosis into carotid plaque risk assessment. Knowledge of the morphologic composition of the plaque allows determination of mechanic stresses exerted on the protective fibrous cap, which may be of importance in the assessment of plaque vulnerability. Based on image processing of transverse MRI scans, longitudinal 2D fluid-structure interaction (FSI) simulations of carotid atherosclerotic plaques were performed facilitating in-vivo estimation of longitudinal internal fibrous cap stresses. The FSI simulation combined finite element analysis (FEA) with computational fluid dynamics (CFD) simulations of blood-flow variables. Preliminary results from two symptomatic patients revealed longitudinal stress levels (max. 254.1 and 143.2 kPa) approaching established criteria for plaque rupture at known predilection sites of plaque rupture. Determination of longitudinal fibrous cap stresses may prove useful in assessing plaque vulnerability and improve risk stratification in patients with carotid atherosclerosis.

Curvature of Synthetic and Natural Surfaces Is an Important Target Feature in Classical Pathway Complement Activation

The binding of Abs to microbial surfaces followed by complement activation constitutes an important line of defense against infections. In this study, we have investigated the relationship between complement activation and the binding of human IgM Abs to surfaces with different curvatures. IgM Abs to dextran were shown to activate complement potently on dextran-coated particles having a diameter around 250 nm, whereas larger (600 nm) particles were less potent activators. This selectivity regarding particle dimension was also found for complement activation by colloidal substances of microbial origin. Peptidoglycan (PGN) is the major chemical component in the cell wall of Gram-positive bacteria. Fragments of purified PGN with sizes of ∼100 nm promoted complement activation effectively through the classical pathway. By contrast, larger or smaller fragments of PGN did not activate complement strongly. A careful analysis of PGN fragments released during planctonic growth of Staphylococcus aureus showed that these include curvatures that would permit strong IgM-mediated complement activation, whereas the curvature of intact cells would be less effective for such activation. Consistently, we found that the suspended PGN fragments were strong activators of complement through the classical pathway. We suggest that these fragments act as decoy targets for complement activation, providing protection for S. aureus against the host immune response to infection.

Self-assembled composite matrix in a hierarchical 3-D scaffold for bone tissue engineering

It is of high clinical relevance in bone tissue engineering that scaffolds promote a high seeding efficiency of cells capable of osteogenic differentiation, such as human bone marrow-derived mesenchymal stem cells (hMSCs). We evaluated the effects of a novel polycaprolactone (PCL) scaffold on hMSC seeding efficiency, proliferation, distribution and differentiation. Porous PCL meshes prepared by fused deposition modeling (FDM) were embedded in matrix of hyaluronic acid, methylated collagen and terpolymer via polyelectrolyte complex coacervation. Scaffolds were cultured statically and dynamically in osteogenic stimulation medium for up to 28 days. Compared to naked PCL scaffolds, embedded scaffolds provided a higher cell seeding efficiency (t-test, P<0.05), a more homogeneous cell distribution and more osteogenically differentiated cells, verified by a more pronounced gene expression of the bone markers alkaline phosphatase, osteocalcin, bone sialoprotein I and bone sialoprotein II. Dynamic culture resulted in higher amounts of DNA (day 14 and day 21) and calcium (day 21 and day 28), compared to static culture. Dynamic culture and the embedding synergistically enhanced the calcium deposition of hMSC on day 21 and day 28. This in vitro study provides evidence that hybrid scaffolds made from natural and synthetic polymers improve cellular seeding efficiency, proliferation, distribution and osteogenic differentiation.

siRNA Nanoparticle Functionalization of Nanostructured Scaffolds Enables Controlled Multilineage Differentiation of Stem Cells

The creation of complex tissues and organs is the ultimate goal in tissue engineering. Engineered morphogenesis necessitates spatially controlled development of multiple cell types within a scaffold implant. We present a novel method to achieve this by adhering nanoparticles containing different small-interfering RNAs (siRNAs) into nanostructured scaffolds. This allows spatial retention of the RNAs within nanopores until their cellular delivery. The released siRNAs were capable of gene silencing BCL2L2 and TRIB2, in mesenchymal stem cells (MSCs), enhancing osteogenic and adipogenic differentiation, respectively. This approach for enhancing a single type of differentiation is immediately applicable to all areas of tissue engineering. Different nanoparticles localized to spatially distinct locations within a single implant allowed two different tissue types to develop in controllable areas of an implant. As a consequence of this, we predict that complex tissues and organs can be engineered by the in situ development of multiple cell types guided by spatially restricted nanoparticles.

Selective isolation and differentiation of a stromal population of human embryonic stem cells with osteogenic potential

The derivation of osteogenic cells from human embryonic stem cells (hESC) has been hampered by the absence of easy and reproducible protocols. hESC grown in feeder-free conditions, often show a sub population of fibroblast-like, stromal cells growing between the colonies. Thus, we examined the possibility that these cells represent a population of stromal (mesenchymal) stem cells (hESC-stromal). Two in house derived hES cell lines (Odense3 and KMEB3) as well as an externally derived cell line (Hues8) were transitioned to feeder-free conditions. A sub population of fibroblast-like cells established between the hESC colonies were isolated by selective adherence to hyaluronic acid-coated plates (100 μg/ml) and were characterized using a combination of FACS analysis and staining. The cells were CD44(+), CD29(+), CD73(+), CD166(+), CD146(+), and CD105(+); and, Oct4⁻, CD34⁻, CD45⁻ and CXCR4⁻. When cultured in osteogenic differentiation media, up regulation of osteoblastic lineage markers (DLX5, MSX2, RUNX2, SPARC, ALP, COL1a1, BGLAP, IBSP, DCN, LOX-L4) and production of in vitro mineralized matrix was detected. hESC-stromal cells loaded on a carrier and implanted either subcutaneously or in a critical size calvarial defect in immune deficient mice for 10 weeks, resulted in new bone formation and partial repair of the calvarial defect. In conclusion, hESC-stromal can be isolated from hESC cultures and represent a good source for obtaining cells with osteogenic differentiation potential suitable for regenerative medicine protocols.

Osteopontin functionalization of hydroxyapatite nanoparticles in a PDLLA matrix promotes bone formation.

We studied the osteoconductive tissue response of hydroxyapatite (HA) nanoparticles functionalized with osteopontin (OPN) in a matrix of poly-D,L-lactic-acid (PDLLA). In a canine endosseus 0.75-mm gap implant model, we tested the osteointegrative impact of the OPN functionalized composite as an implant coating, and a non-functionalized composite was used as reference control. During the four weeks of observation, the OPN functionalized composite coating significantly increased the formation of new bone in the porosities of the implant, but no differences were observed in the gap. The study provides evidence of its potential use either alone or in combination with other osteoconductive compounds.

Fabrication and characterization of a rapid prototyped tissue engineering scaffold with embedded multicomponent matrix for controlled drug release

Bone tissue engineering implants with sustained local drug delivery provide an opportunity for better postoperative care for bone tumor patients because these implants offer sustained drug release at the tumor site and reduce systemic side effects. A rapid prototyped macroporous polycaprolactone scaffold was embedded with a porous matrix composed of chitosan, nanoclay, and β-tricalcium phosphate by freeze-drying. This composite scaffold was evaluated on its ability to deliver an anthracycline antibiotic and to promote formation of mineralized matrix in vitro. Scanning electronic microscopy, confocal imaging, and DNA quantification confirmed that immortalized human bone marrow-derived mesenchymal stem cells (hMSC-TERT) cultured in the scaffold showed high cell viability and growth, and good cell infiltration to the pores of the scaffold. Alkaline phosphatase activity and osteocalcin staining showed that the scaffold was osteoinductive. The drug-release kinetics was investigated by loading doxorubicin into the scaffold. The scaffolds comprising nanoclay released up to 45% of the drug for up to 2 months, while the scaffold without nanoclay released 95% of the drug within 4 days. Therefore, this scaffold can fulfill the requirements for both bone tissue engineering and local sustained release of an anticancer drug in vitro. These results suggest that the scaffold can be used clinically in reconstructive surgery after bone tumor resection. Moreover, by changing the composition and amount of individual components, the scaffold can find application in other tissue engineering areas that need local sustained release of drug.

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.

Can sites prone to flow induced vascular complications in a-v fistulas be assessed using computational fluid dynamics?

Arterio-venous fistulas (shunts between arteries and veins) are the preferred vascular access for hemodialysis. Despite their superior patency, compared with synthetic tubes and grafts, functional problems and inadequate flow rates are the common complications. Local flow conditions, in particular low and oscillating wall shear stresses (WSS), are central to vascular problems and a robust framework for analyzing flow conditions in vascular structures could provide an understanding of the mechanisms leading to vascular complications, such as stenoses, aneurisms, and thromboses. We hypothesize that a validated computational fluid dynamics (CFD) framework can be used to identify critical fistula configurations with elevated risk of complications. Therefore, the aim of the present study was to develop a CFD framework for analyzing fluid flow in complex vascular structures, such as arterio-venous fistulas validated by comparisons of in vitro volume flows with CFD results and flow fields from ultrasound scans with CFD simulations. Volume flows measured in vitro and CFD data differed quantitatively. However, good relative correlations exist between the data using logarithmic scales. Qualitatively, visual comparisons between ultrasound and CFD images showed good agreement between the two methods. In addition, WSS levels and the oscillatory shear index (OSI) were calculated and visualized on the model surface. The method was successfully validated and the method is deemed suitable for more thorough investigations into the field of vascular complications in a-v fistulas.

Hydroxyapatite nanoparticles in poly-D,L-lactic acid coatings on porous titanium implants conducts bone formation.

It is well established in the field of biomaterials that hydroxyapatite (HA) may provide interesting osteoconductive properties. In this study, we investigated the osseointegrational effect of a 50/50 vol % composite of HA nanoparticles and poly-D,L-lactic acid (PDLLA) coated on model titanium bone implants in an in vivo animal model. The aim is to evaluate how the addition of HA to PDLLA may improve the bone formation and initial fixation of the implant. Two titanium implants coated with the PDLLA/HA composite and pure PDLLA, respectively, were implanted bilaterally in proximal part of humeri with a 2-mm peri-implant gap in 10 sheep. After 12 weeks, the remains of the coatings were present on 20.3 and 19.8% of PDLLA/HA composite- and PDLLA-coated implants, respectively. It was observed that newly formed bone (39.3%) and fibrous tissue (58.3%) had replaced the PDLLA/HA composite, whereas pure PDLLA was replaced almost completely by fibrous tissue (96.2%). Consequently, the PDLLA/HA composite-coated implants were better fixated as confirmed by push-out tests. Using quantification of peri-implant tissue and implant fixation as parameters, the present findings, therefore, clearly reveal that the addition of nanoparticulate HA to a PDLLA coating on titanium implants increases osseointegration.