The rupture of an atherosclerotic plaque cap overlying a lipid pool and/or necrotic core can lead to thrombotic cardiovascular events. In essence, the rupture of the plaque cap is a mechanical event, which occurs when the local stress exceeds the local tissue strength. However, due to inter- and intra-cap heterogeneity, the resulting ultimate cap strength varies, causing proper assessment of the plaque at risk of rupture to be lacking. Important players involved in tissue strength include the load-bearing collagenous matrix, macrophages, as major promoters of extracellular matrix degradation, and microcalcifications, deposits that can exacerbate local stress, increasing tissue propensity for rupture. This review summarizes the role of these components individually in tissue mechanics, along with the interplay between them. We argue that to be able to improve risk assessment, a better understanding of the effect of these individual components, as well as their reciprocal relationships on cap mechanics, is required. Finally, we discuss potential future steps, including a holistic multidisciplinary approach, multifactorial 3D in vitro model systems, and advancements in imaging techniques. The obtained knowledge will ultimately serve as input to help diagnose, prevent, and treat atherosclerotic cap rupture.

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Tissue engineering bone via endochondral ossification requires the generation of a cartilage template which undergoes vascularisation and remodelling. While this is a promising route for bone repair, achieving effective cartilage vascularisation remains a challenge. Here, we investigated how mineralisation of tissue-engineered cartilage affects its pro-angiogenic potential. To generate in vitro mineralised cartilage, human mesenchymal stromal cell (hMSC)-derived chondrogenic pellets were treated with β-glycerophosphate (BGP). After optimising this approach, we characterised the changes in matrix components and pro-angiogenic factors by gene expression analysis, histology and ELISA. Human umbilical vein endothelial cells (HUVECs) were exposed to pellet-derived conditioned media, and migration, proliferation and tube formation were assessed. We established a reliable strategy to induce in vitro cartilage mineralisation, whereby hMSC pellets are chondrogenically primed with TGF-β for 2 weeks and BGP is added from week 2 of culture. Cartilage mineralisation determines loss of glycosaminoglycans, reduced expression but not protein abundance of collagen II and X, and decreased VEGFA production. Finally, the conditioned medium from mineralised pellets showed a reduced ability to stimulate endothelial cell migration, proliferation and tube formation. The pro-angiogenic potential of transient cartilage is thus stage-dependent, and this aspect must be carefully considered in the design of bone tissue engineering strategies.

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Macrophage responses following the implantation of orthopaedic implants are essential for successful implant integration in the body, partly through intimate crosstalk with human marrow stromal cells (hMSCs) in the process of new bone formation. Additive manufacturing (AM) and plasma electrolytic oxidation (PEO) in the presence of silver nanoparticles (AgNPs) are promising techniques to achieve multifunctional titanium implants. Their osteoimmunomodulatory properties are, however, not yet fully investigated. Here, we studied the effects of implants with AgNPs on human macrophages and the crosstalk between hMSCs and human macrophages when co-cultured in vitro with biofunctionalised AM Ti6Al4V implants. A concentration of 0.3 g/L AgNPs in the PEO electrolyte was found to be optimal for both macrophage viability and inhibition of bacteria growth. These specimens also caused a decrease of the macrophage tissue repair related factor C-C Motif Chemokine Ligand 18 (CCL18). Nevertheless, co-cultured hMSCs could osteogenically differentiate without any adverse effects caused by the presence of macrophages that were previously exposed to the PEO (±AgNPs) surfaces. Further evaluation of these promising implants in a bony in vivo environment with and without infection is highly recommended to prove their potential for clinical use.@en

Rupture of the cap of an atherosclerotic plaque can lead to thrombotic cardiovascular events. It has been suggested, through computational models, that the presence of microcalcifications in the atherosclerotic cap can increase the risk of cap rupture. However, the experimental confirmation of this hypothesis is still lacking. In this study, we have developed a novel tissue-engineered model to mimic the atherosclerotic fibrous cap with microcalcifications and assess the impact of microcalcifications on cap mechanics. First, human carotid plaque caps were analyzed to determine the distribution, size, and density of microcalcifications in real cap tissue. Hydroxyapatite particles with features similar to real cap microcalcifications were used as microcalcification mimics. Injected clusters of hydroxyapatite particles were embedded in a fibrin gel seeded with human myofibroblasts which deposited a native-like collagenous matrix around the particles, during the 21-day culture period. Second harmonic multiphoton microscopy imaging revealed higher local collagen fiber dispersion in regions of hydroxyapatite clusters. Tissue-engineered caps with hydroxyapatite particles demonstrated lower stiffness and ultimate tensile stress than the control group samples under uniaxial tensile loading, suggesting increased rupture risk in atherosclerotic plaques with microcalcifications. This model supports previous computational findings regarding a detrimental role for microcalcifications in cap rupture risk and can further be deployed to elucidate tissue mechanics in pathologies with calcifying soft tissues.

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Bone Morphogenetic proteins (BMPs) like BMP2 and BMP7 have shown great potential in the treatment of severe bone defects. In recent in vitro studies, BMP9 revealed the highest osteogenic potential compared to other BMPs, possibly due to its unique signaling pathways that differs from other osteogenic BMPs. However, in vivo the bone forming capacity of BMP9-adsorbed scaffolds is not superior to BMP2 or BMP7. In silico analysis of the BMP9 protein sequence revealed that BMP9, in contrast to other osteogenic BMPs such as BMP2, completely lacks so-called heparin binding motifs that enable extracellular matrix (ECM) interactions which in general might be essential for the BMPs' osteogenic function. Therefore, we genetically engineered a new BMP9 variant by adding BMP2-derived heparin binding motifs to the N-terminal segment of BMP9′s mature part. The resulting protein (BMP9 HB) showed higher heparin binding affinity than BMP2, similar osteogenic activity in vitro and comparable binding affinities to BMPR-II and ALK1 compared to BMP9. However, remarkable differences were observed when BMP9 HB was adsorbed to collagen scaffolds and implanted subcutaneously in the dorsum of rats, showing a consistent and significant increase in bone volume and density compared to BMP2 and BMP9. Even at 10-fold lower BMP9 HB doses bone tissue formation was observed. This innovative approach of significantly enhancing the osteogenic properties of BMP9 simply by addition of ECM binding motifs, could constitute a valuable replacement to the commonly used BMPs. The possibility to use lower protein doses demonstrates BMP9 HB's high translational potential.

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Objective: Cartilage is avascular and numerous studies have identified the presence of single anti- and pro-angiogenic factors in cartilage. To better understand the maintenance hyaline cartilage, we assessed the angiogenic potential of complete cartilage releasate with functional assays in vitro and in vivo. Design: We evaluated the gene expression profile of angiogenesis-related factors in healthy adult human articular cartilage with a transcriptome-wide analysis generated by next-generation RNAseq. The effect on angiogenesis of the releasate of cartilage tissue was assessed with a chick chorioallantoic membrane (CAM) assay as well as human umbilical vein endothelial cell (HUVEC) migration and proliferation assays using conditioned media generated from tissue-engineered cartilage derived from human articular and nasal septum chondrocytes as well as explants from bovine articular cartilage and human nasal septum. Experiments were done with triplicate samples of cartilage from 3 different donors. Results: RNAseq data of 3 healthy human articular cartilage donors revealed that the majority of known angiogenesis-related factors expressed in healthy adult articular cartilage are pro-angiogenic. The releasate from generated cartilage as well as from tissue explants, demonstrated at least a 3.1-fold increase in HUVEC proliferation and migration indicating a pro-angiogenic effect of cartilage. Finally, the CAM assay demonstrated that cartilage explants can indeed attract vessels; however, their ingrowth was not observed. Conclusion: Using multiple approaches, we show that cartilage releasate has an inherent pro-angiogenic capacity. It remains vessel free due to anti-invasive properties associated with the tissue itself.

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Additive manufacturing (AM) techniques have provided many opportunities for the rational design of porous metallic biomaterials with complex and precisely controlled topologies that give rise to unprecedented combinations of mechanical, physical, and biological properties. These favorable properties can be enhanced by surface biofunctionalization to enable full tissue regeneration and minimize the risk of implant-associated infections (IAIs). There is, however, an increasing need to investigate the immune responses triggered by surface biofunctionalized AM porous metals. Here, we studied the immunomodulatory effects of AM porous titanium (Ti-6Al-4V) printed using selective laser melting and of two additional groups consisting of AM implants surface biofunctionalized using plasma electrolytic oxidation (PEO) with/without silver nanoparticles. The responses of human primary macrophages and human mesenchymal stromal cells (hMSCs) were studied in terms of cell viability, cell morphology and biomarkers of macrophage polarization. Non-treated AM porous titanium triggered a strong pro-inflammatory response in macrophages, albeit combined with signs of anti-inflammatory effects. The PEO treatment of AM porous titanium implants showed a higher potential to induce polarization towards a pro-repair macrophage phenotype. We detected no cytotoxicity against hMSCs in any of the groups. However, the incorporation of silver nanoparticles resulted in strong cytotoxicity against attached macrophages. The results of this study indicate the potential immunomodulatory effects of the AM porous titanium enhanced with PEO treatment, and point towards caution and further research when using silver nanoparticles for preventing IAIs.@en

Implant-associated infection and limited longevity are two major challenges that orthopedic devices need to simultaneously address. Additively manufactured porous implants have recently shown tremendous promise in improving bone regeneration and osseointegration, but, as any conventional implant, are threatened by infection. In this study, we therefore used rational design and additive manufacturing in the form of selective laser melting (SLM) to fabricate porous titanium implants with interconnected pores, resulting in a 3.75 times larger surface area than corresponding solid implants. The SLM implants were biofunctionalized by embedding silver nanoparticles in an oxide surface layer grown using plasma electrolytic oxidation (PEO) in Ca/P-based electrolytes. The PEO layer of the SLM implants released silver ions for at least 28 days. X-ray diffraction analysis detected hydroxyapatite on the SLM PEO implants but not on the corresponding solid implants. In vitro and ex vivo assays showed strong antimicrobial activity of these novel SLM PEO silver-releasing implants, without any signs of cytotoxicity. The rationally designed SLM porous implants outperformed solid implants with similar dimensions undergoing the same biofunctionalization treatment. This included four times larger amount of released silver ions, two times larger zone of inhibition, and one additional order of magnitude of reduction in numbers of CFU in an ex vivo mouse infection model.

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Additively manufactured Ti-6Al-4V implants were biofunctionalized using plasma electrolytic oxidation. At various time points during this process scanning electron microscopy imaging was performed to analyze the surface morphology (van Hengel et al., 2017) [1]. This data shows the changes in surface morphology during plasma electrolytic oxidation. Data presented in this article are related to the research article “Selective laser melting porous metallic implants with immobilized silver nanoparticles kill and prevent biofilm formation by methicillin-resistant Staphylococcus aureus” (van Hengel et al., 2017) [1].

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