Chondrogenic potential of chondrocytes in hyaluronic acid/PEG-based hydrogels is dependent on the hyaluronic acid concentration

Purpose: Hydrogels based on PEG and methacrylated poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate) (M10P10) are promising biomaterials for Biofabrication of cartilage constructs. Addition of hyaluronic acid (HA) to a hydrogel improves printability by increasing the viscosity. Methacrylating HA (HAMA) can ensure covalent binding in M10P10 hydrogels after UV-cross-linking. Chondrocytes can interact with HAMA via their CD44 receptor, however, the influence of HAMA on chondrogenic potential is unclear. This study aimed to evaluate the influence of different HAMA concentrations on chondrogenesis of chondrocytes in M10P10/HAMA hydrogels. Materials & Methods: Equine chondrocytes were encapsulated in M10P10 hydrogels containing different HAMA concentrations. Cylindrical constructs were cast, UV-cross-linked, and cultured in TGF-β-containing medium. Constructs were analyzed for evidence of cartilage formation. Results: Preliminary data showed an increase in glycosaminoglycan (GAG)/DNA for constructs with low HAMA concentrations (0.1-0.25%) while no differences were found for higher HAMA concentrations, compared to hydrogels without HAMA (Figure 1a). Further, constructs without or with low HAMA concentrations (0.1-0.5%) demonstrated collagen type II positive areas, while this was less pronounced in constructs with 0.5-1% HAMA (n=3, Figure 1b). Conclusion: Preliminary results indicate a dose-dependent effect of HAMA on chondrogenesis of chondrocytes: low concentrations (0.1-0.25%) increase GAG production while higher concentrations (0.5-1%) have no effect on GAG production and reduce collagen type II synthesis. Ongoing evaluations will reveal the extent of chondrogenesis and its association with HAMA concentrations in M10P10/HAMA, and the mechanism responsible for the dose-dependent effect. This study will impact the use of HAMA as viscosity enhancer to improve the printability of hydrogel

Performance evaluation of four cascade impactors for airborne ultrafine-particle (UFP) collection: the influence of particle type, concentration, mass, and chemical nature

Ultrafine particles (UFPs) have aerodynamic diameters of 100 nm or less. As UFPs potentially impact human and environmental health, their chemical composition is of interest. However, their small mass presents challenges for sampling and chemical characterization methods. Therefore, we conducted a comprehensive characterization and comparison of four cascade impactors suitable for separating and collecting UFPs – namely, the 120R Micro-Orifice Uniform Deposit Impactor (120R MOUDI-II), ultraMOUDI, electrical low-pressure impactor (ELPI), and personal nanoparticle sampler (PENS) – under controlled laboratory conditions and in a field application. In the laboratory, we evaluated pressure drops, cutoff diameters, the steepness of the cutoff curve, losses, particle bounce, and transmitted particle mass. We observed that the performance of the impactors varied between 59 and 116 nm in cutoff diameter (electromobility diameter), depending on the impactor's design and the type of test aerosol mixture – salt particles (NaCl), simulated secondary organic aerosol (SimSOA), or soot. All impactors separated UFPs, with the best agreement in cutoff diameters for SimSOA, which showed maximum deviations of about 4 nm. The cutoff curve was steeper for soot compared to SimSOA and NaCl. Pressure drops were measured at 260 ± 1 hPa (PENS), 420 ± 2 hPa (ultraMOUDI), 600 ± 3 hPa (120R MOUDI-II), and 690 ± 3 hPa (ELPI). Losses were assessed as maximum transmissions in the ultrafine fraction at 30 nm, yielding 83 ± 8 % for the PENS, 77 ± 8 % for the ultraMOUDI, 75 ± 8 % for the 120R MOUDI-II, and 69 ± 7 % for the ELPI. We compared two additional impactor-specific factors crucial for mass-based analyses of organic marker compounds: the evaporation of semi-volatile compounds due to a high-pressure drop across the impactor and material addition from larger particles bouncing off upper stages. “Bounce-off” was influenced by the particle number concentration in the sampled air and could be partially mitigated by applying a coating to the upper impaction plates. In the field application, we deployed the four cascade impactors side by side under environmental conditions to sample urban air. We analyzed six markers representing typical UFP sources and various molecular properties using HPLC-MS/FLD (high-performance liquid chromatography with mass spectrometry and fluorescence detection). These markers comprised benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF), levoglucosan (Levo), pinic acid (PA), terpenylic acid (TA), and N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD). The impactors showed the best agreement for BaP and BbF. BaP had an average mass concentration of 175 ± 25 pg m−3 across all impactors and sampling days. However, concentrations were about 29 % higher when sampled with the PENS and 30 % lower when sampled with the 120R MOUDI-II, indicating a maximum disagreement of nearly 60 %. The mass concentrations of the semi-volatile markers (PA, TA, and Levo) decreased on average from the PENS to the ultraMOUDI, then to the 120R MOUDI-II, and finally to the ELPI. We attributed this tendency to the following two effects. (1) Evaporation losses of these markers were likely driven by the pressure drop within the impactor, which increased from the PENS to the ELPI. (2) Despite the applied coating, bounce-off might have affected the smallest impactors (i.e., the PENS and ultraMOUDI) the most as they have fewer stages for retaining larger particles and fragments.</p

The impact of immune response on endochondral bone regeneration

Tissue engineered cartilage substitutes, which induce the process of endochondral ossification, represent a regenerative strategy for bone defect healing. Such constructs typically consist of multipotent mesenchymal stromal cells (MSCs) forming a cartilage template in vitro, which can be implanted to stimulate bone formation in vivo. The use of MSCs of allogeneic origin could potentially improve the clinical utility of the tissue engineered cartilage constructs in three ways. First, ready-to-use construct availability can speed up the treatment process. Second, MSCs derived and expanded from a single donor could be applied to treat several patients and thus the costs of the medical interventions would decrease. Finally, it would allow more control over the quality of the MSC chondrogenic differentiation. However, even though the envisaged clinical use of allogeneic cell sources for bone regeneration is advantageous, their immunogenicity poses a significant obstacle to their clinical application. The aim of this review is to increase the awareness of the role played by immune cells during endochondral ossification, and in particular during regenerative strategies when the immune response is altered by the presence of implanted biomaterials and/or cells. More specifically, we focus on how this balance between immune response and bone regeneration is affected by the implantation of a cartilaginous tissue engineered construct of allogeneic origin

Trained innate immunity modulates osteoblast and osteoclast differentiation

Macrophages are key regulators in bone repair and regeneration. Recent studies have shown that long-term epigenetic changes and metabolic shifts occur during specific immune training of macrophages that affect their functional state, resulting in heightened (trained) or reduced (tolerant) responses upon exposure to a second stimulus. This is known as innate immune memory. Here, we study the impact of macrophages’ memory trait on osteoblast differentiation of human mesenchymal stromal cells (hMSCs) and osteoclast differentiation. An in vitro trained immunity protocol of monocyte-derived macrophages was employed using inactivated Candida albicans and Bacillus Calmette–Guérin (BCG) to induce a ‘trained’ state and Pam3CSK4 (PAM) and Lipopolysaccharides (LPS) to induce a ‘tolerance’ state. Macrophages were subsequently cocultured with hMSCs undergoing osteogenic differentiation during either resting (unstimulated) or inflammatory conditions (restimulated with LPS). Alkaline phosphatase activity, mineralization, and cytokine levels (TNF, IL-6, oncostatin M and SDF-1α) were measured. In addition, macrophages underwent osteoclast differentiation. Our findings show that trained and tolerized macrophages induced opposing results. Under resting conditions, BCG-trained macrophages enhanced ALP levels (threefold), while under inflammatory conditions this was found in the LPS-tolerized macrophages (fourfold). Coculture of hMSCs with trained macrophages showed mineralization while tolerized macrophages inhibited the process under both resting and inflammatory conditions. While osteoclast differentiation was not affected in trained-macrophages, this ability was significantly loss in tolerized ones. This study further confirms the intricate cross talk between immune cells and bone cells, highlighting the need to consider this interaction in the development of personalized approaches for bone regenerative medicine. Graphical Abstract: (Figure presented.)

Local delivery of lipid-based nanoparticles containing microbial nucleic acid for osteoimmunomodulation

Osteoimmunomodulation is a strategy to promote bone regeneration in implants by modifying the immune environment. CpG-containing oligonucleotides type C (CpG ODN C) and Polyinosinic:polycytidylic acid (Poly[I:C]) are analogs of microbial nucleic acids that have been studied for various immunotherapeutic applications. This research investigates the potential of CpG ODN C and Poly(I:C) as an osteoimmunomodulatory agent for bone regenerative purposes. We encapsulated each nucleic acid in a lipid-based nanoparticle to facilitate the delivery into intracellular pathogen recognition receptors in immune cells. The lipid-based nanoparticles were ±250 nm in size with a negative charge (−36 to −40 mV) and an encapsulation efficiency of ±60 %. Lipid-based nanoparticles containing nucleic acids, Lip/CpG ODN C and Lip/Poly(I:C), increased the production of TNF, IL-6, and IL-10 by primary human macrophages compared to free-form nucleic acids. Conditioned medium from macrophages treated with CpG ODN C (10 µg/ml) and Lip/CpG ODN C (0.1, 1, and 10 µg/ml) promoted osteoblast differentiation of human mesenchymal stromal cells by 2.6-fold and 3-fold, respectively; no effect was seen for Lip/Poly(I:C). Bone implants were prepared, consisting of a biphasic calcium phosphate scaffold, bone morphogenetic protein (BMP) 2, and lipid-based nanoparticles suspended in gelatin methacryloyl (GelMA) hydrogel. Implants were evaluated for de novo bone formation in an extra-skeletal implantation model in rabbits for 5 weeks. Based on the particles suspended in GelMA, six groups of implants were prepared: Lip/CpG ODN C, Lip/Poly(I:C), Lip (empty), CpG ODN C, Poly(I:C), and a control group consisting of empty GelMA. After 5 weeks, healthy bone tissue formed in all of the implants with active osteoblast and osteoclast activity, however, the amount of new bone volume and scaffold degradation were similar for all implants. We suggest that the working concentrations of the nucleic acids employed were inadequate to induce a relevant inflammatory response. Additionally, the dosage of BMP-2 used may potentially mask the immune-stimulatory effect. Lip/CpG ODN C holds potential as a bioactive agent for osteoimmunomodulation, although further in vivo demonstration should corroborate the current in vitro findings