Human mesenchymal stromal cells (hMSCs) seeded on calcium phosphate (CaP) bioceramics are extensively explored in bone tissue engineering and have recently shown effective clinical outcomes. In previous pre-clinical studies, hMSCs-CaP-mediated bone formation was preceded by osteoclastogenesis at the implantation site. The current study evaluates to what extent phase composition of CaPs affects the osteoclast response and ultimately influence bone formation. To this end, four different CaP bioceramics were used, hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP) and two biphasic composites of HA/beta- TCP ratios of 60/40 and 20/80 respectively, for in vitro osteoclast differentiation and correlation with in vivo osteoclastogenesis and bone formation. All ceramics allowed osteoclast formation in vitro from mouse and human precursors, except for pure HA, which significantly impaired their maturation. Ectopic implantation alongside hMSCs in subcutis sites of nude mice revealed new bone formation at 8 weeks in all conditions with relative amounts for beta-TCP > biphasic CaPs > HA. Surprisingly, while hMSCs were essential for osteoinduction, their survival did not correlate with bone formation. By contrast, the degree of early osteoclastogenesis (2 weeks) seemed to define the extent of subsequent bone formation. Together, our findings suggest that the osteoclastic response could be used as a predictive marker in hMSC-CaPbased bone regeneration and strengthens the need to understand the underlying mechanisms for future biomaterial development. Statement of significance The combination of mesenchymal stromal cells (MSCs) and calcium phosphate (CaP) materials has demonstrated its safety and efficacy for bone regeneration in clinical trials, despite our insufficient understanding of the underlying biological mechanisms. Osteoclasts were previously suggested as key mediators between the early inflammatory phase following biomaterial implantation and the subsequent bone formation. Here we compared the affinity of osteoclasts for various CaP materials with different ratios of hydroxyapatite to beta-tricalcium phosphate. We found that osteoclast formation, both in vitro and at early stages in vivo, correlates with bone formation when the materials were implanted alongside MSCs in mice. Surprisingly, MSC survival did not correlate with bone formation, suggesting that the number or phenotype of osteoclasts formed was more important. (c) 2024 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

Common ragweed ( Ambrosia artemisiifolia ) is an invasive plant with allergenic pollen. Due to environmental changes, ragweed pollen (RWP) airborne concentrations are predicted to quadruple in Europe by 2050 and more than double allergic sensitization of Europeans by 2060. We developed an experimental RWP model of allergy in BALB/c mice to evaluate how the number of RWP and how RWP collected from different geographical environments influence disease. We administered RWP six times over 3 weeks intranasally to the mice and then evaluated disease parameters 72 h later or allowed the mice to recover for at least 90 days before rechallenging them with RWP to elicit a disease relapse. Doses over 300 pollen grains induced lung eosinophilia. Higher doses of 3,000 and 30,000 pollen grains increased both eosinophils and neutrophils and induced disease relapses. RWP harvested from diverse geographical regions induced a spectrum of allergic lung disease from mild inflammation to moderate eosinophilic and severe mixed eosinophilic-neutrophilic lung infiltrates. After a recovery period, mice rechallenged with pollen developed a robust disease relapse. We found no correlation between Amb a 1 content, the major immunodominant allergen, endotoxin content, or RWP structure with disease severity. These results demonstrate that there is an environmental impact on RWP with clinical consequences that may underlie the increasing sensitization rates and the severity of pollen-induced disease exacerbation in patients. The multitude of diverse environmental factors governing distinctive patterns of disease induced by RWP remains unclear. Further studies are necessary to elucidate how the environment influences the complex interaction between RWP and human health.

(1) Background: Inhibition of osteoclast differentiation is the key approach in treating osteoporosis. However, using state-of-the-art treatments such as bisphosphonates and estrogen-based therapy is usually accompanied by many side effects. As opposed to this, the use of natural products as an osteoporotic remedy delivers promising outcomes with minimal side effects. (2) Methods: In the present study, we implemented a biochemometric workflow comprising (i) chemometric approaches using NMR and mass spectrometry and (ii) cell biological approaches using an osteoclast cytochemical marker (TRAP). The workflow serves as a screening tool to pursue potential in vitro osteoclast inhibitors. (3) Results: The workflow allowed for the selective isolation of two phenylpropanoids (coniferyl alcohol and sinapyl alcohol) from the fruits of neem tree (Azadirachta indica). These two isolated phenylpropanoids showed a very promising dose-dependent inhibition of osteoclast differentiation with negligible effects in terms of cell viability. (4) Conclusion: The presented workflow is an effective tool in the discovery of potential candidates for osteoclast inhibition from complex extracts. The used biochemometric approach saves time, effort and costs while delivering precise hints to selectively isolate bioactive constituents.

Although autografts are considered to be the gold standard treatment for reconstruction of large bone defects resulting from trauma or diseases, donor site morbidity and limited availability restrict their use. Successful bone repair also depends on sufficient vascularization and to address this challenge, novel strategies focus on the development of vascularized biomaterial scaffolds. This pilot study aimed to investigate the feasibility of regenerating large bone defects in sheep using 3D-printed customized calcium phosphate scaffolds with or without surgical vascularization. Pre-operative computed tomography scans were performed to visualize the metatarsus and vasculature and to fabricate customized scaffolds and surgical guides by 3D printing. Critical-sized segmental defects created in the mid-diaphyseal region of the metatarsus were either left empty or treated with the 3D scaffold alone or in combination with an axial vascular pedicle. Bone regeneration was evaluated 1, 2 and 3 months post-implantation. After 3 months, the untreated defect remained non-bridged while the 3D scaffold guided bone regeneration. The presence of the vascular pedicle further enhanced bone formation. Histology confirmed bone growth inside the porous 3D scaffolds with or without vascular pedicle inclusion. Taken together, this pilot study demonstrated the feasibility of precised pre-surgical planning and reconstruction of large bone defects with 3D-printed personalized scaffolds.

A major challenge in orthopedics is the repair of large non-union bone fractures. A promising therapy for this indication is the use of biodegradable bioinspired biomaterials that stabilize the fracture site, relieve pain and initiate bone formation and healing. This study uses a multidisciplinary evaluation strategy to assess immunogenicity, allergenicity, bone responses and physicochemical properties of a novel biomaterial scaffold. Two-photon stereolithography generated personalized custom-built scaffolds with a repeating 3D structure of Schwarz Primitive minimal surface unit cell with a specific pore size of ∼400 μm from three different methacrylated poly(d,l-lactide-co-ε-caprolactone) copolymers with lactide to caprolactone monomer ratios of 16 : 4, 18 : 2 and 9 : 1. Using in vitro and in vivo assays for bone responses, immunological reactions and degradation dynamics, we found that copolymer composition influenced the scaffold physicochemical and biological properties. The scaffolds with the fastest degradation rate correlated with adverse cellular effects and mechanical stiffness correlated with in vitro osteoblast mineralization. The physicochemical properties also correlated with in vivo bone healing and immune responses. Overall these observations provide compelling support for these scaffolds for bone repair and illustrate the effectiveness of a promising multidisciplinary strategy with great potential for the preclinical evaluation of biomaterials.

Major challenges in traditional wound closure methods (e.g. using sutures and skin staplers) remain inadequately unaddressed; these invasive treatments induce extra puncture wounds, anesthetic side effects, and severe scarring. Herein, an oxidized starch/gelatin-based shape memory hydrogel (OSG) was fabricated as a self-contracting wound dressing to facilitate noninvasive wound closure. The self-contracting properties were attributed by introducing crosslink net-points in the hydrogel polymer structure through Schiff base reaction between oxidized starch (OS) and gelatin. We systematically investigated the self-contracting properties to determine the feasibility of the hydrogel to treat wounds and promote wound closure noninvasively. Following elongation, OSGs could be entirely fixed in a temporary shape at 4 °C, and then contracted under infrared irradiation (IR) for shape memory activation near human physiological temperature (38 °C), providing sufficient recovery force (4 kPa) for successful noninvasive wound closure. Additionally, H&E staining revealed that thicker epidermis and dermis layers were achieved upon OSG treatment, confirming that the OSG facilitated tissue reconstruction in an in vivo rabbit model. Moreover, the OSG-treated wounds displayed smoother skin and no visible scarring compared to sutured wounds. Such excellent performance suggests that OSG hydrogel exhibits high potential as an alternative to medical sutures to facilitate noninvasive would closure.

Although tissue engineering has been attracted greatly for healing of critical-sized bone defects, great efforts for improvement are still being made in scaffold design. In particular, bone regeneration would be enhanced if a scaffold precisely matches the contour of bone defects, especially if it could be implanted into the human body conveniently and safely. In this study, polyurethane/hydroxyapatite-based shape memory polymer (SMP) foam was fabricated as a scaffold substrate to facilitate bone regeneration. The minimally invasive delivery and the self-fitting behavior of the SMP foam were systematically evaluated to demonstrate its feasibility in the treatment of bone defects in vivo. Results showed that the SMP foam could be conveniently implanted into bone defects with a compact shape. Subsequently, it self-matched the boundary of bone defects upon shape-recovery activation in vivo. Micro-computed tomography determined that bone ingrowth initiated at the periphery of the SMP foam with a constant decrease towards the inside. Successful vascularization and bone remodeling were also demonstrated by histological analysis. Thus, our results indicate that the SMP foam demonstrated great potential for bone regeneration.

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of β-tricalcium phosphate (β-TCP) disks. We also tested a β-TCP/Collagen (β-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

Novel thermo-sensitive elastin-like recombinamers (ELRs) containing bioactive molecules were created for use as a biomimetic biomaterial for tissue regeneration. For effective use for in vivo applications, it is essential to ensure that they do not induce adverse inflammatory, immune, or allergic responses that inhibit tissue repair. Therefore, we sought to establish a pre-clinical approach to evaluate biocompatibility in experimental mice using ELRs as a prototype biomaterial. First, we measured in vitro proliferation and cytokine production from BALB/c and C57BL/6 mouse splenocytes incubated with ELRs. Second, we used a rapid, high throughput in vivo approach in which inflammatory cells and cytokines were measured following an intraperitoneal implantation. Lastly, a subchronic in vivo approach was used in which ELRs or positive controls were subcutaneously implanted and the implantation sites were assessed for inflammation and gene expression. We found that ELRs induced mild inflammation and minimal fibrosis compared to the intense response to Vitoss. Additionally, implantation increased antigen-specific antibody titers for both groups and gene expression profiling of the implantation sites revealed the upregulation of inflammation, fibrosis, and wound healing-related genes in ELR and positive control-implanted mice compared to sham controls. These data demonstrate that ELRs appear safe for use in tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 924-934, 2018.

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

INTRODUCTION: In the present work, hydrogel/cryogel-polyester combinatory scaffolds for bone tissue engineering were developed and characterized. To this end, gelatin was selected as a cell-interactive, biodegradable hydrogel material to enable the adhesion and the proliferation of mouse calvaria pre-osteoblasts. To secure the mechanical properties for the envisaged application, the hydrogels were combined with biodegradable polyester scaffolds because of their suitable mechanical strength. RESULTS AND DISCUSSION: In order to realize suitable osteoblast carriers, methacrylamide-functionalized gelatin (gel-MOD) was introduced into 3D printed polyester (i.e. poly-e-caprolactone or poly-lactic acid) scaffolds obtained through fused deposition modelling, followed by the application of a cryogenic treatment. The cryogenic treatment resulted in the formation of a porous gelatin microstructure inside the polyester scaffolds. Crosslinking of gel-MOD was performed either via a redox-initiated cryogelation or through a UV-initiated process. In a first part, the efficiency of the crosslinking/cryogelation process was determined using gel fraction experiments and by correlating the results with conventional hydrogel formation at room temperature. Next, the optimal parameters were fed into the combinatory approach and the scaffolds developed were characterized for their structural and mechanical properties using scanning electron microscopy, micro-computed tomography (µ-CT) and compression tests. In a final part, in vitro biocompatibility assays were applied. In parallel, experiments were conducted during which cells were encapsulated into the hydrogel precursor solution prior to UV crosslinking to ensure a homogeneous cell distribution. CONCLUSIONS: The developed combination scaffolds exhibit suitable mechanical properties for hard tissue engineering. Furthermore, µ-CT analysis revealed the presence of a porous micro-sized network throughout the scaffolds both for the redox- initiated system as well as for the UV-initiated setup. However, the cellular assay revealed that cellular infiltration throughout the entire scaffold is sub-optimal. Fortunately, this drawback can elegantly be circumvented by the application of UV-initiation in the presence of cells.

INTRODUCTION: The present work aims at the development of 3D scaffolds using both fused deposition modelling as well as a cryogenic treatment to create a fully interconnected pore network. Two material classes will be applied as starting compounds including polyesters (for their mechanical properties) and crosslinkable gelatin precursors (for their cell-interactive properties). MATERIALS AND METHODS: In a first part, poly(lactic acid) (PLA) was processed using fused deposition modelling (Ultimaker). Next, the porous PLA scaffolds were incubated in aqueous methacrylamide-modified gelatin (gel-MOD) solutions of varying concentrations (i.e. 2-10 w/v%) containing 2 mol% Irgacure 2959. The gel-MOD had a modification degree of 80%. Next, after vacuum application, the scaffolds were exposed to UV-light (365 nm) for 2 hours, placed in a cryo-unit and cooled from room temperature to -30°C during 5.5 hours, applying a temperature gradient of 30°C between top and bottom of the scaffolds. In a final step, the frozen hydrogel-PLA combinatory scaffolds were transferred to a freeze-dryer to remove the ice crystals. The scaffolds were characterized using among other micro-computed tomography (µCT) and scanning electron microscopy (SEM). In addition to assessing the water uptake capacity, the mechanical properties were determined using compression tests. In parallel, similar hydrogel precursor solutions containing MC3T3 cells were introduced into the 3D printed PLA scaffolds, followed by hydrogel crosslinking through UV irradiation. The obtained materials were characterized using confocal laser scanning microscopy and the metabolic activity of the seeded and encapsulated cells was assessed with respectively Presto Blue and gene expression studies. RESULTS AND DISCUSSION: In a first part, the efficiency of the UV crosslinking of gel-MOD was assessed after its introduction in PLA. Interestingly, the results indicated that relatively high gel fractions were obtained which were comparable to the ones obtained in the absence of the PLA. Furthermore, mechanical tests showed that the PLA structure determines the strength of the scaffolds as the elastic moduli of both the filled as well as the native structures were comparable. Currently, cell work is ongoing to determine the optimal cryogelation conditions for sufficient cellular infiltration and proliferation. However, the reference samples consisting of polymerized hydrogels encapsulating MC3T3 cells present in the PLA scaffolds were very promising. After 7 days, a 100% increase of the metabolic activity was observed indicating the occurrence of cell proliferation in the scaffolds. In addition, live-dead stains only showed a limited number of dead cells which did not survive the UV crosslinking. Moreover, an ALP stain came out positive indicating the potential of the scaffolds to support osteogenesis. CONCLUSIONS: The UV crosslinking of cells encapsulated into a cell-interactive hydrogel is a valuable route to introduce cells throughout a 3D PLA scaffold. The combination of a cryogel and a PLA scaffold is a potential candidate for tissue engineering. Yet, the applied polymer-related and cryogenic parameters are crucial aspects which determine the cell seeding efficiency and the possibility to enable cellular infiltration throughout the scaffold. REFERENCES 1. Van Vlierberghe S. et al., Biomacromolecules 8: 331-337, 2007

Hydrogels are widely used as 3D matrices for cell growth owing to similarity of their mechanical and diffusivity properties to the natural extracellular matrix (ECM). Furthermore, encapsulation of living cells within the hydrogel allows to produce constructs with high initial cell loading and intimate cell-matrix contact, similar to that of the natural ECM. In this contribution the results of the seeding of macroporous poly-lactic acid (PLA) based scaffolds with the help of photopolymerizable gelatin as a cell delivery material are presented. The 3D scaffolds were produced from PLA in accordance to computer aided design (CAD) model by means of fused deposition modeling (FDM, Ultimaker). A solution of methacrylamide-modified gelatin (gel-MOD) in cell culture medium was used as a cell carrier material for seeding. A 107/ml suspension of MC3T3 cells in gel-MOD was introduced into the pores of the scaffold and then photopolymerized. Our results indicate that such seeding procedure facilitates delivering cells into the pores of the scaffolds at high density and homogeneous distribution. The proliferation of the cell within the scaffolds was monitored using a Presto Blue assay for the course of three weeks. The data was verified by DNA quantification at different time points. An upregulation of the osteocalcin expression and a downregulation of RUNX2 indicate the osteogenic differentiation of MC3T3s. These results are confirmed by the positive alkaline phosphatase (ALP) staining. Our findings show that photoinduced encapsulation of cells within a cell-interactive hydrogel is a valuable route to introduce cells throughout 3D scaffolds for bone tissue engineering.