Microporous scaffolds support assembly and differentiation of pancreatic progenitors into β-cell clusters Publication date: 15 September 2019 Source: Acta Biomaterialia, Volume 96 Author(s): Richard L. Youngblood, Joshua P. Sampson, Kimberly R. Lebioda, Lonnie D. Shea Abstract Human pluripotent stem cells (hPSCs) represent a promising cell source for the development of β-cells for use in therapies for type 1 diabetes. Current culture approaches provide signals to mimic a temporal control of organogenesis to drive the differentiation towards β-cells. However, spatial control may represent an opportunity to improve the efficiency and manufacturing of β-cells. Herein, we adapted the current culture systems to microporous biomaterials with the hypothesis that the pores can guide the assembly of pancreatic progenitors into clusters of defined size that can influence maturation. The scaffold culture allowed hPSC-derived pancreatic progenitors to form clusters at a consistent size as cells differentiated. By modulating the scaffold pore sizes, we observed 250–425 µm pore size scaffold cultures augmented insulin expression and key β-cell maturation markers compared to cells cultured in suspension. Furthermore, when compared to suspension cultures, the scaffold culture showed increased insulin secretion in response to glucose stimulus indicating the development of functional β-cells. In addition, scaffolds facilitated cell-cell interactions enabled by the scaffold design and supported cell-mediated matrix deposition of extracellular matrix (ECM) proteins associated with the basement membrane of islet cells. We further investigated the influence of ECM on cell development by incorporating an ECM matrix on the scaffold prior to cell seeding; however, their presence did not further enhance maturation. These results suggest the microporous scaffold culture provides a conducive environment that drives in vitro differentiation of hPSC-derived insulin-producing glucose-responsive β-cells and demonstrates the feasibility of these scaffolds as a biomanufacturing platform. Statement of Significance Cell therapy for diabetes is a promising strategy, yet generating limitless insulin-producing mature β-cells from human pluripotent stem cells (hPSCs) remains a challenge. Current hPSC differentiation methods involve media containing signals to drive maturation toward β-cells and spontaneous cluster formation. Herein, we sought to provide spatial cues to guide assembly of cells into 3D structures by culture within the pores of a microporous scaffold. The scaffolds direct cell-cell interactions within the pores and provide a support for cell-mediated matrix deposition that collectively creates a niche to promote functional hPSC-derived β-cell clusters. These scaffolds for 3D culture may contribute to hPSC differentiation methods for the generation of β-cells that can treat patients with diabetes. Graphical abstract

Electrically polarized TiO2 nanotubes on Ti implants to enhance early-stage osseointegration Publication date: 15 September 2019 Source: Acta Biomaterialia, Volume 96 Author(s): Amit Bandyopadhyay, Anish Shivaram, Indranath Mitra, Susmita Bose Abstract Ti is characteristically bioinert and is supplemented with modifications in surface topography and chemistry to find use in biomedical applications. The aim of this study is to understand the effects of surface charge on TiO2 nanotubes (TNT) on Ti implants towards early stage osseointegration. We hypothesize that charge storage on TNT will improve bioactivity and enhance early-stage osseointegration in vivo. Commercially pure Ti surface was altered by growing TNT via anodic oxidation followed by the introduction of surface charge through electrothermal polarization to form bioelectret. Our results indicate a stored charge of 37.15 ± 14 mC/cm2 for TNT surfaces. The polarized TNT (TNT-Ps) samples did not show any charge leakage up to 18 months, and improved wettability with a measured contact angle less than 1°. No cellular toxicity through osteoblast proliferation and differentiation in vitro were shown by the TNT-Ps. Enhanced new bone formation at 5 weeks post-implantation for the TNT-Ps in contrast to TNTs was observed in vivo. Histomorphometric analyses show ∼40% increase in mineralized bone formation around the TNT-P implants than the TNTs at 5 weeks, which is indicative of accelerated bone remodeling cycle. These results show that stored surface charge on TiO2 nanotubes helped to accelerate bone healing due to early-stage osseointegration in vivo. Statement of Significance To improve surface bioactivity of metallic biomaterials, various approaches have been proposed and implemented. Among them, stored surface charge has been explored to enhance biological responses for hydroxyapatite ceramics where charged surfaces show favorable bone tissue ingrowth. However, surface charge effects have not yet been explored as a way to mitigate bio-inertness of titanium. This study intends to understand novel integration of bioactive titania-nanotubes and charge storage as surface modification for titanium implants. Our results show excellent biological response due to surface charge on titania-nanotubes offering possibilities of faster healing particularly for patients with compromised bone health. Graphical abstract

Additively manufactured functionally graded biodegradable porous iron Publication date: 15 September 2019 Source: Acta Biomaterialia, Volume 96 Author(s): Y. Li, H. Jahr, P. Pavanram, F.S.L. Bobbert, U. Puggi, X.-Y. Zhang, B. Pouran, M.A. Leeflang, H. Weinans, J. Zhou, A.A. Zadpoor Abstract Additively manufactured (AM) functionally graded porous metallic biomaterials offer unique opportunities to satisfy the contradictory design requirements of an ideal bone substitute. However, no functionally graded porous structures have ever been 3D-printed from biodegradable metals, even though biodegradability is crucial both for full tissue regeneration and for the prevention of implant-associated infections in the long term. Here, we present the first ever report on AM functionally graded biodegradable porous metallic biomaterials. We made use of a diamond unit cell for the topological design of four different types of porous structures including two functionally graded structures and two reference uniform structures. Specimens were then fabricated from pure iron powder using selective laser melting (SLM), followed by experimental and computational analyses of their permeability, dynamic biodegradation behavior, mechanical properties, and cytocompatibility. It was found that the topological design with functional gradients controlled the fluid flow, mass transport properties and biodegradation behavior of the AM porous iron specimens, as up to 4-fold variations in permeability and up to 3-fold variations in biodegradation rate were observed for the different experimental groups. After 4 weeks of in vitro biodegradation, the AM porous scaffolds lost 5–16% of their weight. This falls into the desired range of biodegradation rates for bone substitution and confirms our hypothesis that topological design could indeed accelerate the biodegradation of otherwise slowly degrading metals, like iron. Even after 4 weeks of biodegradation, the mechanical properties of the specimens (i.e., E = 0.5–2.1 GPa, σy = 8–48 MPa) remained within the range of the values reported for trabecular bone. Design-dependent cell viability did not differ from gold standard controls for up to 48 h. This study clearly shows the great potential of AM functionally graded porous iron as a bone substituting material. Moreover, we demonstrate that complex topological design permits the control of mechanical properties, degradation behavior of AM porous metallic biomaterials. Statement of Significance No functionally graded porous structures have ever been 3D-printed from biodegradable metals, even though biodegradability is crucial both for full tissue regeneration and for the prevention of implant-associated infections in the long term. Here, we present the first report on 3D-printed functionally graded biodegradable porous metallic biomaterials. Our results suggest that topological design in general, and functional gradients in particular can be used as an important tool for adjusting the biodegradation behavior of AM porous metallic biomaterials. The biodegradation rate and mass transport properties of AM porous iron can be increased while maintaining the bone-mimicking mechanical properties of these biomaterials. The observations reported here underline the importance of proper topological design in the development of AM porous biodegradable metals. Graphical abstract

Mineral formation in the primary polyps of pocilloporoid corals Publication date: 15 September 2019 Source: Acta Biomaterialia, Volume 96 Author(s): Maayan Neder, Pierre Philippe Laissue, Anat Akiva, Derya Akkaynak, Marie Albéric, Oliver Spaeker, Yael Politi, Iddo Pinkas, Tali Mass Abstract In reef-building corals, larval settlement and its rapid calcification provides a unique opportunity to study the bio-calcium carbonate formation mechanism involving skeleton morphological changes. Here we investigate the mineral formation of primary polyps, just after settlement, in two species of the pocilloporoid corals: Stylophora pistillata (Esper, 1797) and Pocillopora acuta (Lamarck, 1816). We show that the initial mineral phase is nascent Mg-Calcite, with rod-like morphology in P. acuta, and dumbbell morphology in S. pistillata. These structures constitute the first layer of the basal plate which is comparable to Rapid Accretion Deposits (Centers of Calcification, CoC) in adult coral skeleton. We found also that the rod-like/dumbbell Mg-Calcite structures in subsequent growth step will merge into larger aggregates by deposition of aragonite needles. Our results suggest that a biologically controlled mineralization of initial skeletal deposits occurs in three steps: first, vesicles filled with divalent ions are formed intracellularly. These vesicles are then transferred to the calcification site, forming nascent Mg-Calcite rod/pristine dumbbell structures. During the third step, aragonite crystals develop between these structures forming spherulite-like aggregates. Statement of Significance Coral settlement and recruitment periods are highly sensitive to environmental conditions. Successful mineralization during these periods is vital and influences the coral’s chances of survival. Therefore, understanding the exact mechanism underlying carbonate precipitation is highly important. Here, we used in vivo microscopy, spectroscopy and molecular methods to provide new insights into mineral development. We show that the primary polyp’s mineral arsenal consists of two types of minerals: Mg-Calcite and aragonite. In addition, we provide new insights into the ion pathway by showing that divalent ions are concentrated in intracellular vesicles and are eventually deposited at the calcification site. Graphical abstract

Biomodulation of an implant for enhanced bone-implant anchorage Publication date: 15 September 2019 Source: Acta Biomaterialia, Volume 96 Author(s): Deepak Bushan Raina, David Larsson, Erdem Aras Sezgin, Hanna Isaksson, Magnus Tägil, Lars Lidgren Abstract Aseptic loosening of implants is the major cause for revision surgery. By modulating the bone-implant interface, early bone-implant anchorage could be improved. Implant surface manipulation by the addition of osteopromotive molecules locally and systemically to promote implant integration has been described with limited success. This study describes a novel approach by making the implant capable of biologically modulating its surroundings. It was hypothesized that the early implant fixation would improve by filling the interior of the implant with a carrier providing spatio-temporal release of bone active drugs with known osteogenic effect. The implant consisted of a threaded polyether ether ketone (PEEK) hollow chamber with holes at the bottom. The implant was filled with a calcium sulphate (CaS)/hydroxyapatite (HA) carrier, delivering two bone active molecules; zoledronic acid (ZA) and bone morphogenic protein-2 (BMP-2). At first, a rat abdominal muscle pouch model indicated a sustained in-vivo release of both 125I-rhBMP-2 (57%) and 14C-ZA (22%) from the CaS/HA carrier over a period of 4-weeks. The biomodulated implant was then inserted in the proximal tibia in rats with the following experimental groups: G1) Empty implant, G2) Implant + CaS/HA, G3) Implant + CaS/HA + ZA and G4) Implant + CaS/HA + ZA + rhBMP-2. Significantly higher bone volume (BV) was seen around the implant in groups G3 (3.3 ± 0.7 mm3) and G4 (3.1 ± 0.7 mm3) compared to the control (1.3 ± 0.4 mm3) using micro-computed tomography and qualitative histology. Group G3, also exhibited significantly higher pull-out force and absorbed energy when compared to the control group G1. These findings indicate that a low dose of ZA alone, released in a controlled manner from within a fenestrated implant is enough to improve implant anchorage without the need of adding rhBMP-2. This simple method of using a fenestrated implant containing a ceramic carrier releasing bone active molecules improved bone anchorage and could clinically reduce prosthetic failure. Statement of Significance Aseptic loosening remains as a major cause for implant revisions and early reaction of surrounding bone to the prosthesis is important for longevity. A novel approach to enhance early bone-implant anchorage is presented. The implant is filled with a carrier providing controlled release of bone active molecules. In an animal model, a calcium sulphate (CaS)/hydroxyapatite (HA) carrier was used to provide a spatio-temporal release of bone morphogenic protein-2 (BMP-2) and zoledronic acid (ZA). Significantly better bone-implant integration was achieved using ZA alone, thereby eliminating the need for adding BMP-2. The developed method of implant biomodulation holds potential to prevent implant loosening and is an alternative to prosthetic coatings or systemic drug treatment. Importantly, all constituents are approved for clinical use. Graphical abstract

The effect of biomimetic calcium deficient hydroxyapatite and sintered β-tricalcium phosphate on osteoimmune reaction and osteogenesis Publication date: 15 September 2019 Source: Acta Biomaterialia, Volume 96 Author(s): Joanna M. Sadowska, Fei Wei, Jia Guo, Jordi Guillem-Marti, Zhengmei Lin, Maria-Pau Ginebra, Yin Xiao Abstract Biomaterial implantation triggers inflammatory reactions. Understanding the effect of physicochemical features of biomaterials on the release of inflammatory cytokines from immune cells would be of great interest in view of designing bone graft materials to enhance the healing of bone defects. The present work investigated the interactions of two chemically and texturally different calcium phosphate (CaPs) substrates with macrophages, one of the main innate immune cells, and its further impact on osteogenic differentiation of bone forming cells. The behaviour of macrophages seeded on biomimetic calcium deficient hydroxyapatite (CDHA) and sintered β-tricalcium phosphate (β-TCP) was assessed in terms of the release of inflammatory cytokines and osteoclastogenic factors. The osteogenic differentiation of bone progenitor cells (bone marrow stromal cells (BMSCs) and osteoblastic cell line (SaOS-2)) were subsequently studied by incubating with the conditioned medium induced by macrophage-CaPs interaction in order to reveal the effect of immune cell reaction to CaPs on osteogenic differentiation. It was found that the incubation of macrophages with CaPs substrates caused a decrease of pro-inflammatory cytokines, more pronounced for β-TCP compared with CDHA showing significantly decreased IL-6, TNF-a, and iNOS. However, the macrophage-CDHA interaction resulted in a more favourable environment for osteogenic differentiation of osteoblasts with more collagen type I production and osteogenic genes (Runx2, BSP) expression, suggesting that osteogenic differentiation of bone cells is not only determined by the nature of biomaterials, but also significantly influenced by the inflammatory environment generated by the interaction of immune cells and biomaterials. Statement of Significance The field of osteoimmunology highlights the importance of the cross-talk between immune and bone cells for effective bone regeneration. This tight interaction opens the door to new strategies that encompass the development of smart cell-instructive biomaterials which performance covers the events from early inflammation to osteogenesis. The present work links the anti-inflammatory and osteoimmunomodulatory features of synthetic bone grafts to their chemistry and texture, focussing on the cross-talk between macrophages and two major orchestrators of bone healing, namely primary mesenchymal stem cells and osteoblasts. The results emphasize the importance of the microenvironment created through the interaction between the substrate and the immune cells as it can stimulate osteogenic events and subsequently foster bone healing. Graphical abstract

Lithium-containing surface pre-reacted glass fillers enhance hDPSC functions and induce reparative dentin formation in a rat pulp capping model through activation of Wnt/β-catenin signaling Publication date: 15 September 2019 Source: Acta Biomaterialia, Volume 96 Author(s): Manahil Ali, Motoki Okamoto, Shungo Komichi, Masakatsu Watanabe, Hailing Huang, Yusuke Takahashi, Mikako Hayashi Abstract Surface pre-reacted glass (S-PRG) fillers are new bioactive molecules used in dental clinic work to fill tooth defects. These fillers release various types of ions (Al+3, BO−3, Na+, SiO3−2, Sr+2 and F−) and exhibit high biocompatibility, antibacterial capability, reduced plaque accumulation, and enhanced osteoblast differentiation. We previously showed that cement of S-PRG fillers could induce tertiary dentin formation in rat models. Previous work also showed that lithium ions can activate the Wnt/β-catenin signaling pathway in vitro and induce dentin formation in pulpotomized teeth in vivo. In the current study, we sought to enhance the effect of S-PRG cement by incorporating LiCl. We show that treatment of human dental pulp stem cells with eluates from S-PRG/LiCl combination cements leads to an upregulation in cell migration, differentiation, and mineralization in vitro. In pulp-capping animal trials, we found that S-PRG/LiCl cements could induce tertiary dentin formation 28-days post-capping. At 7 days post-capping, we identified both β-catenin and Axin2 expression using immunofluorescence, indicative of Wnt/β-catenin signaling activity. In conclusion, S-PRG/LiCl cement is highly effective in promoting human dental pulp stem cells profiles and in enhancing reparative dentin formation in rat teeth through activation of the Wnt/β-catenin canonical signaling pathway. Statement of Significance This is the first study to assess the behavior of S-PRG fillers containing lithium ions on human dental pulp stem cells. We show that this new combination cement promotes positive cell responses by activating the endogenous Wnt/β-catenin signaling pathway in the pulp. The Wnt/β-catenin canonical signaling pathway is involved in many developmental and wound healing processes. The released lithium ions from the S-PRG cement were systematically detected <0.01 mmol/L in our rat model. But it was efficient to induce tertiary dentin formation at the defect site. Since this novel bioactive cement is potentially a promising material for clinical pulp regenerative therapy, future human clinical trials will be needed. Graphical abstract

Interfacial characterization of poly (vinyl alcohol) fibers embedded in a calcium phosphate cement matrix: An experimental and numerical investigation Publication date: 15 September 2019 Source: Acta Biomaterialia, Volume 96 Author(s): Ali Paknahad, Daniela G. Petre, Sander C.G. Leeuwenburgh, Lambertus J. Sluys Abstract Because of their chemical similarity to the mineral phase of bone and teeth, calcium phosphate cements (CPCs) are extensively investigated for applications in biomedicine. Nevertheless, their applicability in load-bearing anatomical sites is restricted by their brittleness. Reinforcement of calcium phosphate cements with polymeric fibers can overcome this mechanical limitation provided that the affinity between these fibers and the surrounding matrix is optimal. To date, the effects of the fiber-matrix affinity on the mechanical properties of fiber-reinforced calcium phosphate cements are still poorly understood. The goal of this study is therefore to investigate the interfacial properties and bond-slip response between the CPC matrix and polymeric fibers. To this end, we selected poly (vinyl alcohol) (PVA) fibers as reinforcing agents because of their high strength and stiffness and their effective reinforcement of cementitious matrices. Micromechanical pull-out experiments were combined with numerical simulations based on an dedicated constitutive interfacial law to characterize the interfacial properties of PVA fibers embedded in a CPC matrix at the single fiber pull-out level. The computational model developed herein is able to predict all three main phases of pull-out response, i.e.the elastic, debonding and frictional pull-out phases. The resulting interfacial constitutive law is validated experimentally and predicts the pull-out response of fibers with different diameters and embedded lengths. Statements of Significance To date, the effects of the fiber-matrix affinity on the mechanical properties of fiber-reinforced calcium phosphate cements are still poorly understood. In this study, we present a novel experimental protocol to investigate the affinity between poly (vinyl alcohol) PVA fibers and the calcium phosphate cement (CPC) matrix by means of single-fiber pull out tests. We determine the critical embedded length for PVA fibers with two different diameters; and we design a numerical FE model including a distinct representation of fiber, matrix and interface with a predictive interfacial constitutive law which is capable of capturing all three main phases of single-fiber pull-out, i.e. elastic, debonding and frictional stages. The resulting interfacial constitutive law is validated experimentally and predicts the pull-out response of fibers with different diameters and embedded lengths. Graphical abstract

Plasma deposited poly-oxazoline nanotextured surfaces dictate osteoimmunomodulation towards ameliorative osteogenesis Publication date: 15 September 2019 Source: Acta Biomaterialia, Volume 96 Author(s): Zetao Chen, Rahul Madathiparambil Visalakshan, Jia Guo, Fei Wei, Linjun Zhang, Lingling Chen, Zhengmei Lin, Krasimir Vasilev, Yin Xiao Abstract Developing “osteoimmune-smart” bone substitute materials have become the forefront of research in bone regeneration. Biocompatible polymer coatings are applied widely to improve the bioactivity of bone substitute materials. In this context, polyoxazolines (Pox) have attracted substantial attention recently due to properties such as biocompatibility, stability, and low biofouling. In view of these useful properties, it is interesting to explore the capacity of Pox as an osteoimmunomodulatory agent to generate a favorable osteoimmune environment for osteogenesis. We applied a technique called plasma polymerization and succeeded in preparing Pox-like coatings (Ppox) and engineered their nanotopography at the nanoscale. We found that Ppox switched macrophages towards M2 extreme, thus inhibiting the release of inflammatory cytokines. The underlying mechanism may be related to the suppression of TLR pathway. The generated osteoimmune environment improved osteogenesis while inhibited osteoclastogenesis. This may be related to the release of osteogenic factors, especially Wnt10b from macrophages. The addition of nanotopography (16 nm, 38 nm, 68 nm) can tune the Ppox-mediated inhibition on inflammation and osteoclastic activities, while no significant effects were observed within the tested nano sizes on the Ppox-mediated osteogenesis. These results collectively suggest that Ppox can be useful as an effective osteoiumunomodulatory agent to endow bone substitute materials with favourable osteoimmunomodulatory property. Statement of Significance In this study, we succeeded in preparing plasma deposited Pox-like nano-coatings (Ppox) via plasma polymerization and found that Ppox nanotopographies are useful osteoimmunomodulatory tools. Their osteoimmunodolatory effects and underlying mechanisms are unveiled. It is the first investigation into the feasibility of applying poly-oxazoline as an osteoimmunomodulatory agent. This expand the application of poly-oxazoline into the forefront in bone regeneration area for the development of advanced “osteoimmune-smart” bone substitute materials. Graphical abstract

Guiding mesenchymal stem cell differentiation using mesoporous silica nanoparticle-based films Publication date: 15 September 2019 Source: Acta Biomaterialia, Volume 96 Author(s): Lea Andrée, David Barata, Pichaporn Sutthavas, Pamela Habibovic, Sabine van Rijt Abstract The development of smart interfaces that can guide tissue formation is of great importance in the field of regenerative medicine. Nanoparticles represent an interesting class of materials that can be used to enhance regenerative treatments by enabling close control over surface properties and directing cellular responses. Moreover, nanoparticles can be used to provide temporally controlled delivery of (multiple) biochemical compounds. Here, we exploited the cargo loading and surface functionalization properties of mesoporous silica nanoparticles (MSNs) to design films that can guide human mesenchymal stem cell (hMSC) differentiation towards the osteogenic lineage. We developed biocompatible MSN-based films that support stem cell adhesion and proliferation and demonstrated that these MSN films simultaneously allowed efficient local delivery of biomolecules without effecting film integrity. Films loaded with the osteogenesis-stimulating drug dexamethasone (Dex) were able to induce osteogenic differentiation of hMSCs in vitro. Dex delivery from the films led to increased alkaline phosphatase levels and matrix mineralization compared to directly supplementing Dex to the medium. Furthermore, we demonstrated that Dex release kinetics can be modulated using surface modifications with supported lipid bilayers. Together, these data demonstrate that MSN films represent an interesting approach to create biomaterial interfaces with controllable biomolecule release and surface properties to improve the bioactivity of biomaterials. Statement of significance Engineering surfaces that can control cell and tissue responses is one of the major challenges in biomaterials-based regenerative therapies. Here, we demonstrate the potential of mesoporous silica nanoparticles (MSNs) as drug-delivering surface coatings. First, we show differentiation of mesenchymal stem cells towards the bone lineage when in contact with MSN films loaded with dexamethasone. Furthermore, we demonstrate that modification of MSNs with supported lipid bilayer allows control over drug release dynamics and cell shape. Given the range of loadable cargos and the tunability of release kinetics, MSN coatings can be used to mimic the sequential appearance of bioactive factors during tissue regeneration, which will ultimately lead to biomaterials with improved bioactivity. Graphical abstract