Influence of Scaffold Microarchitecture on Angiogenesis and Regulation of Cell Differentiation during the Early Phase of Bone Healing: A Transcriptomics and Histological Analysis.

The early phase of bone healing is a complex and poorly understood process. With additive manufacturing, we can generate a specific and customizable library of bone substitutes to explore this phase. In this study, we produced tricalcium phosphate-based scaffolds with microarchitectures composed of filaments of 0.50 mm in diameter, named Fil050G, and 1.25 mm named Fil125G, respectively. The implants were removed after only 10 days in vivo followed by RNA sequencing (RNAseq) and histological analysis. RNAseq results revealed upregulation of adaptive immune response, regulation of cell adhesion, and cell migration-related genes in both of our two constructs. However, significant overexpression of genes linked to angiogenesis, regulation of cell differentiation, ossification, and bone development was observed solely in Fil050G scaffolds. Moreover, quantitative immunohistochemistry of structures positive for laminin revealed a significantly higher number of blood vessels in Fil050G samples. Furthermore, µCT detected a higher amount of mineralized tissue in Fil050G samples suggesting a superior osteoconductive potential. Hence, different filament diameters and distances in bone substitutes significantly influence angiogenesis and regulation of cell differentiation involved in the early phase of bone regeneration, which precedes osteoconductivity and bony bridging seen in later phases and as consequence, impacts the overall clinical outcome.

Effect of N-Vinyl-2-Pyrrolidone (NVP), a Bromodomain-Binding Small Chemical, on Osteoblast and Osteoclast Differentiation and Its Potential Application for Bone Regeneration.

The human skeleton is a dynamic and remarkably organized organ system that provides mechanical support and performs a variety of additional functions. Bone tissue undergoes constant remodeling; an essential process to adapt architecture/resistance to growth and mechanical needs, but also to repair fractures and micro-damages. Despite bone's ability to heal spontaneously, certain situations require an additional stimulation of bone regeneration, such as non-union fractures or after tumor resection. Among the growth factors used to increase bone regeneration, bone morphogenetic protein-2 (BMP2) is certainly the best described and studied. If clinically used in high quantities, BMP2 is associated with various adverse events, including fibrosis, overshooting bone formation, induction of inflammation and swelling. In previous studies, we have shown that it was possible to reduce BMP2 doses significantly, by increasing the response and sensitivity to it with small molecules called "BMP2 enhancers". In the present study, we investigated the effect of N-Vinyl-2-pyrrolidone (NVP) on osteoblast and osteoclast differentiation in vitro and guided bone regeneration in vivo. We showed that NVP increases BMP2-induced osteoblast differentiation and decreases RANKL-induced osteoclast differentiation in a dose-dependent manner. Moreover, in a rabbit calvarial defect model, the histomorphometric analysis revealed that bony bridging and bony regenerated area achieved with NVP-loaded poly (lactic-co-glycolic acid (PLGA) membranes were significantly higher compared to unloaded membranes. Taken together, our results suggest that NVP sensitizes BMP2-dependent pathways, enhances BMP2 effect, and inhibits osteoclast differentiation. Thus, NVP could prove useful as "osteopromotive substance" in situations where a high rate of bone regeneration is required, and in the management of bone diseases associated with excessive bone resorption, like osteoporosis.

Microporosities in 3D-Printed Tricalcium-Phosphate-Based Bone Substitutes Enhance Osteoconduction and Affect Osteoclastic Resorption.

Additive manufacturing is a key technology required to realize the production of a personalized bone substitute that exactly meets a patient's need and fills a patient-specific bone defect. Additive manufacturing can optimize the inner architecture of the scaffold for osteoconduction, allowing fast and reliable defect bridging by promoting rapid growth of new bone tissue into the scaffold. The role of scaffold microporosity/nanoarchitecture in osteoconduction remains elusive. To elucidate this relationship, we produced lithography-based osteoconductive scaffolds from tricalcium phosphate (TCP) with identical macro- and microarchitecture, but varied their nanoarchitecture/microporosity by ranging maximum sintering temperatures from 1000 °C to 1200 °C. After characterization of the different scaffolds' microporosity, compression strength, and nanoarchitecture, we performed in vivo studies that showed that ingrowth of bone as an indicator of osteoconduction significantly decreased with decreasing microporosity. Moreover, at the 1200 °C peak sinter temperature and lowest microporosity, osteoclastic degradation of the material was inhibited. Thus, even for wide-open porous TCP-based scaffolds, a high degree of microporosity appears to be essential for optimal osteoconduction and creeping substitution, which can prevent non-unions, the major complication during bone regeneration procedures.

Regenerative Dentistry: Animal Model for Regenerative Endodontology.

BACKGROUND: Ectopic tissue has been observed frequently in human root canal specimens when cell homing studies were performed at the dorsum of rodents. In contrast, pulp-like tissue formed when immature teeth were implanted on top of the rat calvaria. It was surmised, yet not tested, that the implantation site might affect tissue ingrowth. METHODS: Four root sections from human immature molars cleaned with 5% sodium hypochlorite (NaOCl) followed by 17% ethylenediaminetetraacetic acid (EDTA) were implanted per rat (n = 5). Two specimens were placed at the dorsum (control), while the other two specimens were implanted at the calvaria. After 6 weeks, the specimens were investigated for histological structure, immunoreactivity to dentine sialoprotein (DSP) and bone sialoprotein (BSP), per-area percentage of tissue ingrowth, and gene expression (DSPP, COL1, NGF and VEGF). Data were statistically compared. RESULTS: Tooth specimens placed at the calvaria generally showed pulp-like tissue and odontoblast-like cells at the dentinal wall where DSP and BSP immunoreactivity were intense. The area of tissue ingrowth was significantly larger in the specimens placed at the calvaria compared to those placed at the dorsum. DSPP was the only gene that was upregulated significantly when specimens were implanted at the calvaria. CONCLUSION: Our findings suggest that the calvarial site is superior to the dorsum to study pulp regeneration in human teeth in the rat.

Enhanced bone regeneration in rat calvarial defects through BMP2 release from engineered poly(ethylene glycol) hydrogels.

The clinical standard therapy for large bone defects, typically addressed through autograft or allograft donor tissue, faces significant limitations. Tissue engineering offers a promising alternative strategy for the regeneration of substantial bone lesions. In this study, we harnessed poly(ethylene glycol) (PEG)-based hydrogels, optimizing critical parameters including stiffness, incorporation of arginine-glycine-aspartic acid (RGD) cell adhesion motifs, degradability, and the release of BMP2 to promote bone formation. In vitro we demonstrated that human bone marrow derived stromal cell (hBMSC) proliferation and spreading strongly correlates with hydrogel stiffness and adhesion to RGD peptide motifs. Moreover, the incorporation of the osteogenic growth factor BMP2 into the hydrogels enabled sustained release, effectively inducing bone regeneration in encapsulated progenitor cells. When used in vivo to treat calvarial defects in rats, we showed that hydrogels of low and intermediate stiffness optimally facilitated cell migration, proliferation, and differentiation promoting the efficient repair of bone defects. Our comprehensive in vitro and in vivo findings collectively suggest that the developed hydrogels hold significant promise for clinical translation for bone repair and regeneration by delivering sustained and controlled stimuli from active signaling molecules.

Triply Periodic Minimal Surface-Based Scaffolds for Bone Tissue Engineering: A Mechanical, In Vitro and In Vivo Study.

Triply periodic minimal surfaces (TPMSs) are found to be promising microarchitectures for bone substitutes owing to their low weight and superior mechanical characteristics. However, existing studies on their application are incomplete because they focus solely on biomechanical or in vitro aspects. Hardly any in vivo studies where different TPMS microarchitectures are compared have been reported. Therefore, we produced hydroxyapatite-based scaffolds with three types of TPMS microarchitectures, namely Diamond, Gyroid, and Primitive, and compared them with an established Lattice microarchitecture by mechanical testing, 3D-cell culture, and in vivo implantation. Common to all four microarchitectures was the minimal constriction of a sphere of 0.8 mm in diameter, which earlier was found superior in Lattice microarchitectures. Scanning by µCT revealed the precision and reproducibility of our printing method. The mechanical analysis showed significantly higher compression strength for Gyroid and Diamond samples compared with Primitive and Lattice. After in vitro culture with human bone marrow stromal cells in control or osteogenic medium, no differences between these microarchitectures were observed. However, from the TPMS microarchitectures, Diamond- and Gyroid-based scaffolds showed the highest bone ingrowth and bone-to-implant contact in vivo. Therefore, Diamond and Gyroid designs appear to be the most promising TPMS-type microarchitectures for scaffolds produced for bone tissue engineering and regenerative medicine. Impact Statement Extensive bone defects require the application of bone grafts. To match the existing requirements, scaffolds based on triply periodic minimal surface (TPMS)-based microarchitectures could be used as bone substitutes. This work is dedicated to the investigation of mechanical and osteoconductive properties of TPMS-based scaffolds to determine the influencing factors on differences in their behavior and choose the most promising design to be used in bone tissue engineering.

Pulp-Derived Exosomes in a Fibrin-Based Regenerative Root Filling Material.

Regenerative endodontics has been described as a paradigm shift in dentistry, despite its current limitation to immature teeth and reparative rather than regenerative outcomes. Cell-free treatments are favored because of regulatory issues. However, the recruitment of host-derived stem cells to the desired site remains challenging. We investigated whether dental pulp-derived exosomes, which are extracellular vesicles that contain proteins, lipids, RNA, and DNA and thus mirror their parental cells, may be used for this purpose. The use of exosomes may present appreciable advantages over the direct use of transplanted stem cells due to a higher safety profile, easier isolation, preservation, and handling. Here we harvested exosomes from a cultured third-molar pulp cell and assessed them by transmission electron microscopy and Western blotting. Human mesenchymal stem cells (MSCs) were exposed to these exosomes to assess exosome uptake, cell migration, and proliferation. In addition, a fibrin gel (i.e., a diluted fibrin sealant), was assessed as a delivery system for the exosomes. Our results show that exosomes attracted MSCs, and the fibrin gel enhanced their effect. Moreover, exosomes improved the proliferation of MSCs. Therefore, we propose that pulp-derived exosomes in combination with a fibrin gel could be a powerful combination for clinical translation towards improved cell-free regenerative endodontics and thus represent a new way to fill dental hard tissues.

Antimicrobial peptide gene expression in medication-related osteonecrosis of the jaw.

Bisphosphonates and denosumab are commonly used antiresorptive therapies in patients with bone metastasis and osteoporosis. Medication-related osteonecrosis of the jaw (MRONJ) is a serious side effect of these drugs, and infection has been recognized as a contributing factor. Current therapeutic options for MRONJ show limited effectiveness, therefore necessitating novel treatment strategies. Bisphosphonates have recently been reported to induce the expression of antimicrobial peptides (AMPs), an inherent component of the immune system. Therefore, the aim of the present study was to investigate and compare the influence of the anti-RANKL antibody denosumab and bisphosphonates on the gene expression of selected AMPs: human α-defensin-1, human α-defensin-3, human ß-defensin-1, and human ß-defensin-3. Bone specimens were collected from patients with MRONJ who had been treated with bisphosphonates (n = 6) or denosumab (n = 6), and from healthy subjects (n = 6) with no history of treatment with bone metabolism-influencing drugs. Reverse transcription-quantitative polymerase chain reaction was used to quantify the expression levels of selected AMPs. Samples from patients treated with denosumab showed significantly higher mRNA expression of human α-defensin-3 and human ß-defensin-3 than those from healthy subjects. This finding is similar to previously described upregulated expression of human defensins in patients with MRONJ after bisphosphonates treatment. This suggests that the elevated expression of defensins may be at least a part of the mechanism underlying the pathogenesis of osteonecrosis induced by antiresorptive therapies, which can serve as a new target for potential treatment of MRONJ.

Biomimetic Conditioning of Human Dentin Using Citric Acid.

INTRODUCTION: In carious teeth, transforming growth factor beta 1 (TGF-ß1) is released from the dentin matrix and possibly activated in an acidic environment. Conversely, EDTA solutions with a neutral to slightly alkaline pH are used in clinics to promote cell homing in regenerative endodontic procedures. We hypothesized that citric acid (CA) might be more beneficial. METHODS: TGF-ß1 release from human dentin disks conditioned with either 10% CA (pH = 2) or 17% EDTA (pH = 8) and the behavior of human stem cells toward such pretreated dentin were studied. The protein concentration in conditioning solutions after 10 minutes of dentin exposure was determined using a pH-independent slot blot technique. RESULTS: There was a 5-fold higher concentration of the target protein in CA (382 ± 30 ng/disk) compared with EDTA (66 ± 3 ng/disk, P < .005). Using confocal laser scanning microscopy on immunofluorescent-labeled disks, we identified a high density of TGF-ß1 in peritubular dentin after CA treatment. A migration assay showed that CA conditioning attracted significantly more stem cells toward the dentin after 24 hours compared with EDTA (P < .05) or phosphate-buffered saline (P < .005). To investigate whether the cell response to these dentin surfaces could be affected by different pretreatments, we cultured stem cells on conditioned dentin disks and found that CA had a significantly (P < .05) better effect than EDTA on cell attachment and cell survival. CONCLUSIONS: CA conditioning could be useful and may have significant benefits over current treatments.

Comparative study of NMP-preloaded and dip-loaded membranes for guided bone regeneration of rabbit cranial defects.

Guided bone regeneration (GBR) has been utilized for several decades for the healing of cranio-maxillofacial bone defects and, particularly in the dental field, by creating space with a barrier membrane to exclude soft tissue and encourage bone growth in the membrane-protected volume. Although the first membranes were non-resorbable, a new generation of GBR membranes aims to biodegrade and provide bioactivity for better overall results. The Inion GTR™ poly(lactide-co-glycolide) (PLGA) membrane is not only resorbable but also bioactive, since it includes N-methylpyrrolidone (NMP), which has been shown to promote bone regeneration. In this study, the effects of loading different amounts of NMP onto the membrane through chemical vapour deposition or dipping have been explored. In vitro release demonstrated that lower levels of NMP led to lower NMP concentrations and slower release, based on total NMP loaded in the membrane. The dipped membrane released almost all of the NMP within 15 min, leading to a high NMP concentration. For the in vivo studies in rabbits, 6 mm calvarial defects were created and left untreated or covered with an ePTFE membrane or PLGA membranes dipped in, or preloaded with, NMP. Evaluation of the bony regeneration revealed that the barrier membranes improved bony healing and that a decrease in NMP content improved the performance. Overall, we have demonstrated the potential of these PLGA membranes with a more favourable NMP release profile and the significance of exploring the effect of NMP on these PLGA membranes with regard to bone ingrowth. Copyright © 2014 John Wiley & Sons, Ltd.

Effects of Stem Cell Factor on Cell Homing During Functional Pulp Regeneration in Human Immature Teeth.

Conventional root canal treatment in immature permanent teeth can lead to early tooth loss in children because root formation is discontinued. We investigated whether the stem cell factor (SCF) could facilitate cell homing in the pulpless immature root canal and promote regeneration of a functional pulp. In vitro, human mesenchymal stem cells (hMSCs) were exposed to SCF at various concentrations for assessing cell migration, proliferation, and differentiation toward odonto/osteoblasts by 3D-chemotaxis slides, WST-1 assay, and alkaline phosphatase activity, respectively. Fibrin gels were used to deliver 15 µg/mL SCF for in vivo experiments. The release kinetic of SCF was assessed in vitro. Two corresponding human immature premolars, with or without SCF, were placed at rat calvariae for 6 and 12 weeks. All tooth specimens were either analyzed histologically and the percentage of tissue ingrowth determined or the cells were extracted from the pulp space, and the mRNA level of DMP1, DSPP, Col1, NGF, and VEGF were assessed by quantitative polymerase chain reaction. In the presence of SCF, we saw an increase in hMSCs directional migration, proliferation, and odonto/osteogenic differentiation. SCF also increased the extent of tissue ingrowth at 6 weeks but not at 12 weeks. However, at this time point, the formed tissue appeared more mature in samples with SCF. In terms of gene transcription, DMP1, Col1, and VEGF were the significantly upregulated genes, while DSPP and NGF were not affected. Our results suggest that SCF can accelerate cell homing and the maturation of the pulp-dentin complex in human immature teeth.

Modular poly(ethylene glycol) matrices for the controlled 3D-localized osteogenic differentiation of mesenchymal stem cells.

The in vitro formation of physiologically relevant engineered tissues is still limited by the availability of adequate growth-factor-presenting cell-instructive biomaterials, allowing simultaneous and three-dimensionally localized differentiation of multiple tissue progenitor cells. Together with ever improving technologies such as microfluidics, printing, or lithography, these biomaterials could provide the basis for generating provisional cellular constructs, which can differentiate to form tissue mimetics. Although state-of-the-art biomaterials are endowed with sophisticated modules for time- and space-controlled positioning and release of bioactive molecules, reports on 3D arrangements of differentiation-inducing growth factors are scarce. This paper describes the stable and localized immobilization of biotinylated bioactive molecules to a modular, Factor XIII-cross-linked poly(ethylene glycol) hydrogel platform using a genetically engineered streptavidin linker. Linker incorporation is demonstrated by Western blot, and streptavidin functionality is confirmed by capturing biotinylated alkaline phosphatase (ALP). After optimizing bone morphogenetic protein 2 (BMP-2) biotinylation, streptavidin-modified hydrogels are able to bind and present bioactive BMP-2-biotin. Finally, with this immobilization scheme for BMP-2, the specific osteogenic differentiation of mesenchymal stem cells is demonstrated by inducing ALP expression in confined 3D areas. In future, this platform together with other affinity-based strategies will be useful for the local incorporation of various growth factors for engineering cell-responsive constructs.

Delivery of BMP-2 by two clinically available apatite materials: in vitro and in vivo comparison.

Bone morphogenetic proteins (BMPs) are deposited in bone and responsible for osteoinduction. The interplay between delivery system and BMP, resulting in a characteristic release profile, is crucial for clinical success. We here report on two apatite based commercially available granules which could potentially be used in a combination product with recombinant human BMP-2 (rhBMP-2). Regardless of their similar chemistry, their interaction with rhBMP-2 differs. Deproteinized bovine bone matrix (DBBM), a clinically well-established bone substitute, has a high affinity to rhBMP-2 and releases only 50% of the growth factor during the first 2 weeks in vitro. Activity of the physio-adsorbed rhBMP-2 is indicated by an enhanced bone augmentation in vivo. In contrast, all rhBMP-2 delivered in combination with synthetic hydroxyapatite/ß-tricalcium phosphate (HA/TCP) granules is released during the first 24 h. For both HA/TCP and DBBM, the released rhBMP-2 is active in vitro. Our results suggest that the different release behavior from these two apatite granules is due to the 1000-fold higher specific surface area of DBBM compared to HA/TCP.

N-methyl pyrrolidone/bone morphogenetic protein-2 double delivery with in situ forming implants.

Bone morphogenetic proteins (BMPs) are growth and differentiation factors involved during development in morphogenesis, organogenesis and later mainly in regeneration processes, in particular in bone where they are responsible for osteoinduction. For more than a decade, recombinant human (rh)BMP-2 has been used in the clinic for lumbar spinal fusion at non-physiological high dosages that appear to be causative for side effects, like male sterility. A possible strategy to reduce the effective amount of rhBMP-2 in the clinic is the co-delivery with an enhancer of BMPs' activity. In an earlier study, we showed that N-methylpyrrolidone (NMP) enhances BMP activity in vitro and in vivo. Here we report on the development of a slow and sustained double delivery of rhBMP-2 and NMP via an in situ forming implant based on poly(lactide-co-glycolide). The results showed that the release of NMP can be adjusted by varying the lactide/glycolide ratio and the polymer's molecular weight. The same applied to rhBMP-2, with release rates that could be sustained from two to three weeks. In the in vivo model of a critical size defect in the calvarial bone of rabbits, the implant containing 50mol% lactide performed better than the one having 75mol% lactide in terms of defect bridging and extent of bony regenerated area. In situ forming implants for the double delivery of the BMP enhancer NMP and rhBMP-2 appear to be promising delivery systems in bone regeneration.