In vitro evaluation of shape-memory hydrogels for removable dental prostheses and optimization of phase-transition temperature for intraoral use.

STATEMENT OF PROBLEM: Removable dental prostheses require periodic relining with the loss of intaglio surface fit because of mucosal shape changes over time. Therefore, a new material with high adaptability to tissue changes over time would be beneficial. PURPOSE: This study focused on a shape-memory gel (SMG) that softens when heated, retains its shape when cooled, and returns to its original shape when reheated. The purpose was to optimize SMG for intraoral use by controlling the ratio of 2 acrylate monomers and to evaluate the changes in the shape memory and physical properties of SMG with temperature and to evaluate biocompatibility. MATERIAL AND METHODS: SMG specimens were synthesized using the following mixing ratios of 2 monomers, docosyl acrylate (DA) and stearyl acrylate (SA): 0:100, 25:75, 50:50, 75:25, and 100:0. SMG specimens were photopolymerized using a fluorescent light-polymerizing unit. To evaluate shape memory as a function of temperature, permanent deformation was measured based on the standardized compression set test for thermoplastic rubber. For evaluation of the physical properties and cytotoxicity, a 3-dimensionally printed denture base material was used as the control material. All assessments were compared between the groups by using 1-way analysis of variance followed by the Tukey-Kramer multiple comparison test (α=.05). RESULTS: SMGs with a higher amount of DA maintained their compressed shape at room and intraoral temperatures. However, the SMG matrices softened and recovered their original shapes above 60 °C. SMGs showed Shore A hardness equivalent to that of the denture-base polymer material at intraoral temperatures because of the high phase-transition temperature. The low water solubility of SMGs supported the biocompatibility test results. CONCLUSIONS: SMG, in which the phase-transition temperature was controlled by mixing acrylate monomers with different melting points, exhibited shape memory in the intraoral environment. The results indicate the feasibility of applying SMG for the fabrication of removable dental prostheses because of its high adaptability to tissue changes over time and biocompatibility.

Innate immune regulation in dental implant osseointegration.

PURPOSE: Dental implant osseointegration comprises two types of bone formation-contact and distance osteogenesis-which result in bone formation originating from the implant surface or bone edges, respectively. The physicochemical properties of the implant surface regulate initial contact osteogenesis by directly tuning the osteoprogenitor cells in the peri-implant environment. However, whether these implant surface properties can regulate osteoprogenitor cells distant from the implant remains unclear. Innate immune cells, including neutrophils and macrophages, govern bone metabolism, suggesting their involvement in osseointegration and distance osteogenesis. This narrative review discusses the role of innate immunity in osseointegration and the effects of implant surface properties on distant osteogenesis, focusing on innate immune regulation. STUDY SELECTION: The role of innate immunity in bone formation and the effects of implant surface properties on innate immune function were reviewed based on clinical, animal, and in vitro studies. RESULTS: Neutrophils and macrophages are responsible for bone formation during osseointegration, via inflammatory mediators. The microroughness and hydrophilic status of titanium implants have the potential to alleviate this inflammatory response of neutrophils, and induce an anti-inflammatory response in macrophages, to tune both contact and distance osteogenesis through the activation of osteoblasts. Thus, the surface micro-roughness and hydrophilicity of implants can regulate the function of distant osteoprogenitor cells through innate immune cells. CONCLUSIONS: Surface modification of implants aimed at regulating innate immunity may be useful in promoting further osteogenesis and overcoming the limitations encountered in severe situations, such as early loading protocol application.

Mouse gingival single-cell transcriptomic atlas identified a novel fibroblast subpopulation activated to guide oral barrier immunity in periodontitis.

Periodontitis, one of the most common non-communicable diseases, is characterized by chronic oral inflammation and uncontrolled tooth supporting alveolar bone resorption. Its underlying mechanism to initiate aberrant oral barrier immunity has yet to be delineated. Here, we report a unique fibroblast subpopulation activated to guide oral inflammation (AG fibroblasts) identified in a single-cell RNA sequencing gingival cell atlas constructed from the mouse periodontitis models. AG fibroblasts localized beneath the gingival epithelium and in the cervical periodontal ligament responded to the ligature placement and to the discrete topical application of Toll-like receptor stimulants to mouse maxillary tissue. The upregulated chemokines and ligands of AG fibroblasts linked to the putative receptors of neutrophils in the early stages of periodontitis. In the established chronic inflammation, neutrophils, together with AG fibroblasts, appeared to induce type 3 innate lymphoid cells (ILC3s) that were the primary source of interleukin-17 cytokines. The comparative analysis of Rag2-/- and Rag2-/-Il2rg-/- mice suggested that ILC3 contributed to the cervical alveolar bone resorption interfacing the gingival inflammation. We propose the AG fibroblast-neutrophil-ILC3 axis as a previously unrecognized mechanism which could be involved in the complex interplay between oral barrier immune cells contributing to pathological inflammation in periodontitis.

Mouse gingival single-cell transcriptomic atlas: An activated fibroblast subpopulation guides oral barrier immunity in periodontitis.

Periodontitis, one of the most common non-communicable diseases, is characterized by chronic oral inflammation and uncontrolled tooth supporting alveolar bone resorption. Its underlying mechanism to initiate aberrant oral barrier immunity has yet to be delineated. Here, we report a unique fibroblast subpopulation activated to guide oral inflammation (AG fibroblasts) identified in a single-cell RNA sequencing gingival cell atlas constructed from the mouse periodontitis models. AG fibroblasts localized beneath the gingival epithelium and in the cervical periodontal ligament responded to the ligature placement and to the discrete application of Toll-like receptor stimulants to mouse maxillary tissue. The upregulated chemokines and ligands of AG fibroblasts linked to the putative receptors of neutrophils in the early stages of periodontitis. In the established chronic inflammation, neutrophils together with AG fibroblasts appeared to induce type 3 innate lymphoid cells (ILC3s) that were the primary source of interleukin-17 cytokines. The comparative analysis of Rag2-/- and Rag2γc-/- mice suggested that ILC3 contributed to the cervical alveolar bone resorption interfacing the gingival inflammation. We propose that AG fibroblasts function as a previously unrecognized surveillant to initiate gingival inflammation leading to periodontitis through the AG fibroblast-neutrophil-ILC3 axis.

Incomplete Polymerization of Dual-Cured Resin Cement Due to Attenuated Light through Zirconia Induces Inflammatory Responses.

Zirconia restorations are becoming increasingly common. However, zirconia reduces the polymerization of dual-cured resin cement owing to light attenuation, resulting in residual resin monomers. This study investigated the effects of dual-cured resin cement, with incomplete polymerization owing to attenuated light through zirconia, on the inflammatory response in vitro. The dual-cured resin cement (SA Luting Multi, Kuraray) was light-irradiated through zirconia with three thickness diameters (1.0, 1.5, and 2.0 mm). The light transmittance and the degree of conversion (DC) of the resin cement significantly decreased with increasing zirconia thickness. The dual-cured resin cement in 1.5 mm and 2.0 mm zirconia and no-irradiation groups showed significantly higher amounts of hydroxyethylmethacrylate and triethyleneglycol dimethacrylate elution and upregulated gene expression of proinflammatory cytokines IL-1ß and IL-6 from human gingival fibroblasts (hGFs) and TNFα from human monocytic cells, compared with that of the 0 mm group. Dual-cured resin cement with lower DC enhanced intracellular reactive oxygen species (ROS) levels and activated mitogen-activated protein (MAP) kinases in hGFs and monocytic cells. This study suggests that dual-cured resin cement with incomplete polymerization induces inflammatory responses in hGFs and monocytic cells by intracellular ROS generation and MAP kinase activation.

Cyclic pressure-induced cytokines from gingival fibroblasts stimulate osteoclast activity: Clinical implications for alveolar bone loss in denture wearers.

Purpose The involvement of oral mucosa cells in mechanical stress-induced bone resorption is unclear. The aim of this study was to investigate the effects of cyclic pressure-induced cytokines from oral mucosal cells (human gingival fibroblasts: hGFs) on osteoclast activity in vitro.Methods Cyclic pressure at 50 kPa, which represents high physiologic occlusal force of dentures on the molar area, was applied to hGFs. NFAT-reporter stable RAW264.7 preosteoclasts (NFAT/Luc-RAW cells) were cultured in conditioned medium collected from hGF cultures under cyclic pressure or static conditions. NFAT activity and osteoclast formation were determined by luciferase reporter assay and TRAP staining, respectively. Cyclic pressure-induced cytokines in hGF culture were detected by ELISA, real-time RT-PCR, and cytokine array analyses.Results Conditioned media from hGFs treated with 48 hours of cyclic pressure significantly induced NFAT activity and increased multinucleated osteoclast formation. Furthermore, the cyclic pressure significantly increased the bone resorption activity of RAW264.7 cells. Cyclic pressure significantly increased the expression of major inflammatory cytokines including IL-1ß/IL-1ß, IL-6/IL-6, IL-8/IL-8 and MCP-1/CCL2 in hGFs compared to hGFs cultured under static conditions, and it suppressed osteoprotegerin (OPG/OPG) expression. A cytokine array detected 12 cyclic pressure-induced candidates. Among them, IL-8, decorin, MCP-1 and ferritin increased, whereas IL-28A and PDGF-BB decreased, NFAT activation of NFAT/Luc-RAW cells.Conclusions These results suggest that cyclic pressure-induced cytokines from hGFs promote osteoclastogenesis, possibly including up-regulation of IL-1ß, IL-6, IL-8 and MCP-1, and down-regulation of OPG. These findings introduce the possible involvement of GFs in mechanical stress-induced alveolar ridge resorption, such as in denture wearers.

Current perspectives of residual ridge resorption: Pathological activation of oral barrier osteoclasts.

PURPOSE: Tooth extraction is a last resort treatment for resolving pathological complications of dentition induced by infection and injury. Although the extraction wound generally heals uneventfully, resulting in the formation of an edentulous residual ridge, some patients experience long-term and severe residual ridge reduction. The objective of this review was to provide a contemporary understanding of the molecular and cellular mechanisms that may potentially cause edentulous jawbone resorption. STUDY SELECTION: Clinical, in vivo, and in vitro studies related to the characterization of and cellular and molecular mechanisms leading to residual ridge resorption. RESULTS: The alveolar processes of the maxillary and mandibular bones uniquely juxtapose the gingival tissue. The gingival oral mucosa is an active barrier tissue that maintains homeostasis of the internal organs through its unique barrier immunity. Tooth extraction not only generates a bony socket but also injures oral barrier tissue. In response to wounding, the alveolar bone socket initiates regeneration and remodeling through coupled bone formation and osteoclastic resorption. Osteoclasts are also found on the external surface of the alveolar bone, interfacing the oral barrier tissue. Osteoclasts in the oral barrier region are not coupled with osteoblastic bone formation and often remain active long after the completion of wound healing, leading to a net decrease in the alveolar bone structure. CONCLUSIONS: The novel concept of oral barrier osteoclasts may provide important clues for future clinical strategies to maintain residual ridges for successful prosthodontic and restorative therapies.

Oral microbial extracellular DNA initiates periodontitis through gingival degradation by fibroblast-derived cathepsin K in mice.

Periodontitis is a highly prevalent disease leading to uncontrolled osteoclastic jawbone resorption and ultimately edentulism; however, the disease onset mechanism has not been fully elucidated. Here we propose a mechanism for initial pathology based on results obtained using a recently developed Osteoadsorptive Fluogenic Sentinel (OFS) probe that emits a fluorescent signal triggered by cathepsin K (Ctsk) activity. In a ligature-induced mouse model of periodontitis, a strong OFS signal is observed before the establishment of chronic inflammation and bone resorption. Single cell RNA sequencing shows gingival fibroblasts to be the primary cellular source of early Ctsk. The in vivo OFS signal is activated when Toll-Like Receptor 9 (TLR9) ligand or oral biofilm extracellular DNA (eDNA) is topically applied to the mouse palatal gingiva. This previously unrecognized interaction between oral microbial eDNA and Ctsk of gingival fibroblasts provides a pathological mechanism for disease initiation and a strategic basis for early diagnosis and treatment of periodontitis.

Fluorescent risedronate analogue 800CW-pRIS improves tooth extraction-associated abnormal wound healing in zoledronate-treated mice.

Background: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a rare but serious side effect of nitrogen-containing bisphosphonate drugs (N-BPs) frequently prescribed to reduce skeletal-related events in bone malignancies and osteoporosis. BRONJ is associated with abnormal oral wound healing after dentoalveolar surgery and tooth extraction. We previously found that N-BP chemisorbed to bone mineral hydroxyapatite was dissociated by secondary applied N-BP. This study investigated the effect of the surface equilibrium-based removal of N-BP from jawbone on tooth extraction wound healing of zoledronate (ZOL)-treated mice. Methods: A pharmacologically inactive N-BP derivative (the 4-pyridyl isomer of risedronate equipped with a near-infrared 800CW fluorescent imaging dye, 800CW-pRIS) was designed and synthesized. 800CW-pRIS was intra-orally injected or topically applied in a deformable nano-scale vesicle formulation (DNV) to the palatal tissue of mice pretreated with ZOL, a potent N-BP. The female C56BL6/J mice were subjected to maxillary molar extraction and oral wound healing was compared for 800CW-pRIS/ZOL, ZOL and untreated control groups. Results: 800CW-pRIS is confirmed to be inactive in inhibiting prenylation in cultured osteoclasts while retaining high affinity for hydroxyapatite. ZOL-injected mice exhibit delayed tooth extraction wound healing with osteonecrosis relative to the untreated controls. 800CW-pRIS applied topically to the jaw one week before tooth extraction significantly reduces gingival oral barrier inflammation, improves extraction socket bone regeneration, and prevents development of osteonecrosis in ZOL-injected mice. Conclusions: Topical pre-treatment with 800CW-RIS in DNV is a promising approach to prevent the complication of abnormal oral wound healing associated with BRONJ while retaining the anti-resorptive benefit of legacy N-BP in appendicular or vertebrate bones.

Effect of Calcium Ion Supplementation on Oral Microbial Composition and Biofilm Formation In Vitro.

The oral cavity contains a variety of ecological niches with very different environmental conditions that shape biofilm structure and composition. The space between the periodontal tissue and the tooth surface supports a unique anaerobic microenvironment that is bathed in the nutrient-rich gingival crevicular fluid (GCF). During the development of periodontitis, this environment changes and clinical findings reported a sustained level of calcium ion concentration in the GCF collected from the periodontal pockets of periodontitis patients. Here, we report the effect of calcium ion supplementation on human oral microbial biofilm formation and community composition employing an established SHI medium-based in vitro model system. Saliva-derived human microbial biofilms cultured in calcium-supplemented SHI medium (SHICa) exhibited a significant dose-dependent increase in biomass and metabolic activity. The effect of SHICa medium on the microbial community composition was evaluated by 16S rRNA gene sequencing using saliva-derived microbial biofilms from healthy donors and periodontitis subjects. In this study, intracellular microbial genomic DNA (iDNA) and extracellular DNA (eDNA) were analyzed separately at the genus level. Calcium supplementation of SHI medium had a differential impact on iDNA and eDNA in the biofilms derived from healthy individuals compared to those from periodontitis subjects. In particular, the genus-level composition of the eDNA portion was distinct between the different biofilms. This study demonstrated the effect of calcium in a unique microenvironment on oral microbial complex supporting the dynamic transformation and biofilm formation.

Mechanism of bisphosphonate-related osteonecrosis of the jaw (BRONJ) revealed by targeted removal of legacy bisphosphonate from jawbone using competing inert hydroxymethylene diphosphonate.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) presents as a morbid jawbone lesion in patients exposed to a nitrogen-containing bisphosphonate (N-BP). Although it is rare, BRONJ has caused apprehension among patients and healthcare providers and decreased acceptance of this antiresorptive drug class to treat osteoporosis and metastatic osteolysis. We report here a novel method to elucidate the pathological mechanism of BRONJ by the selective removal of legacy N-BP from the jawbone using an intra-oral application of hydroxymethylene diphosphonate (HMDP) formulated in liposome-based deformable nanoscale vesicles (DNV). After maxillary tooth extraction, zoledronate-treated mice developed delayed gingival wound closure, delayed tooth extraction socket healing and increased jawbone osteonecrosis consistent with human BRONJ lesions. Single cell RNA sequencing of mouse gingival cells revealed oral barrier immune dysregulation and unresolved proinflammatory reaction. HMDP-DNV topical applications to nascent mouse BRONJ lesions resulted in accelerated gingival wound closure and bone socket healing as well as attenuation of osteonecrosis development. The gingival single cell RNA sequencing demonstrated resolution of chronic inflammation by increased anti-inflammatory signature gene expression of lymphocytes and myeloid-derived suppressor cells. This study suggests that BRONJ pathology is related to N-BP levels in jawbones and demonstrates the potential of HMDP-DNV as an effective BRONJ therapy.

Rapid and efficient generation of cartilage pellets from mouse induced pluripotent stem cells by transcriptional activation of BMP-4 with shaking culture.

Induced pluripotent stem cells (iPSCs) offer an unlimited source for cartilage regeneration as they can generate a wide spectrum of cell types. Here, we established a tetracycline (tet) controlled bone morphogenetic protein-4 (BMP-4) expressing iPSC (iPSC-Tet/BMP-4) line in which transcriptional activation of BMP-4 was associated with enhanced chondrogenesis. Moreover, we developed an efficient and simple approach for directly guiding iPSC-Tet/BMP-4 differentiation into chondrocytes in scaffold-free cartilaginous pellets using a combination of transcriptional activation of BMP-4 and a 3D shaking suspension culture system. In chondrogenic induction medium, shaking culture alone significantly upregulated the chondrogenic markers Sox9, Col2a1, and Aggrecan in iPSCs-Tet/BMP-4 by day 21. Of note, transcriptional activation of BMP-4 by addition of tet (doxycycline) greatly enhanced the expression of these genes. The cartilaginous pellets derived from iPSCs-Tet/BMP-4 showed an oval morphology and white smooth appearance by day 21. After day 21, the cells presented a typical round morphology and the extracellular matrix was stained intensively with Safranin O, alcian blue, and type II collagen. In addition, the homogenous cartilaginous pellets derived from iPSCs-Tet/BMP-4 with 28 days of induction repaired joint osteochondral defects in immunosuppressed rats and integrated well with the adjacent host cartilage. The regenerated cartilage expressed the neomycin resistance gene, indicating that the newly formed cartilage was generated by the transplanted iPSCs-Tet/BMP-4. Thus, our culture system could be a useful tool for further investigation of the mechanism of BMP-4 in regulating iPSC differentiation toward the chondrogenic lineage, and should facilitate research in cartilage development, repair, and osteoarthritis.

Therapeutic downregulation of neuronal PAS domain 2 (Npas2) promotes surgical skin wound healing.

Attempts to minimize scarring remain among the most difficult challenges facing surgeons, despite the use of optimal wound closure techniques. Previously, we reported improved healing of dermal excisional wounds in circadian clock neuronal PAS domain 2 (Npas2)-null mice. In this study, we performed high-throughput drug screening to identify a compound that downregulates Npas2 activity. The hit compound (Dwn1) suppressed circadian Npas2 expression, increased murine dermal fibroblast cell migration, and decreased collagen synthesis in vitro. Based on the in vitro results, Dwn1 was topically applied to iatrogenic full-thickness dorsal cutaneous wounds in a murine model. The Dwn1-treated dermal wounds healed faster with favorable mechanical strength and developed less granulation tissue than the controls. The expression of type I collagen, Tgfß1, and α-smooth muscle actin was significantly decreased in Dwn1-treated wounds, suggesting that hypertrophic scarring and myofibroblast differentiation are attenuated by Dwn1 treatment. NPAS2 may represent an important target for therapeutic approaches to optimal surgical wound management.

In vitro assessment of Neuronal PAS domain 2 mitigating compounds for scarless wound healing.

Background: The core circadian gene Neuronal PAS domain 2 (NPAS2) is expressed in dermal fibroblasts and has been shown to play a critical role in regulating collagen synthesis during wound healing. We have performed high throughput drug screening to identify genes responsible for downregulation of Npas2 while maintaining cell viability. From this, five FDA-approved hit compounds were shown to suppress Npas2 expression in fibroblasts. In this study, we hypothesize that the therapeutic suppression of Npas2 by hit compounds will have two effects: (1) attenuated excessive collagen deposition and (2) accelerated dermal wound healing without hypertrophic scarring. Materials and methods: To test the effects of each hit compound (named Dwn1, 2, 3, 4, and 5), primary adult human dermal fibroblasts (HDFa) were treated with either 0, 0.1, 1, or 10 µM of a single hit compound. HDFa behaviors were assessed by picrosirius red staining and quantitative RT-PCR for in vitro collagen synthesis, cell viability assay, in vitro fibroblast-to-myofibroblast differentiation test, and cell migration assays. Results: Dwn1 and Dwn2 were found to significantly affect collagen synthesis and cell migration without any cytotoxicity. Dwn3, Dwn4, and Dwn5 did not affect collagen synthesis and were thereby eliminated from further consideration for their role in mitigation of gene expression or myofibroblast differentiation. Dwn1 also attenuated myofibroblast differentiation on HDFa. Conclusion: Dwn1 and Dwn2 may serve as possible therapeutic agents for future studies related to skin wound healing.

Point-of-care antimicrobial coating protects orthopaedic implants from bacterial challenge.

Implant related infections are the most common cause of joint arthroplasty failure, requiring revision surgeries and a new implant, resulting in a cost of $8.6 billion annually. To address this problem, we created a class of coating technology that is applied in the operating room, in a procedure that takes less than 10 min, and can incorporate any desired antibiotic. Our coating technology uses an in situ coupling reaction of branched poly(ethylene glycol) and poly(allyl mercaptan) (PEG-PAM) polymers to generate an amphiphilic polymeric coating. We show in vivo efficacy in preventing implant infection in both post-arthroplasty infection and post-spinal surgery infection mouse models. Our technology displays efficacy with or without systemic antibiotics, the standard of care. Our coating technology is applied in a clinically relevant time frame, does not require modification of implant manufacturing process, and does not change the implant shelf life.

Shaking culture enhances chondrogenic differentiation of mouse induced pluripotent stem cell constructs.

Mechanical loading on articular cartilage induces various mechanical stresses and strains. In vitro hydrodynamic forces such as compression, shear and tension impact various cellular properties including chondrogenic differentiation, leading us to hypothesize that shaking culture might affect the chondrogenic induction of induced pluripotent stem cell (iPSC) constructs. Three-dimensional mouse iPSC constructs were fabricated in a day using U-bottom 96-well plates, and were subjected to preliminary chondrogenic induction for 3 days in static condition, followed by chondrogenic induction culture using a see-saw shaker for 17 days. After 21 days, chondrogenically induced iPSC (CI-iPSC) constructs contained chondrocyte-like cells with abundant ECM components. Shaking culture significantly promoted cell aggregation, and induced significantly higher expression of chondrogenic-related marker genes than static culture at day 21. Immunohistochemical analysis also revealed higher chondrogenic protein expression. Furthemore, in the shaking groups, CI-iPSCs showed upregulation of TGF-ß and Wnt signaling-related genes, which are known to play an important role in regulating cartilage development. These results suggest that shaking culture activates TGF-ß expression and Wnt signaling to promote chondrogenic differentiation in mouse iPSCs in vitro. Shaking culture, a simple and convenient approach, could provide a promising strategy for iPSC-based cartilage bioengineering for study of disease mechanisms and new therapies.

Size-Optimized Microspace Culture Facilitates Differentiation of Mouse Induced Pluripotent Stem Cells into Osteoid-Rich Bone Constructs.

Microspace culture is promising for self-organization of induced pluripotent stem cells (iPSCs). However, the optimal size of microspaces for osteogenic differentiation is unclear. We hypothesized that a specific microspace size could facilitate self-organizing iPSC differentiation to form bone-like tissue in vitro. The objectives of this study were to investigate such effects of microspace size and to evaluate bone regeneration upon transplantation of the resulting osteogenic constructs. Dissociated mouse gingival fibroblast-derived iPSCs were plated in ultra-low-attachment microspace culture wells containing hundreds of U-bottom-shaped microwell spots per well to form cell aggregates in growth medium. The microwells had different aperture diameters/depths (400/560 µm (Elp400), 500/700 µm (Elp500), and 900/700 µm (Elp900)) (Kuraray; Elplasia). After 5 days of aggregation, cells were maintained in osteogenic induction medium for 35 days. Only cells in the Elp500 condition tightly aggregated and maintained high viability during osteogenic induction. After 10 days of induction, Elp500 cell constructs showed significantly higher gene expression of Runx2, Osterix, Collagen 1a1, Osteocalcin, Bone sialoprotein, and Osteopontin compared to constructs in Elp400 and Elp900. In methylene blue-counterstained von Kossa staining and Movat's pentachrome staining, only Elp500 constructs showed robust osteoid formation on day 35, with high expression of type I collagen (a major osteoid component) and osteocalcin proteins. Cell constructs were transplanted into rat calvarial bone defects, and micro-CT analysis after 3 weeks showed better bone repair with significantly higher bone mineral density in the Elp500 group compared to the Elp900 group. These results suggest that microspace size affects self-organized osteogenic differentiation of iPSCs. Elp500 microspace culture specifically induces mouse iPSCs into osteoid-rich bone-like tissue possessing high bone regeneration capacity.

In Vitro Fabrication of Hybrid Bone/Cartilage Complex Using Mouse Induced Pluripotent Stem Cells.

Cell condensation and mechanical stimuli play roles in osteogenesis and chondrogenesis; thus, they are promising for facilitating self-organizing bone/cartilage tissue formation in vitro from induced pluripotent stem cells (iPSCs). Here, single mouse iPSCs were first seeded in micro-space culture plates to form 3-dimensional spheres. At day 12, iPSC spheres were subjected to shaking culture and maintained in osteogenic induction medium for 31 days (Os induction). In another condition, the osteogenic induction medium was replaced by chondrogenic induction medium at day 22 and maintained for a further 21 days (Os-Chon induction). Os induction produced robust mineralization and some cartilage-like tissue, which promoted expression of osteogenic and chondrogenic marker genes. In contrast, Os-Chon induction resulted in partial mineralization and a large area of cartilage tissue, with greatly increased expression of chondrogenic marker genes along with osterix and collagen 1a1. Os-Chon induction enhanced mesodermal lineage commitment with brachyury expression followed by high expression of lateral plate and paraxial mesoderm marker genes. These results suggest that combined use of micro-space culture and mechanical stimuli facilitates hybrid bone/cartilage tissue formation from iPSCs, and that the bone/cartilage tissue ratio in iPSC constructs could be manipulated through the induction protocol.

Preconditioning of bone marrow-derived mesenchymal stem cells with N-acetyl-L-cysteine enhances bone regeneration via reinforced resistance to oxidative stress.

Oxidative stress on transplanted bone marrow-derived mesenchymal stem cells (BMSCs) during acute inflammation is a critical issue in cell therapies. N-acetyl-L cysteine (NAC) promotes the production of a cellular antioxidant molecule, glutathione (GSH). The aim of this study was to investigate the effects of pre-treatment with NAC on the apoptosis resistance and bone regeneration capability of BMSCs. Rat femur-derived BMSCs were treated in growth medium with or without 5 mM NAC for 6 h, followed by exposure to 100 µM H2O2 for 24 h to induce oxidative stress. Pre-treatment with NAC significantly increased intracellular GSH levels by up to two fold and prevented H2O2-induced intracellular redox imbalance, apoptosis and senescence. When critical-sized rat femur defects were filled with a collagen sponge containing fluorescent-labeled autologous BMSCs with or without NAC treatment, the number of apoptotic and surviving cells in the transplanted site after 3 days was significantly lower and higher in the NAC pre-treated group, respectively. By the 5th week, significantly enhanced new bone formation was observed in the NAC pre-treated group. These data suggest that pre-treatment of BMSCs with NAC before local transplantation enhances bone regeneration via reinforced resistance to oxidative stress-induced apoptosis at the transplanted site.

Binding of PICK1 PDZ domain with calcineurin B regulates osteoclast differentiation.

The calcineurin/nuclear factor of activated T cell (NFAT) signaling pathway plays a major role in osteoclast differentiation; however, the proteins that react with the calcineurin-NFAT complex in osteoclasts to regulate osteoclastogenesis remain unclear. Here, we present evidence that PICK1 also positively regulates calcineurin B in osteoclasts to activate NFAT to promote osteoclastogenesis. mRNA and protein expression of PICK1 in murine primary bone marrow macrophages (BMMs) was significantly increased during RANKL-induced osteoclast differentiation. The interaction of PICK1 with calcineurin B in BMMs was confirmed by co-immunoprecipitation. An inhibitor of the PICK1 PDZ domain significantly decreased osteoclastogenesis marker gene expression and the number of TRAP-positive multinucleated cells among RAW264.7 osteoclast progenitor cells. Overexpression of PICK1 in RAW264.7 cells significantly increased the number of TRAP-positive mature osteoclasts. Increased NFAT activation with transcriptional activation of PICK1 during RAW264.7 osteoclastogenesis was also confirmed in a tetracycline-controlled PICK1 expression system. These results suggest that the PDZ domain of PICK1 directly interacts with calcineurin B in osteoclast progenitor cells and promotes osteoclast differentiation through activation of calcineurin-NFAT signaling.