Enhancing osteoblast differentiation through small molecule-incorporated engineered nanofibrous scaffold.

OBJECTIVE: This study aimed to investigate the effect of small molecules incorporated into the engineered nanofibrous scaffold to enhance the osteoblast differentiation MATERIALS AND METHODS: Poly-ε-caprolactone (PCL) nanofiber matrices with lithium chloride (LiCl) were fabricated using the electrospinning technique. Scaffolds were characterized using scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX). Scaffolds were seeded with MC3T3-E1 cells and assessed using Western blots (ß-catenin), alamarBlue assay (proliferation), qPCR (osteoblast differentiation), and mineralization (Alizarin Red staining). RESULTS: We observed LiCl nanofiber scaffolds induced concentration-dependent cell proliferation that correlated with an increased ß-catenin expression indicating sustained Wnt signaling. Next, we examined osteoblast differentiation markers such as osteocalcin (OCN) and Runt-related transcription factor 2 (Runx2) and noted increased expression in LiCl nanofiber scaffolds. We also noted increased bone morphogenetic protein (BMP-2, 4, and 7) expressions suggesting activated Wnt can promote cures to further osteogenic differentiation. Finally, Alizarin Red staining demonstrated increased mineral deposition in LiCl-incorporated nanofiber scaffolds. CONCLUSIONS: Together, these results indicated that LiCl-incorporated nanofiber scaffolds enhance osteoblast differentiation. CLINICAL RELEVANCE: Small molecule-incorporated nanofibrous scaffolds are an innovative clinical tool for bone tissue engineering.

Fibrin-Based Biomaterial Applications in Tissue Engineering and Regenerative Medicine.

The fibrin matrix is fundamentally formed by the polymerization of fibrinogen and thrombin in blood plasma. It is a natural biopolymeric material to widely investigate for various tissue regenerations due to good biocompatibility, rapid biodegradability, and easy fabrication. In particular, the conjugated bioactive molecules with fibrinogen can promote tissue morphogenesis or maturation after cell adhesion on the matrices, migration, proliferation, or differentiation. Using these physiological properties with cell-material interactions, the fibrin matrices have been utilized in tissue engineering applications such as skin tissue, cardiovascular tissue, musculoskeletal tissue, or nerve tissue in preclinical and clinical situations. This chapter demonstrates the fibrin material and its tissue engineering applications as the therapeutic strategies.

Fibrin-Enhanced Canonical Wnt Signaling Directs Plasminogen Expression in Cementoblasts.

Cementum is a mineralized layer on the tooth's root surface and facilitates the biomechanical anchoring of fibrous connective tissues as a part of tooth-supportive complexes. Previously, we observed that OCCM30 cementoblasts cultured on fibrin matrices underwent apoptosis due to fibrin degradation through the expression of proteases. Here, we demonstrated that OCCM30 on fibrin matrices (OCCM30-fibrin) enhanced canonical Wnt signaling, which directed to plasminogen expression. The OCCM30-fibrin showed higher levels of Wnt3a expression, nuclear translocation of ß-catenin, and T-cell factor (TCF) optimal motif (TOP) reporter activity than the cells on tissue culture dishes (OCCM30-TCD), indicating that the OCCM30-fibrin enhanced canonical Wnt/ß-catenin signaling. Also, OCCM30-fibrin expressed biomineralization-associated markers at higher levels than OCCM30-TCD, of which levels were further increased with LiCl, a Wnt signaling activator. The OCCM30 cementoblasts simultaneously showed that high levels of plasminogen, a critical component of fibrinolysis, were expressed in the OCCM30-fibrin. Activation of canonical Wnt signaling with LiCl treatment or with forced lymphoid enhancer factor 1 (LEF1)-expression increased the expression of plasminogen. On the contrary, the inhibition of canonical Wnt signaling with siRNAs against Wnt3a or ß-catenin abrogated fibrin-enhanced plasminogen expression. Furthermore, there are three conserved putative response elements for the LEF1/ß-catenin complex in the plasminogen proximal promoter regions (-900 to +54). Site-directed mutations and chromatin immunoprecipitation indicated that canonical Wnt signaling directed plasminogen expression. Taken together, this study suggests that fibrin-based materials can modulate functional periodontal formations in controlling cementoblast differentiation and fibrin degradation.

Time-dependent periimplant bone reaction of acidic monomer-treated injection molded zirconia implants in rabbit tibiae.

PURPOSE: The aim of this study was to evaluate and compare the osseointegration of zirconia implants in rabbit tibiae with and without 10-methacryloyloxydecyl dihydrogen phosphate (MDP) treatment. MATERIALS AND METHODS: Twenty-eight rabbits received a total of 112 external hex-type implants made by the powder injection molding technique with and without MDP treatment before installation in the tibiae. The contact angles on the zirconia implants and zirconia discs before and after MDP application were evaluated. Removal torque (RT) and bone-implant contact (BIC) ratios were measured. RESULTS: The MDP treatment markedly enhanced the hydrophilicity and seemed to alter the topography and chemical composition of the implant and disc surface. As the healing time increases, the BIC and RT were increased in both groups. The MDP-treated implants exhibited higher BIC values than the control implants after 2 and 4 weeks of healing. The RT was higher in MDP-treated implants after 2 weeks of healing but not after 4 weeks of healing. CONCLUSION: The 10-MDP treatments made the surface more hydrophilic and enhanced the osseointegration of the implants in the early healing phase.

Tideglusib-incorporated nanofibrous scaffolds potently induce odontogenic differentiation.

Pulp-Dentin regeneration is a key aspect of maintain tooth vitality and enabling good oral-systemic health. This study aimed to investigate a nanofibrous scaffold loaded with a small molecule i.e. Tideglusib to promote odontogenic differentiation. Tideglusib (GSK-3ß inhibitor) interaction with GSK-3ß was determined using molecular docking and stabilization of ß-catenin was examined by confocal microscopy. 3D nanofibrous scaffolds were fabricated through electrospinning and their physicochemical characterizations were performed. Scaffolds were seeded with mesenchymal stem cells or pre-odontoblast cells to determine the cells proliferation and odontogenic differentiation. Our results showed that Tideglusib (TG) binds with GSK-3ß at Cys199 residue. Stabilization and nuclear translocation of ß-catenin was increased in the odontoblast cells treated with TG. SEM analysis revealed that nanofibers exhibited controlled architectural features that effectively mimicked the natural ECM. UV-Vis spectroscopy demonstrated that TG was incorporated successfully and released in a controlled manner. Both kinds of biomimetic nanofibrous matrices (PCLF-TG100, PCLF-TG1000) significantly stimulated cells proliferation. Furthermore, these scaffolds significantly induced dentinogenic markers (ALP, and DSPP) expression and biomineralization. In contrast to current pulp capping material driving dentin repair, the sophisticated, polymeric scaffold systems with soluble and insoluble spatiotemporal cues described here can direct stem cell differentiation and dentin regeneration. Hence, bioactive small molecule-incorporated nanofibrous scaffold suggests an innovative clinical tool for dentin tissue engineering.

Nanofibrous topography-driven altered responsiveness to Wnt5a mediates the three-dimensional polarization of odontoblasts.

Cell differentiation with the proper three-dimensional (3-D) structure is critical for cells to carry out their cellular functions in tissues. Odontoblasts derived from neural crest cells are elongated and polarized with the cell process, which is decisive for one directional tubular dentin formation. Here, we report that the fibrous topography of scaffolds directs odontoblast-lineage cells to differentiate to have the 3-D structure of odontoblasts through an altered responsiveness to Wnt family member 5A (Wnt5a). In a pulp-exposure animal model, the scaffolds with the nanofibrous topography supported the regeneration of tubular dentin with odontoblast processes. In cultures of pre-odontoblast cells, the nanofibrous topography heightened the cells on the z-axis. The cells on nanofibrous substrate (FIBER) formed stress fiber cytoskeletons on a conventional tissue culture plate (TCP). Differential activation of Cell division control protein 42 (Cdc42) on FIBER and Ras homolog family member A (RhoA) on TCP led to these differences. The signal from Wnt5a-Cdc42 in the cells on FIBER mediated the phosphorylation of JNK and the polarity growth signaling. Taken together, the nanofibrous topography of the scaffolds led to the 3-D structural differentiation of odontoblasts in vitro and in vivo, implying its application for dentin regeneration. Furthermore, the results on the altered activation of Cdc42 by Wnt5a on FIBER provide evidence that the topography of the scaffolds can cause a distinctive cell responsiveness to their micro-environments.

The canonical BMP signaling pathway plays a crucial part in stimulation of dentin sialophosphoprotein expression by BMP-2.

Dentin sialophosphoprotein (DSPP), a typical dentin-specific protein, is mainly expressed in the dentin extracellular matrix and plays a role in dentin mineralization. BMP-2 provides a strong signal for differentiation and mineralization of odontoblasts and osteoblasts. Previously, BMP-2 treatment is reported to stimulate Dspp expression in the MD10-F2 pre-odontoblast cells through activation of the heterotrimeric transcription factor Y (NF-Y). The canonical BMP signaling pathway is known to contribute greatly to biomineralization, however, it is not known whether it is involved in Dspp expression. Here, we investigated this question. Activation of the canonical BMP-2 signaling pathway in MDPC-23, preodontoblast cell, by overexpression of constitutively active Smad1/5 or downstream transcription factors Dlx5 and Runx2 stimulated Dspp expression. Conversely, knockdown of each element with siRNA significantly blocked the BMP-2-induced Dspp expression. To test whether these transcription factors downstream of BMP-2 are directly involved in regulating Dspp, we analyzed the mouse Dspp promoter. There are 5 well conserved homeodomain binding elements, H1 to H5, in Dspp proximal promoter regions (-791 to +54). A serial deletion of H1 and H2 greatly changed basal promoter activity and responsiveness to Dlx5 or Msx2. However, further deletions did not change the responsiveness to Dlx5 or Msx2. H1 and H2 sites can be suggested as specific response elements of Dlx5 and Msx2, respectively, based on their promoter activity modulation. Thus, the canonical BMP-2 signaling pathway plays a crucial part in the regulation of Dspp expression through the action of Smads, Dlx5, Runx2, and Msx2.

Physicochemical properties of dentinogenesis imperfecta with a known DSPP mutation.

AIM: To investigate the chemical and mechanical properties of teeth affected by a 1-bp deletion (c.2688delT) in the DSPP gene. METHODS AND MATERIALS: Maxillary first premolars were extracted from the affected individual at age 9 years due to the orthodontic reason for crowding. A sample was imbedded in epoxy resin and sectioned buccolingually, after micro-computerized tomography (µCT) images were taken. Scanning Electron Microscopy (SEM), Energy Dispersive Spectrometry (EDS) and Vickers microhardness testing were also performed. RESULTS: µCT reconstruction and analysis showed an irregularly obliterated pulp chamber and an extremely small pulpal volume in the DGI-II sample. The mineral density and microhardness scores were smaller in the dentin of the DGI-II sample compared to the wild-type. Mg content was lower in the dentin of the DGI-II sample compared to the wild-type. CONCLUSION: This study shows that dentin affected by a 1-bp deletion in DSPP has a reduced mineral density, diminished microhardness and reduced Mg content.

Effects of pentoxifylline and tocopherol on an osteoradionecrosis animal model.

PURPOSE: Osteoradionecrosis (ORN) is known to be a refractory disease in the oral and maxillofacial field. The purpose of this study was to examine the effects of pentoxifylline (PTX) and tocopherol (TP) on an ORN animal model focused on bone healing. MATERIALS AND METHODS: A total of 48 Sprague-Dawley rats were used: 40 received a single irradiation dose of 35 Gy on the left mandible, and eight were used as the nonirradiated control group. The rats received PTX (T1, C1), TP (T2, C2), a combination of PTX and TP (T3, C3), or normal saline (T4, C4). Three weeks after irradiation, the mandibular posterior teeth were extracted. The rats were sacrificed 4 weeks after extraction. RESULTS: In the T3 group, bone volume/tissue volume was 19.62 ± 16.03 (%), bone mineral density was as 0.31 ± 0.16 (g/cm3) in the micro-CT analysis, which were higher than that of other groups (p = 0.025, p = 0.012, respectively). In the histological analysis, bone regeneration was the most prominent in the T3 group. The ratio of empty lacunae was the highest in the T4 group, 68.77 ± 15.47 (%, p = 0.004). Immunohistochemistry showed that the expression of TNF-α was relatively lower in the T3 than in the T4 or T2 groups. The RT-qPCR showed the expression level of PECAM, VEGF-A, and osteocalcin was more than twofold as high as in the T3 group compared to the other groups. CONCLUSION: The combination of PTX and TP appears to promote angiogenesis and osteogenesis in a rat ORN model. Therefore, PTX and TP might be useful in the treatment and prevention of ORN.

Development of a standardized mucositis and osteoradionecrosis animal model using external radiation.

OBJECTIVES: Although the side effects of radiation therapy vary from mucositis to osteomyelitis depending on the dose of radiation therapy, to date, an experimental animal model has not yet been proposed. The aim of this study was to develop an animal model for assessing complications of irradiated bone, especially to quantify the dose of radiation needed to develop a rat model. MATERIALS AND METHODS: Sixteen Sprague-Dawley rats aged seven weeks with a mean weight of 267.59 g were used. Atraumatic extraction of a right mandibular first molar was performed. At one week after the extraction, the rats were randomized into four groups and received a single dose of external radiation administered to the right lower jaw at a level of 14, 16, 18, or 20 Gy, respectively. Clinical alopecia with body weight changes were compared and bony volumetric analysis with micro-computed tomography (CT), histologic analysis with H&E were performed. RESULTS: The progression of the skin alopecia was different depending on the irradiation dose. Micro-CT parameters including bone volume, bone volume/tissue volume, bone mineral density, and trabecular spaces, showed no significant differences. The progression of osteoradionecrosis (ORN) along with that of inflammation, fibrosis, and bone resorption, was found with increased osteoclast or fibrosis in the radiated group. As the radiation dose increases, osteoclast numbers begin to decrease and osteoclast tends to increase. Osteoclasts respond more sensitively to the radiation dose, and osteoblasts are degraded at doses above 18 Gy. CONCLUSION: A standardized animal model clinically comparable to ORN of the jaw is a valuable tool that can be used to examine the pathophysiology of the disease and trial any potential treatment modalities. We present a methodology for the use of an experimental rat model that incorporates a guideline regarding radiation dose.

Dimethyloxalylglycine-embedded Poly(ε-caprolactone) Fiber Meshes Promote Odontoblastic Differentiation of Human Dental Pulp-derived Cells.

INTRODUCTION: The in vivo effect of prolyl hydroxylase inhibitors on the regeneration of the pulp-dentin complex is unclear. The purpose of this study was to investigate the effect of dimethyloxalylglycine (DMOG)-embedded poly(ε-caprolactone) fiber (PCLF/DMOG) on odontoblastic differentiation of human dental pulp-derived cells (hDPCs) by transplantation of the dentin slice model. METHODS: The hDPCs were seeded onto electrospun PCLF and PCLF/DMOG in dentin slices and then transplanted into nude mice. The surface topography was evaluated for both PCLFs, and DMOG release from the PCLF/DMOG was examined. The effects of the PCLF/DMOG were assessed by histology and quantitative reverse transcription polymerase chain reaction. RESULTS: The PCLF/DMOG-treated dentin slices showed higher cellularity with a palisading arrangement of hDPCs and organized collagen fibers. We found that the PCLF/DMOG significantly stimulated the expression of vascular endothelial growth factor, dentin sialoprotein, and bone sialoprotein in the hDPCs (P < .05) and mouse vascular endothelial growth factor A, mouse platelet endothelial cell adhesion molecule 1, and mouse neurofilament light polypeptide in the surrounding host cells (P < .05). CONCLUSIONS: These results show that PCLF/DMOG has potential in pulp-dentin complex regeneration by promoting odontoblastic differentiation of hDPCs and by enhancing host cell recruitment, angiogenesis, and neurogenesis through the released DMOG-mediated cell responses.

Fibrous Topography-Potentiated Canonical Wnt Signaling Directs the Odontoblastic Differentiation of Dental Pulp-Derived Stem Cells.

Nanofibrous engineered matrices have significant potential in cellular differentiation and tissue regeneration. Stem cells require specific extracellular signals that lead to the induction of different lineages. However, the mechanisms by which the nanofibrous matrix promotes mesenchymal stem cell (MSC) differentiation are largely unknown. Here, we investigated the mechanisms that underlie nanofibrous matrix-induced odontoblastic differentiation of human dental pulp MSCs (DP-MSCs). An electrospun polystyrene nanofibrous (PSF) matrix was prepared, and the cell responses to the PSF matrix were assessed in comparison with those on conventional tissue culture dishes. The PSF matrix promoted the expression of Wnt3a, Wnt5a, Wnt10a, BMP2, BMP4, and BMP7 in the DP-MSCs, concomitant with the induction of odontoblast/osteoblast differentiation markers, dentin sialophosphoprotein (DSPP), osteocalcin, and bone sialoprotein, whose levels were further enhanced by treatment with recombinant Wnt3a. The DP-MSCs cultured on the PSF matrix also exhibited a high alkaline phosphatase activity and intense Alizarin Red staining, indicating that the PSF matrix promotes odontoblast differentiation. Besides inducing the expression of Wnt3a, the PSF matrix maintained high levels of ß-catenin protein and enhanced its translocation to the nucleus, leading to its transcriptional activity. Forced expression of LEF1 or treatments with LiCl further enhanced the DSPP expression. Blocking the Wnt3a-initiated signaling abrogated the PSF-induced DSPP expression. Furthermore, the cells on the PSF matrix increased the DSPP promoter activity. The ß-catenin complex was bound to the conserved motifs on the DSPP promoter dictating its transcription. Transplantations of the preodontoblast-seeded PSF matrix to the subcutaneous tissues of nude mice confirmed the association of the PSF matrix with the Wnt3a and DSPP expressions in vivo. Taken together, these results demonstrate the nanofibrous engineered matrix strongly supports odontoblastic differentiation of DP-MSCs by enhancing Wnt/ß-catenin signaling.

Suppression of apoptosis by enhanced protein adsorption on polymer/hydroxyapatite composite scaffolds.

Bone tissue engineering is a promising alternative to bone grafting. Scaffolds play a critical role in tissue engineering. Composite scaffolds made of biodegradable polymers and bone mineral-like inorganic compounds have been reported to be advantageous over plain polymer scaffolds by our group and others. In this study, we compared cellular and molecular events during the early periods of osteoblastic cell culture on poly(l-lactic acid)/hydroxyapatite (PLLA/HAP) composite scaffolds with those on plain PLLA scaffolds, and showed that PLLA/HAP scaffolds improved cell survival over plain PLLA scaffolds. Most cells (MC3T3-E1) on PLLA/HAP scaffolds survived the early culture. In contrast, about 50% of the cells initially adhered to the plain PLLA scaffolds were detached within the first 12h and showed characteristics of apoptotic cell death, which was confirmed by TUNEL staining and caspase-3 activation. To investigate the mechanisms, we examined the adsorption of serum protein and adhesion molecules to the scaffolds. The PLLA/HAP scaffold adsorbed more than 1.4 times of total serum protein and much greater amounts of serum fibronectin and vitronectin than pure PLLA scaffolds. Similarly, significantly larger amounts of individual adhesion proteins and peptides (fibronectin, vitronectin, RGD, and KRSR) were adsorbed on the PLLA/HAP scaffolds than on the PLLA scaffolds, which resulted in higher cell density on the PLLA/HAP scaffolds. Furthermore, beta1 and beta3 integrins and phosphorylation of Fak and Akt proteins in the cells on the PLLA/HAP scaffolds were significantly more abundent than those on PLLA scaffolds, which suggest that enhanced adsorption of serum adhesion proteins to PLLA/HAP scaffolds protect the cells from apoptosis possibly through the integrin-FAK-Akt pathway. These results demonstrate that biomimetic composite scaffolds are advantageous for bone tissue engineering.

Nano-fibrous scaffolding promotes osteoblast differentiation and biomineralization.

Nano-fibrous poly(L-lactic acid) (PLLA) scaffolds with interconnected pores were developed under the hypothesis that nano-fibrous scaffolding would mimic a morphological function of collagen fibrils to create a more favorable microenvironment for cells versus solid-walled scaffolds. In this study, an in vitro system was used to examine biological properties of the nano-fibrous scaffolds compared with those of solid-walled scaffolds for their potential use in bone tissue engineering. Biomineralization was enhanced substantially on the nano-fibrous scaffolds compared to solid-walled scaffolds, and this was confirmed by von Kossa staining, measurement of calcium contents, and transmission electron microscopy. In support of this finding, osteoblasts cultured on the nano-fibrous scaffolds exhibited higher alkaline phosphatase activity and an earlier and enhanced expression of the osteoblast phenotype versus solid-walled scaffolds. Most notable were the increases in runx2 protein and in bone sialoprotein mRNA in cells cultured on nano-fibrous scaffolds versus solid-walled scaffolds. At the day 1 of culture, alpha2 and beta1 integrins as well as alphav and beta3 integrins were highly expressed on the surface of cells seeded on nano-fibrous scaffolds, and linked to this were higher levels of phospho-Paxillin and phospho-FAK in cell lysates. In contrast, cells seeded on solid-walled scaffolds expressed significantly lower levels of these integrins, phospho-Paxillin, and phospho-FAK. To further examine the role of nano-fibrous architecture, we inhibited the formation of collagen fibrils by adding 3,4-dehydroproline to cultures and then assayed cells for expression of alpha2 integrin. Cells seeded on nano-fibrous scaffolds sustained expression of alpha2 integrin in the presence of dehydroproline, while suppression of alpha2 integrin was evident in cells seeded on solid-walled scaffolds. These results provide initial evidence that synthetic nano fibers may exhibit certain properties that are comparable to natural collagen fibers, and thus, the nano-fibrous architecture may serve as a superior scaffolding versus solid-walled architecture for promoting osteoblast differentiation and biomineralization.

N-acetylcysteine prevents LPS-induced pro-inflammatory cytokines and MMP2 production in gingival fibroblasts.

Periodontitis is an inflammatory process that ultimately results in tooth loss. Although the primary etiologic agent for periodontitis is bacteria, the majority of periodontal tissue destruction is thought to be caused by an inappropriate host response. Reactive oxygen species (ROS) have been known to be involved in periodontal tissue destruction. We treated human gingival fibroblasts with lipopolysaccharide (LPS) obtained from E. coli and the periodontopathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, and examined their inflammatory responses in the presence and absence of the antioxidant N-acetylcysteine (NAC). LPS enhanced ROS production, as well as, expression of pro-inflammatory cytokines such as interleukin-1beta, interleukin-6, interleukin-8 and tumor necrosis factor-alpha, and the production and activation of MMP2. NAC suppressed all LPS-induced inflammatory responses examined, suggesting that LPS-induced ROS may play a major regulatory role in these responses in gingival fibroblasts. In addition, NAC prevented LPS-induced activation of p38 MAPK and JNK but not phosphorylation and subsequent degradation of IkB. These results indicate that NAC exerts anti-inflammatory effects in LPS-stimulated gingival fibroblasts, functioning at least in part via down-regulation of JNK and p38 MAPK activation. Furthermore, this work suggests that antioxidants may be useful in adjunctive therapies that complement conventional periodontal treatments.

Sinus augmentation using a histone deacetylase inhibitor in a calcium sulfate carrier in rabbit: A pilot study.

Histone deacetylase inhibitors such as sodium butyrate (SB) have been suggested to be promising candidate small molecules for bone regeneration. In this study, the capacity of SB loaded onto calcium sulfate (CaS) to enhance bone formation was investigated in a rabbit sinus model. Following preparation of the sinus access window on a randomly selected side, SB loaded onto CaS (CaS/SB) was grafted in five rabbits, and CaS alone (control) was grafted in another five rabbits. The animals were euthanized after 4 weeks for radiographic, histometric, and immunohistochemical analyses. There was a statistically significant difference in the total augmented volume between the groups in the radiographic analysis (158.22 ± 39.31 mm3 and 107.09 ± 39.69 mm3 , respectively, p = 0.040). The CaS/SB group showed a larger portion of mature lamellar bone and a higher level of mineralization of bone trabeculae, characterized by more intense labeling with osteocalcin compared with the control group in the immunohistochemical analysis. The number of osteocalcin-positive cells within the central area of the augmented sinus was significantly higher in the CaS/SB group than in the control group (179 ± 26.0 mm2 and 123 ± 33.2 mm2 , respectively, p = 0.027). In conclusion, CaS/SB exhibited superior osteogenic potential, especially in the central portion of the augmented sinus as well as improvement of the volume maintenance for sinus augmentation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1916-1923, 2017.

Effects of Dimethyloxalylglycine-Embedded Poly(ε-caprolactone) Fiber Meshes on Wound Healing in Diabetic Rats.

Impaired wound healing in diabetic patients is associated with altered inflammatory responses, poor angiogenesis, deficient extracellular matrix (ECM) component, and peripheral neuropathy. To develop a wound dressing that is capable of the controlled delivery of bioactive small molecules that can improve diabetic wound healing, dimethyloxalylglycine (DMOG)-embedded poly(ε-caprolactone) (PCL) fiber (PCLF/DMOG) meshes are fabricated by electrospinning, and the effects of the PCLF/DMOG meshes on wound healing in diabetic rats are evaluated. Electrospun PCLF/DMOG meshes increase not only the wound closure, re-epithelialization ratio, epithelial maturation (K-10-positive epidermis), and collagen-positive area but also the numbers of angiogenic marker (CD-31)-positive and neuronal marker (neurofilament)-positive cells compared to PCLF (p < 0.05). In in vitro examinations, RAW264.7 macrophages grown on PCLF/DMOG meshes enhance the expression of growth factors (IGF-1, HB-EGF, and NGF) and anti-inflammatory factors (TGF-ß1 and IL-4) but decrease that of pro-inflammatory factors (IL-1ß and IL-6). Keratinocyte migration is increased by conditioned media from the cultures of the macrophages grown either in the presence of DMOG or on PCLF/DMOG. Collectively, these results indicate that PCLF/DMOG meshes promote impaired wound healing in diabetic rats by modulating macrophage responses, enhancing angiogenesis and nerve innervation, and improving ECM synthesis.

Fibroin particle-supported cationic lipid layers for highly efficient intracellular protein delivery.

Directly delivering therapeutic proteins into cells has promise as an intervention without side effects for protein deficiencies caused by genetic defects. However, as negatively charged macromolecules, proteins require carriers for achieving cellular uptake and maintaining their activity in the cytoplasm. The biodegradable natural polymer silk fibroin has demonstrated outstanding advantages as a protein drug scaffold in vitro and in vivo, but its usage has been limited in the extracellular space because of its negatively charged character. Here, we present an intracellular protein delivery system based on fibroin particles coated with cationic lipid layers, denoted as Fibroplex, the surface charge of which can be modulated. Fibroplex showed higher delivery efficiency than conventional delivery methods as well as long-term cargo release in the cytoplasm without toxicity. Furthermore, in vivo experiments showed that Fibroplex efficiently delivered tyrosinase and horseradish peroxidase, which led to hyper-pigmentation and tumor regression, respectively, suggesting its potential for therapeutic protein applications in hereditary diseases or cancer.

Effects of the fibrous topography-mediated macrophage phenotype transition on the recruitment of mesenchymal stem cells: An in vivo study.

Host responses to a biomaterial critically influence its in vivo performance. Biomaterial architectures that can recruit endogenous host stem cells could be beneficial in tissue regeneration or integration. Here, we report that the fibrous topography of biomaterials promotes the recruitment of host mesenchymal stem cells (MSCs) by facilitating the macrophage phenotype transition from M1-to-M2. Electrospun poly (ε-caprolactone) fiber (PCL-fiber) films were implanted into the subcutaneous tissues of rats, and the response of host cells to the PCL-fiber was evaluated and compared with those of solid ones (PCL-solid). During the initial post-implantation period, greater numbers of cells were recruited and adhered to the PCL-fiber compared to the PCL-solid, and the cells exhibited the M1 phenotype, which was supported by the enhanced adsorption of complement C3a to the implanted PCL-fiber. Subsequently, the PCL-fiber supported the macrophage phenotype transition from M1-to-M2, which was confirmed by the ratio of M2/M1 marker (CD163/CCR7)-positive cells and by the expression of M2/M1 markers (arginase-1/iNOS). The PCL-fiber also reduced the formation of foreign body giant cells. MSC marker (CD29, CD44, and CD90)-positive cells began to appear as early as day 4 on the PCL-fiber, while few MSCs were observed on the PCL-solid. The MSCs migration ex vivo assay showed that MSCs substantially migrated across the trans-wells toward the implanted PCL-fiber. The cells on the implanted PCL-fiber expressed and secreted substantial levels of SDF-1 (CXCL-12), while anti-SDF-1 neutralizing antibody abrogated the MSCs migration. Taken together, these results provide evidence that the fibrous topography of biomaterials enhances the recruitment of MSCs by promoting macrophage recruitment, facilitating M1-to-M2 transition, and enhancing SDF-1 secretion.

Effects of the incorporation of ε-aminocaproic acid/chitosan particles to fibrin on cementoblast differentiation and cementum regeneration.

Cementum formation on the exposed tooth-root surface is a critical process in periodontal regeneration. Although various therapeutic approaches have been developed, regeneration of integrated and functional periodontal complexes is still wanting. Here, we found that the OCCM30 cementoblasts cultured on fibrin matrix express substantial levels of matrix proteinases, leading to the degradation of fibrin and the apoptosis of OCCM30 cells, which was reversed upon treatment with a proteinase inhibitor, ε-aminocaproic acid (ACA). Based on these findings, ACA-releasing chitosan particles (ACP) were fabricated and ACP-incorporated fibrin (fibrin-ACP) promoted the differentiation of cementoblasts in vitro, as confirmed by bio-mineralization and expressions of molecules associated with mineralization. In a periodontal defect model of beagle dogs, fibrin-ACP resulted in substantial cementum formation on the exposed root dentin in vivo, compared to fibrin-only and enamel matrix derivative (EMD) which is used clinically for periodontal regeneration. Remarkably, the fibrin-ACP developed structural integrations of the cementum-periodontal ligament-bone complex by the Sharpey's fiber insertion. In addition, fibrin-ACP promoted alveolar bone regeneration through increased bone volume of tooth roof-of-furcation defects and root coverage. Therefore, fibrin-ACP can promote cementogenesis and osteogenesis by controlling biodegradability of fibrin, implicating the feasibility of its therapeutic use to improve periodontal regeneration. STATEMENT OF SIGNIFICANCE: Cementum, the mineralized layer on root dentin surfaces, functions to anchor fibrous connective tissues on tooth-root surfaces with the collagenous Sharpey's fibers integration, of which are essential for periodontal functioning restoration in the complex. Through the cementum-responsible fiber insertions on tooth-root surfaces, PDLs transmit various mechanical responses to periodontal complexes against masticatory/occlusal stimulations to support teeth. In this study, periodontal tissue regeneration was enhanced by use of modified fibrin biomaterial which significantly promoted cementogenesis within the periodontal complex with structural integration by collagenous Sharpey's fiber insertions in vivo by controlling fibrin degradation and consequent cementoblast apoptosis. Furthermore, the modified fibrin could improve repair and regeneration of tooth roof-of-furcation defects, which has spatial curvatures and geometrical difficulties and hardly regenerates periodontal tissues.