PAI-1 transfected-conditioned media promotes osteogenic differentiation of hBMSCs.

Reconstruction of injured bone remains challenging in the clinic owing to the lack of suitable bone grafts. The utilization of PAI-1 transfected-conditioned media (P-CM) has demonstrated its ability to facilitate the differentiation process of mesenchymal stem cells (MSCs), potentially serving as a crucial mediator in tissue regeneration. This research endeavored to explore the therapeutic potential of P-CM concerning the differentiation of human bone marrow mesenchymal stem cells (hBMSCs). To assess new bone formation, a rat calvaria critical defect model was employed, while in vitro experiments involved the use of the alizarin Red-S mineral induction test. In the rat calvaria critical defect model, P-CM treatment resulted in significan new bone formation. In vitro, P-CM treated hBMSCs displayed robust osteogenesis compared to the control group, as demonstrated by the mineral induction test using alizarin Red-S. P-CM with hydroxyapatite/ß-tricalcium phosphate/fibrin gel treatment significantly exhibited new bone formation, and the expression of osteogenic associated markers was enhanced in the P-CM-treated group. In conclusion, results demonstrate that P-CM treatment significantly enhanced the osteogenic differantiation efficiency and new bone formation, thus could be used as an ideal therapeutic biomolecule for constructing bone-specific implants, especially for orthopedic and dental applications.

Magnetic field-assisted aligned patterning in an alginate-silk fibroin/nanocellulose composite for guided wound healing.

This study was focused on utilizing the magneto-responsiveness of cellulose nanocrystals (CNCs) in an alginate-silk fibroin (ASF) matrix under a low-strength (0.28 T) magnetic field (MF) for fabrication of a magnetically aligned, anisotropic, three-dimensional wound healing scaffold. The effect of the MF on three different concentrations of CNCs (0.5%, 1%, and 2%) was studied to control the alignment of the ASF scaffold. The as-fabricated scaffolds exhibited a concentration-dependent anisotropy with respect to the CNCs. The SEM, AFM, and, SAXS analysis indicated a higher degree of anisotropy of the MF-treated scaffolds with significant enhancement of Young's modulus vis-à-vis control, demonstrating their mechanical stability. Skin fibroblasts, keratinocytes, and endothelial cells cultured on the magnetically aligned scaffolds showed enhanced proliferation in vitro and demonstrated rapid wound closure under in vivo conditions. Hence, the magnetic property of CNCs could be useful for developing biomimetic anisotropic constructs for wound healing applications.

A Modified Minimally Invasive Approach for the Treatment of Mandibular Condylar Fracture.

ABSTRACT: In this study, we introduced a small "V" shaped incision around the ear lobe modified from fact-lift approach to treat the mandibular condylar neck and subcondylar fractures to further minimize and hide the postsurgical scars. A total of 15 cases with mandibular condylar fractures from low levels of condylar neck to high levels of subcondylar region were treated by this approach. An incision was designed from the posterior rim of the tragus down to the anterior skin wrinkle to the ear lobe, surrounding it, continually up and backward for a little distance, forming a "V"-like shape. After cutting and elevation of the skin flap, the remaining steps were the same as those in trans-parotid mini-invasive approaches. The surgical scars introduced in this study were more hidden than those in other mini-invasive approaches and all patients were satisfied with their aesthetic appearances. We suggested that the modified "V" shaped mini-invasive approach has an advantage in achieving aesthetic results and could be an alternative incision in treating some types of mandibular condylar and subcondylar fractures.

Human Teeth-Derived Bioceramics for Improved Bone Regeneration.

Hydroxyapatite (HAp, Ca10(PO4)6(OH)2) is one of the most promising candidates of the calcium phosphate family, suitable for bone tissue regeneration due to its structural similarities with human hard tissues. However, the requirements of high purity and the non-availability of adequate synthetic techniques limit the application of synthetic HAp in bone tissue engineering. Herein, we developed and evaluated the bone regeneration potential of human teeth-derived bioceramics in mice's defective skulls. The developed bioceramics were analyzed by X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectroscopy, and scanning electron microscopy (FE-SEM). The developed bioceramics exhibited the characteristic peaks of HAp in FTIR and XRD patterns. The inductively coupled plasma mass spectrometry (ICP-MS) technique was applied to determine the Ca/P molar ratio in the developed bioceramics, and it was 1.67. Cytotoxicity of the simulated body fluid (SBF)-soaked bioceramics was evaluated by WST-1 assay in the presence of human alveolar bone marrow stem cells (hABMSCs). No adverse effects were observed in the presence of the developed bioceramics, indicating their biocompatibility. The cells adequately adhered to the bioceramics-treated media. Enhanced bone regeneration occurred in the presence of the developed bioceramics in the defected skulls of mice, and this potential was profoundly affected by the size of the developed bioceramics. The bioceramics-treated mice groups exhibited greater vascularization compared to control. Therefore, the developed bioceramics have the potential to be used as biomaterials for bone regeneration application.

Evaluation of Osteogenic/Cementogenic Modulating Potential of PAI-1 Transfected Media for Stem Cells.

AIM OF THE STUDY: In vitro evaluation of the effects of plasminogen activator inhibitor-1 (PAI-1) transfected-conditioned media (P-CM) on the differentiation of human periodontal ligament stem cells (hPDLSCs) and human periapical follicular stem cells (hPAFSCs). MATERIALS AND METHODS: The hPDLSCs and hPAFSCs received from impacted third molars were treated with P-CM and viability, as well as differentiation of the cells were evaluated. Plasmids were constructed according to standard techniques, and all sequences were validated by proper enzyme digestion and sequencing. Chinese hamster ovarian (CHO) cells were transfected with pcDNA3.1-hPAI-1 plasmid to obtain P-CM, followed by western blotting and PAI-1-specific ELISA kit to evaluate the proteins of P-CM. The cell viability of hPDLSCs and hPAFSCs were analyzed using MTT assay after 48 h of incubation. Alizarin red S staining was performed to evaluate the differentiation of hPDLSCs and hPAFSCs. The reverse transcription-polymerase chain reaction was used to observe the expression levels of osteogenic/cementogenic marker genes. The human cytokine antibody array was applied for further analysis of cytokine expression in P-CM. RESULTS: P-CM significantly promoted the differentiation of hPDLSCs and hPAFSCs and upregulated the expression of osteogenic/cementogenic marker genes in vitro. Furthermore, rhPAI-1 promoted mineralized nodules formation of hPDLSCs and hPAFSCs, and we identified that other proteins, RANTES and IL-6, were highly expressed in P-CM. CONCLUSIONS: P-CM promoted the differentiation of hPDLSCs and hPAFSCs by upregulating the expression of RANTES and IL-6, and interaction between PAI-1 and RANTES/IL-6 signaling may be involved in P-CM-induced osteogenic/cementogenic differentiation.

Three-Dimensional Computed Tomography Analysis of Pharynx in Juvenile Patients With Unrepaired Isolated Cleft Palate.

Previous research showed that pharynx in adult patients with unrepaired isolated cleft palate (UICP) significantly enlarged. However, the literature lacks studies in juvenile patients. Thus, this study aimed to conduct a 3-dimensional evaluation of pharynx in juvenile patients with UICP using cone beam computed tomography. Cone beam computed tomography images of 58 nonsyndromic patients with UICP and 60 noncleft controls aged from 4 to 13 years were acquired. Both patients and controls were divided into 3 groups according to their ages. Image processing and analyses were performed using Mimics and ZWCAD. Linear, angular, planar, and volumetric measurements and comparisons were made. Statistical analyses comparing patients with controls were performed using independent-samples t test, with the threshold of significance set at P = 0.05. Results showed that, in all age groups, anterior height (P = 0.002, P = 0.004, P = 0.004) and length of the floor (P = 0.023, P = 0.000, P = 0.024) of bony nasopharynx; transverse (P = 0.028, P = 0.031, P = 0.043) and posteroanterior (P = 0.000, P = 0.000, P = 0.000) diameters of pharyngeal airway at palatal plane; cross-sectional area of pharyngeal airway at palatal plane (P = 0.006, P = 0.001, P = 0.001); total volume (P = 0.005, P = 0.003, P = 0.038), volume above palatal plane (P = 0.001, P = 0.000, P = 0.005), and volume between palatal plane and C2 plane (P = 0.047, P = 0.025, P = 0.048) were larger in UICP patients. Based on this study, the authors can conclude that pharynx in juvenile UICP patients around the palatal plane was significantly enlarged, and narrowing of velopharyngeal orifice in palatoplasty was seemed important.

Physical Stimulation-Based Osteogenesis: Effect of Secretion In Vitro on Fluid Dynamic Shear Stress of Human Alveolar Bone-Derived Mesenchymal Stem Cells.

Human alveolar bone-derived mesenchymal stem cells (hABMSCs) are promising candidates for bone therapies, which have the capacity to differentiate into osteoblasts. Recently, secretion of inducible cytokines and growth factors from mesenchymal stem cells (MSCs) has been discovered, and we also have reported the osteogenic effects of cell physical stimulation. In this study, we investigated the effects of hABMSCs-conditioned secretion media (B-CSM) on osteogenic differentiation of hABMSCs in vitro. Furthermore, we analyzed the B-CSM by proteomics array to identify inducible factors which facilitate osteogenic differentiation. To determine optimal concentration, B-CSM was firstly added at varying amounts (5, 10, 20, 40, and 60%) relative to culture medium. The viability and proliferation of hABMSCs were higher after treating with 5-20% B-CSM to the cells, compared to 40-60%. In addition, the expression of stem cells markers CD146 and STRO-1 was increased in the cells treated with 5-20% B-CSM, but decreased with 40-60%. We also found that B-CSM promoted osteogenic differentiation of hABMSCs such as mineralized nodules were strongly generated by 5-20%. B-CSM was most effective in increasing the expression of Vinculin and osteocalcin (OCN) in osteogenic differentiation of hABMSCs. Taken together, the results of our study ultimately indicate that B-CSM from hABMSCs induced by physical stimulation induce the proliferation and osteogenic differentiation of hABMSCs.

Pulsed-Electromagnetic-Field-Assisted Reduced Graphene Oxide Substrates for Multidifferentiation of Human Mesenchymal Stem Cells.

Electromagnetic fields (EMFs) can modulate cell proliferation, DNA replication, wound healing, cytokine expression, and the differentiation of mesenchymal stem cells (MSCs). Graphene, a 2D crystal of sp(2) -hybridized carbon atoms, has entered the spotlight in cell and tissue engineering research. However, a combination of graphene and EMFs has never been applied in tissue engineering. This study combines reduced graphene oxide (RGO) and pulsed EMFs (PEMFs) on the osteogenesis and neurogenesis of MSCs. First, the chemical properties of RGO are measured. After evaluation, the RGO is adsorbed onto glass, and its morphological and electrical properties are investigated. Next, an in vitro study is conducted using human alveolar bone marrow stem cells (hABMSCs). Their cell viability, cell adhesion, and extracellular matrix (ECM) formation are increased by RGO and PEMFs. The combination of RGO and PEMFs enhances osteogenic differentiation. Together, RGO and PEMFs enhance the neurogenic and adipogenic differentiation of hABMSCs. Moreover, in a DNA microarray analysis, the combination of RGO and PEMFs synergically increases ECM formation, membrane proteins, and metabolism. The combination of RGO and PEMFs is expected to be an efficient platform for stem cell and tissue engineering.

Recombinant Human Plasminogen Activator Inhibitor-1 Promotes Cementogenic Differentiation of Human Periodontal Ligament Stem Cells.

The periodontium, consisting of gingiva, periodontal ligament (PDL), cementum, and alveolar bone, is necessary for the maintenance of tooth function. Specifically, the regenerative abilities of cementum with inserted PDL are important for the prevention of tooth loss. Periodontal ligament stem cells (PDLSCs), which are located in the connective tissue PDL between the cementum and alveolar bone, are an attractive candidate for hard tissue formation. We investigated the effects of recombinant human plasminogen activator inhibitor-1 (rhPAI-1) on cementogenic differentiation of human PDLSCs (hPDLSCs) in vitro and in vivo. Untreated and rhPAI-1-treated hPDLSCs mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and dentin matrix were transplanted subcutaneously into the dorsal surface of immunocompromised mice to assess their capacity for hard tissue formation at 8 and 10 weeks posttransplantation. rhPAI-1 accelerated mineral nodule formation and increased the mRNA expression of cementoblast-associated markers in hPDLSCs. We also observed that rhPAI-1 upregulated the levels of osterix (OSX) and cementum protein 1 (CEMP1) through Smad2/3 and p38 pathways, whereas specific inhibitors of Smad3 and p38 inhibited the enhancement of mineralization of hPDLSCs by rhPAI-1. Furthermore, transplantation of hPDLSCs with rhPAI-1 showed a great ability to promote cementogenic differentiation. Notably, rhPAI-1 induced hPDLSCs to regenerate cementum-like tissue with PDL fibers inserted into newly formed cementum-like tissue. These results suggest that rhPAI-1 may play a key role in cementogenic differentiation of hPDLSCs. rhPAI-1 with hPDLSCs may be a good candidate for future clinical applications in periodontal tissue regeneration and possibly in tooth root bioengineering.

Hypoxia promotes CEMP1 expression and induces cementoblastic differentiation of human dental stem cells in an HIF-1-dependent manner.

Cementum covering the tooth root provides attachment for the tooth proper to the surrounding alveolar bone via non-mineralized periodontal ligament (PDL). Cementum protein 1 (CEMP1) has been shown to induce a cementoblastic phenotype in cementoblast precursors cells of PDL. Oxygen availability is a critical signal for correct development of many tissues; however, its role in tooth root and periodontium development remains poorly understood. In this study, we demonstrated that reduced oxygen tension increased CEMP1 expression, mineral deposition, and alkaline phosphatase activity in human dental stem cells such as PDL stem cells and periapical follicular stem cells. Since an oxemic state is transduced by the transcription factor, hypoxia-inducible factor-1 (HIF-1), we performed experiments to determine whether this protein was responsible for the observed changes. We noted that when HIF-1 was activated by gene introduction or chemically, CEMP1 expression and mineralization increased. In contrast, when HIF-1α was silenced, CEMP1 expression and mineralization did not increase in vitro. Furthermore, we showed for the first time that mouse tooth root and periodontium development occurs partly under hypoxic conditions, particularly at the apical part and latently at the PDL space in vivo. Desferrioxamine, an HIF-1 stimulator, enhances CEMP1 expression in the mouse PDL space, suggesting that hypoxia affects cementogenesis of PDL cells lining the surface of the developing tooth root in an HIF-1-dependent manner. These results suggest that HIF-1 activators may have the ability to stimulate regeneration of the tooth root and cementum formation.

HDAC inhibitor trichostatin A promotes proliferation and odontoblast differentiation of human dental pulp stem cells.

Trichostatin A (TSA) is a potent histone deacetylase (HDAC) inhibitor with a broad spectrum of epigenetic activities known to regulate diverse cellular mechanisms, including differentiation of mesenchymal stem cells. In this study, we demonstrate that TSA promotes proliferation and odontoblast differentiation of human dental pulp stem cells (hDPSCs) in vitro and has the ability to enhance dentin formation and odontoblast differentiation in vivo during tooth development. We observed that TSA increased the expression of proliferating cell nuclear antigen and cyclin D1 in hDPSCs at a certain concentration and the activation of JNK/c-Jun pathway was essential for TSA-dependent hDPSC proliferation. Further, TSA accelerated mineral nodule formation in vitro and increased gene expression of dentin sialophosphoprotein, dentin matrix protein 1, bone sialoprotein, and osteocalcin. In addition, TSA significantly upregulated the levels of phospho-Smad2/3, Smad4, and nuclear factor I-C, while the specific inhibitor of Smad3 inhibits TSA enhancing mineralization differentiation of hDPSCs. HDAC3 is downregulated by TSA treatment, suggesting a possible mediator of TSA-dependent pathways among the members of HDAC family. Moreover, TSA-injected embryos exhibited increased dentin thickness, larger dentin areas, and higher odontoblast numbers in their postnatal molars with stronger dentin sialoprotein expression in immunohistochemical staining. These findings indicate that TSA may serve a key role in proliferation and odontoblast differentiation of hDPSCs in dental developmental stages and can be used as an accelerator in dental hard tissue engineering.

Epigallocatechin-3-gallate inhibits osteoclastogenesis by down-regulating c-Fos expression and suppressing the nuclear factor-kappaB signal.

Epigallocatechin-3-gallate (EGCG), the major anti-inflammatory compound in green tea, has been shown to suppress osteoclast differentiation. However, the precise molecular mechanisms underlying the inhibitory action of EGCG in osteoclastogenesis and the effect of EGCG on inflammation-mediated bone destruction remain unclear. In this study, we found that EGCG inhibited osteoclast formation induced by osteoclastogenic factors in bone marrow cell-osteoblast cocultures but did not affect the ratio of receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) to osteoprotegerin induced by osteoclastogenic factors in osteoblasts. We also found that EGCG inhibited osteoclast formation from bone marrow macrophages (BMMs) induced by macrophage colony-stimulating factor plus RANKL in a dose-dependent manner without cytotoxicity. Pretreatment with EGCG significantly inhibited RANKL-induced the gene expression of c-Fos and nuclear factor of activated T-cells (NFATc1), essential transcription factors for osteoclast development. EGCG suppressed RANKL-induced activation of c-Jun N-terminal protein kinase (JNK) pathway, among the three well known mitogen-activated protein kinases and also inhibited RANKL-induced phosphorylation of the NF-kappaB p65 subunit at Ser276 and NF-kappaB transcriptional activity without affecting the degradation of IkappaBalpha and NF-kappaB DNA-binding in BMMs. The inhibitory effect of EGCG on osteoclast formation was somewhat reversed by retroviral c-Fos overexpression, suggesting that c-Fos is a downstream target for antiosteoclastogenic action of EGCG. In addition, EGCG treatment reduced interleukin-1-induced osteoclast formation and bone destruction in mouse calvarial bone in vivo. Taken together, our data suggest that EGCG has an antiosteoclastogenic effect by inhibiting RANKL-induced the activation of JNK/c-Jun and NF-kappaB pathways, thereby suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors.