Therapeutic Effect of HDAC5 Binding and Cell Penetrating Peptide for the Treatment of Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) is an incurable disease that negatively influences the quality of life of patients. Current and emerging therapies target proinflammatory cytokines and/or receptors to downregulate proinflammatory responses, but insufficient remission requires other therapeutic agents. Herein, we report that the synthetic anti-inflammatory peptide 15 (SAP15) is capable of cell penetration and anti-inflammatory activity in human macrophages. METHODS: SAP15 was labeled with fluorescence and administered to human leukemia monocytic cells (THP-1) cells for cell penetration analysis. Using biolayer interferometry analysis, the binding affinity of SAP15 with histone deacetylase 5 (HDAC5) was measured. SAP15-treated THP-1 cells were analyzed by protein phosphorylation assay, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). In addition, in vivo analysis of the therapeutic effect on IBD was observed in a dextran sulfate sodium (DSS)-induced model. Samples from SAP15-treated mice were analyzed at both the macroscopic and microscopic levels using ELISA, myeloperoxidase (MPO) assays, and histological evaluations. RESULTS: SAP15 was internalized within the cytosol and nucleus of THP-1 cells and bound to the HDAC5 protein. SAP15-treated macrophages were assessed for protein phosphorylation and showed inhibited phosphorylation of HDAC5 and other immune-related proteins, which led to increased M2-like macrophage markers and decreased M1-like macrophage markers and tumor necrosis factor-α and interleukin-6 cytokine levels. The SAP15 treatment on IBD model showed significant recovery of colon length. Further histological analysis of colon demonstrated the therapeutic effect of SAP15 on mucosal layer. Moreover, proinflammatory cytokine levels and MPO activity from the plasma show that SAP15 is effective in reduced proinflammatory responses. CONCLUSION: These findings suggest that SAP15 is a novel peptide with a novel cell-penetrating peptide with anti-inflammatory property that can be used as a therapeutic agent for IBD and other inflammatory diseases.

The Synthetic Collagen-Binding Peptide NIPEP-OSS Delays Mouse Myeloma Progression.

Multiple myeloma (MM) is the second most common hematological malignancy. It is a clonal B-cell disorder characterized by the proliferation of malignant plasma cells in the bone marrow, the presence of monoclonal serum immunoglobulin, and osteolytic lesions. An increasing amount of evidence shows that the interactions of MM cells and the bone microenvironment play a significant role, suggesting that these interactions may be good targets for therapy. The osteopontin-derived collagen-binding motif-bearing peptide NIPEP-OSS stimulates biomineralization and enhances bone remodeling dynamics. Due to its unique targeted osteogenic activity with a broad safety margin, we evaluated the potential of NIPEP-OSS for anti-myeloma activity using MM bone disease (MMBD) animal models. In a 5TGM1-engrafted NSG model, the survival rates of the control and treated groups were significantly different (p = 0.0014), with median survival times of 45 and 57 days, respectively. The bioluminescence analyses showed that myeloma slowly developed in the treated mice compared to the control mice in both models. NIPEP-OSS enhanced bone formation by increasing biomineralization in the bone. We also tested NIPEP-OSS in a well-established 5TGM1-engrafted C57BL/KaLwRij model. Similar to the previous model, the median survival times of the control and treated groups were significantly different (p = 0.0057), with 46 and 63 days, respectively. In comparison with the control, an increase in p1NP was found in the treated mice. We concluded that NIPEP-OSS delays mouse myeloma progression via bone formation in MMBD mouse models.

Tonsil-derived mesenchymal stem cells incorporated in reactive oxygen species-releasing hydrogel promote bone formation by increasing the translocation of cell surface GRP78.

Controlling the senescence of mesenchymal stem cells (MSCs) is essential for improving the efficacy of MSC-based therapies. Here, a model of MSC senescence was established by replicative subculture in tonsil-derived MSCs (TMSCs) using senescence-associated ß-galactosidase, telomere-length related genes, stemness, and mitochondrial metabolism. Using transcriptomic and proteomic analyses, we identified glucose-regulated protein 78 (GRP78) as a unique MSC senescence marker. With increasing cell passage number, GRP78 gradually translocated from the cell surface and cytosol to the (peri)nuclear region of TMSCs. A gelatin-based hydrogel releasing a sustained, low level of reactive oxygen species (ROS-hydrogel) was used to improve TMSC quiescence and self-renewal. TMSCs expressing cell surface-specific GRP78 (csGRP78+), collected by magnetic sorting, showed better stem cell function and higher mitochondrial metabolism than unsorted cells. Implantation of csGRP78+ cells embedded in ROS-hydrogel in rats with calvarial defects resulted in increased bone regeneration. Thus, csGRP78 is a promising biomarker of senescent TMSCs, and the combined use of csGRP78+ cells and ROS-hydrogel improved the regenerative capacity of TMSCs by regulating GRP78 translocation.

Cell-Permeable Oct4 Gene Delivery Enhances Stem Cell-like Properties of Mouse Embryonic Fibroblasts.

Direct conversion of one cell type into another is a trans-differentiation process. Recent advances in fibroblast research revealed that epithelial cells can give rise to fibroblasts by epithelial-mesenchymal transition. Conversely, fibroblasts can also give rise to epithelia by undergoing a mesenchymal to epithelial transition. To elicit stem cell-like properties in fibroblasts, the Oct4 transcription factor acts as a master transcriptional regulator for reprogramming somatic cells. Notably, the production of gene complexes with cell-permeable peptides, such as low-molecular-weight protamine (LMWP), was proposed to induce reprogramming without cytotoxicity and genomic mutation. We designed a complex with non-cytotoxic LMWP to prevent the degradation of Oct4 and revealed that the positively charged cell-permeable LMWP helped condense the size of the Oct4-LMWP complexes (1:5 N:P ratio). When the Oct4-LMWP complex was delivered into mouse embryonic fibroblasts (MEFs), stemness-related gene expression increased while fibroblast intrinsic properties decreased. We believe that the Oct4-LMWP complex developed in this study can be used to reprogram terminally differentiated somatic cells or convert them into stem cell-like cells without risk of cell death, improving the stemness level and stability of existing direct conversion techniques.

Engineered synthetic cell penetrating peptide with intracellular anti-inflammatory bioactivity: An in vitro and in vivo study.

Various biomaterials have been used for bone and cartilage regeneration, and inflammation associated with biomaterial implantation is also increased. A 15-mer synthetic anti-inflammatory peptide (SAP15) was designed from human ß-defensin 3 to penetrate cells and induce intracellular downregulation of inflammation. The downregulation of inflammation was achieved by the binding of SAP15 to intracellular histone deacetylase (HDAC5). SAP15-mediated inhibition of inflammation was examined in vitro and in vivo using murine macrophages, human articular chondrocytes, and a collagen-induced arthritis (CIA) rat model. Surface plasmon resonance and immunoprecipitation assays indicated that SAP15 binds to HDAC5. SAP15 inhibited the lipopolysaccharide (LPS)-induced phosphorylation of intracellular HDAC5 and NF-κB p65 in murine macrophages. SAP15 treatment increased aggrecan and type II collagen expression and decreased osteocalcin expression in LPS-induced chondrocytes. Subcutaneous injection of SAP15-loaded sodium hyaluronic acid (HA) solution significantly decreased hind paw swelling, joint inflammation, and serum cytokine levels in CIA rats compared with the effects of sodium HA solution alone. The SAP15-loaded HA group exhibited preservation of cartilage and bone structure in CIA rat joints. Moreover, a more robust anti-inflammatory effect of the SAP15 loaded HA was observed than that of etanercept (an anti-tumor necrosis factor-alpha [TNF-α] antibody)-loaded HA. These findings suggest that SAP15 has an anti-inflammatory effect that is not controlled by sodium HA and is mediated by inhibiting HDAC5, unlike the anti-inflammatory mechanism of etanercept. These results demonstrate that SAP15 is useful as an inflammatory regulator of biomaterials and can be developed as a therapeutic for the treatment of inflammation.

A Synthetic Cell-Penetrating Heparin-Binding Peptide Derived from BMP4 with Anti-Inflammatory and Chondrogenic Functions for the Treatment of Arthritis.

We report dual therapeutic effects of a synthetic heparin-binding peptide (HBP) corresponding to residues 15-24 of the heparin binding site in BMP4 in a collagen-induced rheumatic arthritis model (CIA) for the first time. The cell penetrating capacity of HBP led to improved cartilage recovery and anti-inflammatory effects via down-regulation of the iNOS-IFNγ-IL6 signaling pathway in inflamed RAW264.7 cells. Both arthritis and paw swelling scores were significantly improved following HBP injection into CIA model mice. Anti-rheumatic effects were accelerated upon combined treatment with Enbrel® and HBP. Serum IFNγ and IL6 concentrations were markedly reduced following intraperitoneal HBP injection in CIA mice. The anti-rheumatic effects of HBP in mice were similar to those of Enbrel®. Furthermore, the combination of Enbrel® and HBP induced similar anti-rheumatic and anti-inflammatory effects as Enbrel®. We further investigated the effect of HBP on damaged chondrocytes in CIA mice. Regenerative capacity of HBP was confirmed based on increased expression of chondrocyte biomarker genes, including aggrecan, collagen type II and TNFα, in adult human knee chondrocytes. These findings collectively support the utility of our cell-permeable bifunctional HBP with anti-inflammatory and chondrogenic properties as a potential source of therapeutic agents for degenerative inflammatory diseases.

Synthetic Human ß Defensin-3-C15 Peptide in Endodontics: Potential Therapeutic Agent in Streptococcus gordonii Lipoprotein-Stimulated Human Dental Pulp-Derived Cells.

Human ß defensin-3-C15, an epithelium-derived cationic peptide that has antibacterial/antifungal and immuno-regulatory properties, is getting attention as potential therapeutic agent in endodontics. This study aimed to investigate if synthetic human ß defensin-3-C15 (HBD3-C15) peptides could inhibit inflammatory responses in human dental pulp cells (hDPCs), which had been induced by gram-positive endodontic pathogen. hDPC explant cultures were stimulated with Streptococcus gordonii lipoprotein extracts for 24 h to induce expression of pro-inflammatory mediators. The cells were then treated with either HBD3-C15 (50 µg/mL) or calcium hydroxide (CH, 100 µg/mL) as control for seven days, to assess their anti-inflammatory effects. Quantitative RT-PCR analyses and multiplex assays showed that S. gordonii lipoprotein induced the inflammatory reaction in hDPCs. There was a significant reduction of IL-8 and MCP-1 within 24 h of treatment with either CH or HBD3-C15 (p < 0.05), which was sustained over 1 week of treatment. Alleviation of inflammation in both medications was related to COX-2 expression and PGE2 secretion (p < 0.05), rather than TLR2 changes (p > 0.05). These findings demonstrate comparable effects of CH and HDB3-C15 as therapeutic agents for inflamed hDPCs.

Identification of cell-penetrating osteogenic peptide from copine-7 protein and its delivery system for enhanced bone formation.

Peptide and proteins are recognized as highly selective and therapeutically active biomaterials, as well as relatively safe in clinical application. A calcium phospholipid-binding protein, copine 7 (CPNE7), has been recently identified to induce hard tissue regeneration, including bone and dentin by internalizing into the cells. However, the clinical application of the full length of CPNE7 has limited due to its large size with short half-life. Herein, as an alternative to CPNE7, six bioactive synthetic peptides are designed from CPNE7 (CPNE7-derived peptides, CDP1-CDP6) and investigated their osteogenic potential. Among the CDPs, CDP4 have the highest level of cell-penetrating activity as well as osteogenic efficiency in dental pulp stem cells (DPSCs). CDP4 increased the expression of osteogenesis-related genes and proteins, which was comparable to that by BMP-2. The cell penetration capacity of CDP4 may synergistically induce the osteogenic potential of DPSCs. Moreover, the implantation of the mixture of CDP4 with injectable collagen gel increased bone formation with recovery in the mouse calvarial defect model, comparable to full-length CPNE7 and even BMP-2. In conclusion, these results suggest that our synthetic peptide, CDP4, can be applied in combination with biomaterial to provide high osteogenic efficacy in the field of bone tissue engineering.

A novel calcium-accumulating peptide/gelatin in situ forming hydrogel for enhanced bone regeneration.

Bioactive agents, including proteins and peptides, can be loaded into hydrogels to improve bone regenerative capacity with their controlled release. However, the current loading method has focused on physical mixing, which has limited release control. Therefore, alternative conjugation of bioactive agents with hydrogels is highly recommended. Direct chemical conjugation of synthetic peptides containing a functional moiety with a hydrogel would be ideal. Here, we synthesized a bioactive calcium accumulating peptide (CAP) containing a collagen binding motif, which can induce osteogenic differentiation. A tyrosine residue in CAP was used to directly chemically conjugate the peptide with a gelatin-based enzymatically crosslinked hydroxyphenyl propionic acid hydrogel under H2 O2 /Horse radish peroxidase conditions. To test the acceleration of bone formation, human periodontal ligament stem cells (PDLSCs) were loaded into a chemically conjugated CAP hydrogel. The CAP hydrogel induced bone mineralization around the PDLSCs and increased osteogenic marker expressions in vitro. It also recovered a bone layer in a calvarial defect 4 weeks postimplantation. In summary, an injectable CAP hydrogel scaffold system was developed as a potentially useful engineered microenvironment to enhance bone restoration, and it could be utilized as a vehicle for bioactive delivery of stem cells in tissue regenerative therapy. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 531-542, 2018.

Comparative Study of rhPDGF-BB Plus Equine-Derived Bone Matrix Versus rhPDGF-BB Plus ß-TCP in the Treatment of Periodontal Defects.

The purpose of this study was to evaluate the efficacy and safety of equine-derived bone matrix as a carrier for recombinant human platelet-derived growth factor BB (rhPDGF-BB) versus beta-tricalcium phosphate (ß-TCP) for the treatment of intraosseous periodontal defects in adult patients. This study was performed on 32 adults with advanced periodontal disease. Eligible subjects were randomized in 1:1 ratio into a test (rhPDGF-BB-coated equine-derived bone matrix) or control group (rhPDGF-BB-coated ß-TCP). Probing pocket depth (PD), clinical attachment level (CAL), gingival recession (GR), and defect depth on radiographs were measured at 2 weeks before surgery, on the day of surgery (DOS), and 6 months postsurgery (6MPS). The clinical and radiographic data were analyzed over the test period. Statistically significant PD reductions and CAL gain between baseline and 6MPS and between ODS and 6MPS were seen in both groups (P < .01). No statistically significant differences in PD reduction were found between groups. However, the test group showed significant CAL gain between DOS and 6MPS. The radiographic bone level change was statistically significant compared to baseline (P < .01) in both groups. The results suggested that equine-derived bone matrix is a viable, effective, and safe carrier scaffold for rhPDGF in periodontal defects.

The Study on the Lives and Health Conditions of Internees in Santo Thomas Camp of Philippines - Based on McAnlis's The War in Manila (1941-1945).

When Japan invaded the Philippines, two missionary dentists (Dr. McAnlis and Dr. Boots) who were forced to leave Korea were captured and interned in the Santo Thomas camp in Manila. Japan continued to bombard and plunder the Philippines in the wake of the Pacific War following the Great East Asia policy, leading to serious inflation and material deficiency. More than 4,000 Allied citizens held in Santo Thomas camp without basic food and shelter. Santo Thomas Camp was equipped with the systems of the Japanese military medical officers and Western doctors of captivity based on the Geneva Conventions(1929). However, it was an unsanitary environment in a dense space, so it could not prevent endemic diseases such as dysentery and dengue fever. With the expansion of the war in Japan, prisoners in the Shanghai and Philippine prisons were not provided with medicines, cures and food for healing diseases. In May 1944, the Japanese military ordered the prisoners to reduce their ration. The war starting in September 1944, internees received 1000 kcal of food per day, and since January 1945, they received less than 800 kcal of food. This was the lowest level of food rationing in Japan's civilian prison camps. They suffered beriberi from malnutrition, and other endemic diseases. An averaged 24 kg was lost by adult men due to food shortages, and 10 percent of the 390 deaths were directly attributable to starvation. The doctors demanded food increases. The Japanese Military forced the prisoner to worship the emperor and doctors not to record malnourishment as the cause of death. During the period, the prisoners suffered from psychosomatic symptoms such as headache, diarrhea, acute inflammation, excessive smoking, and alcoholism also occurred. Thus, the San Thomas camp had many difficulties in terms of nutrition, hygiene and medical care. The Japanese military had unethical and careless medical practices in the absence of medicines. Dr. McAnlis and missionary doctors handled a lot of patients focusing mainly on examination, emergency treatment and provided the medical services needed by Philippines and foreigners as well as prisoners. Through out the war in the Great East Asia, the prisoners of Santo Thomas camp died of disease and starvation due to inhumane Japanese Policy. Appropriate dietary prescriptions and nutritional supplements are areas of medical care that treat patients' malnutrition and disease. It is also necessary to continue research because it is a responsibility related to the professionalism and ethics of medical professionals to urge them to observe the Geneva Convention.

Selective osteogenesis by a synthetic mineral inducing peptide for the treatment of osteoporosis.

Mineralization in mammalian cells is accomplished by concerted regulation of protein-based extracellular matrix (ECM) components, such as non-collagenous proteins and collagen fibrils. In this study, we investigated the ability of a collagen-binding motif (CBM) peptide derived from osteopontin to selectively affect osteogenic or adipogenic differentiation in vitro and in vivo. In particular, increased osteogenic differentiation and decreased adipogenic differentiation were observed in human mesenchymal stem cells (hMSCs). Osteocalcin (OCN) protein expression in MC3T3-E1 cells without osteogenic inducers was then investigated following treatment with the CBM peptide. In ovariectomized (OVX) mice, estrogen deficiency induced osteoporosis and increased fat tissue deposition. However, after the CBM peptide or estradiol was injected into the OVX mice for 2 months, the increased serum OCN concentration and alkaline phosphate (ALP) activity were decreased in the estradiol-treated group (OVX-E) and the high-concentration CBM peptide-treated group (OVX-HP). Significant bone loss was also observed in the ovariectomized mice (OVX-PBS). In particular, the bone volume per total volume (BV/TV) and bone mineral density (BMD) were significantly decreased in the OVX mice; however, both of these markers were restored in the OVX-HP group, which also had significantly well-developed bone structure and bone formation. In contrast to the bone structural change, adipose tissue was increased in the OVX-PBS. However, a significant decrease in total fat and subcutaneous fat was observed in the low-concentration CBM peptide-treated group (OVX-LP) and the estradiol-treated group (OVX-E). Taken together, these results suggest that the CBM peptide could be an effective therapeutic agent for osteoporosis due to its selective stimulation of osteogenic differentiation, rather than adipogenesis.

Comparative study of two collagen membranes for guided tissue regeneration therapy in periodontal intrabony defects: a randomized clinical trial.

PURPOSE: THE PURPOSE OF THIS STUDY WAS TO ASSESS AND COMPARE THE CLINICAL AND RADIOGRAPHIC OUTCOMES OF GUIDED TISSUE REGENERATION THERAPY FOR HUMAN PERIODONTAL INTRABONY DEFECTS USING TWO DIFFERENT COLLAGEN MEMBRANES: a porous nonchemical cross-linking collagen membrane (NC) and a bilayer collagen membrane (BC). METHODS: Thirty subjects were randomly assigned and divided into the following 3 groups: a test group (NC+BM), in which a NC was used with xenograft bone mineral (BM), a positive control group (BC+BM), in which a BC was used with xenograft BM, and a negative control group (BM), in which only xenograft BM was used. The following clinical measurements were taken at baseline and 3 months after surgery: plaque index, gingival index, probing pocket depth, gingival recession, and clinical attachment level. Radiographic analysis was performed at baseline, 1 week and 3 months after surgery. RESULTS: Membrane exposure was not observed in any cases. Significant probing depth reduction, attachment-level gain and bone fill were observed for both test and control groups compared to baseline at 3 months after surgery (P<0.05). However, there were no statistically significant differences in clinical improvement and radiographic bone fill between treatment protocols (P>0.05). CONCLUSIONS: Within the limitations of this study, the results suggest that both NC and BC were comparable in terms of clinical and radiographic outcomes for the treatment of periodontal intrabony defects in human subjects.

The cell-penetrating peptide domain from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) has anti-inflammatory activity in vitro and in vivo.

A heparin-binding peptide (HBP) sequence from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified and was shown to exhibit cell penetration activity. This cell penetration induced an anti-inflammatory reaction in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. HBP penetrated the cell membrane during the 10 min treatment and reduced the LPS-induced production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cytokines (TNF-α and IL-6) in a concentration-dependent manner. Additionally, HBP inhibited the LPS-induced upregulation of cytokines, including TNF-α and IL-6, and decreased the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. HBP inhibited NF-κB-dependent inflammatory responses by directly blocking the phosphorylation and degradation of IκBα and by subsequently inhibiting the nuclear translocation of the p65 subunit of NF-κB. Taken together, this novel HBP may be potentially useful candidate for anti-inflammatory treatments and can be combined with other drugs of interest to transport attached molecules into cells.

Biomimetic surface modification using synthetic oligopeptides for enhanced guided bone regeneration in beagles.

BACKGROUND: In previous studies, oligopeptide corresponding to the cell-binding domains of bone morphogenetic proteins that bind to bone morphogenetic protein receptor enhanced the bone regenerative capacity of bovine bone minerals (BBM). The aim of this study is to evaluate the ability of BBM coated with oligopeptide to promote periodontal regeneration in a 1-wall intrabony defect model in dogs. METHODS: The second and third mandibular premolars and first molars of six adult beagles were extracted bilaterally, and the extraction sites were allowed to heal for 10 weeks. The 1-wall intrabony defects were prepared bilaterally on the mesial and distal side of the fourth mandibular premolars. Twenty-four intrabony defects were assigned to four treatment groups: 1) open flap debridement; 2) guided tissue regeneration (GTR); 3) GTR with a collagen membrane and BBM; and 4) GTR with a collagen membrane and BBM coated with the oligopeptide (Pep-BBM). The animals were sacrificed 10 weeks after surgery. For the histometric analysis, defect height, junctional epithelium migration, new cementum, new bone height, and new bone area were measured. New bone volume was measured using microcomputed tomography. RESULTS: Wound healing was generally uneventful. For junctional epithelium migration, the BBM and Pep-BBM groups exhibited mean (± SE) values of 0.53 ± 0.41 and 0.48 ± 0.30 mm, and for new cementum height, 1.71 ± 0.46 and 2.50 ± 2.00 mm, respectively. For junctional epithelium migration and cementum regeneration, there were no significant differences between the two groups. The mean (± SE) values of new bone height and new bone volume in the Pep-BBM group (3.88 ± 0.31 mm and 32.35% ± 9.60%) were significantly greater than the mean values for the BBM group (2.60 ± 0.41 mm and 20.56% ± 1.89%). For bone regeneration, the Pep-BBM group showed superior results compared to the BBM group with statistically significant differences. CONCLUSIONS: Through various parameters to evaluate periodontal regeneration, this oligopeptide coating influenced only the ability of BBM to promote bone regeneration in 1-wall intrabony defects in beagles. Junctional epithelium migration and cementum regeneration were not affected by this oligopeptide coating, and further investigations with special focus on regeneration of the periodontal ligament are necessary.

Bioactive peptide-modified biomaterials for bone regeneration.

Bioactive biomaterials are desirable as tissue engineering scaffolds by virtue of their capability to mimic the natural environment of the extracellular matrix. Bioactive biomaterials have been achieved by incorporating synthetic short peptide sequences into suitable materials either by surface modification or by bulk incorporation. The goal is to enhance cell attachment and other basic functions. Bioactive peptides can be obtained from biological or chemically synthesized sources, increasing their specific cellular responses for tissue growth and development. Compared to using an entire growth factor in regenerative therapy, these peptides demonstrate potential advantages such as overcoming possible immunogenicity, being less susceptible to degradation, and producing fewer tumor-related side effects. Biomaterial scaffolds modified with peptides can provide biological ligands for cell-scaffold interactions that promote cell attachment, proliferation, and differentiation. Peptide-based biomaterial scaffolds can be fabricated to form two- and three-dimensional structures. This review discusses cell-binding, biominerailization inducing peptides, and receptor-binding peptides for bone regeneration. This review also addresses issues related to peptide immobilization as well as potential complications that may develop as a result of using these versatile bioactive peptides. The development of self-assembled peptide amphiphiles with the goal of generating new three-dimensional scaffolds for tissue engineering is also summarized.

Cell-penetrating chitosan/doxorubicin/TAT conjugates for efficient cancer therapy.

In this study, a cell-penetrating peptide, the transactivating transcriptional factor (TAT) domain from HIV, was linked to a chitosan/doxorubicin (chitosan/DOX) conjugate to form a chitosan/DOX/TAT hybrid. The synthesized chitosan/DOX/TAT conjugate showed a different intracellular distribution pattern from a conjugate without TAT. Unlike both free DOX and the conjugate without TAT, the chitosan/DOX/TAT conjugate was capable of efficient cell entry. The chitosan/DOX/TAT conjugate was found to be highly cytotoxic, with an IC(50) value of approximately 480 nM, 2 times less than that of chitosan/DOX (980 nM). The chitosan/DOX/TAT provided decreases in tumor volume of 77.4 and 57.5% compared to free DOX and chitosan/DOX, respectively, in tumor-bearing mice. Therefore, this study suggests that TAT-mediated chitosan/DOX conjugate delivery is effective in slowing tumor growth.

Biological effects of a porcine-derived collagen membrane on intrabony defects.

PURPOSE: To prolong the degradation time of collagen membranes, various cross-linking techniques have been developed. For cross-linking, chemicals such as formaldehyde and glutaraldehyde are added to collagen membranes, but these chemicals could adversely affect surrounding tissues. The aim of this study is to evaluate the ability of porous non-chemical cross-linking porcine-derived collagen nanofibrous membrane to enhance bone and associated tissue regeneration in one-wall intrabony defects in beagle dogs. METHODS: The second and third mandibular premolars and the first molars of 2 adult beagles were extracted bilaterally and the extraction sites were allowed to heal for 10 weeks. One-wall intrabony defects were prepared bilaterally on the mesial and distal side of the fourth mandibular premolars. Among eight defects, four defects were not covered with membrane as controls and the other four defects were covered with membrane as the experimental group. The animals were sacrificed 10 weeks after surgery. RESULTS: Wound healing was generally uneventful. For all parameters evaluating bone regeneration, the experimental group showed significantly superior results compared to the control. In new bone height (NBh), the experimental group exhibited a greater mean value than the control (3.04 ± 0.23 mm/1.57 ± 0.59, P = 0.003). Also, in new bone area (NBa) and new bone volume (NBv), the experimental group showed superior results compared to the control (NBa, 34.48 ± 10.21% vs. 5.09 ± 5.76%, P = 0.014; and NBv, 28.04 ± 12.96 vs. 1.55 ± 0.57, P = 0.041). On the other hand, for parameters evaluating periodontal tissue regeneration, including junctional epithelium migration and new cementum height, there were no statistically significant differences between two groups. CONCLUSIONS: Within the limitations of this study, this collagen membrane enhanced bone regeneration at one-wall intrabony defects. On the other hand, no influence of this membrane on periodontal tissue regeneration could be ascertained in this study.

Biological effects of a root conditioning agent for dentin surface modification in vitro.

PURPOSE: Connective tissue reattachment to periodontally damaged root surfaces is one of the most important goals of periodontal therapy. The aim of this study was to develop a root conditioning agent that can demineralize and detoxify the infected root surface. METHODS: Dentin slices obtained from human teeth were treated with a novel root planing agent for 2 minutes and then washed with phosphate-buffered saline. Smear layer removal and type I collagen exposure were observed by scanning electron microscopy (SEM) and type I collagen immunostaining, respectively. Cell attachment and lipopolysaccharides (LPS) removal demonstrated the efficiency of the root conditioning agent. RESULTS: SEM revealed that the smear layer was entirely removed and the dentinal tubules were opened by the experimental gel. Type I collagen was exposed on the surfaces of the dentin slices treated by the experimental gel, which were compared with dentin treated with other root planing agents. Dentin slices treated with the experimental gel showed the highest number of attached fibroblasts and flattened cell morphology. The agar diffusion assay demonstrated that the experimental gel also has effective antimicrobial activity. Escherichia coli LPS were effectively removed from well plates by the experimental gel. CONCLUSIONS: These results demonstrated that this experimental gel is a useful tool for root conditioning of infected root surfaces and can also be applied for detoxification of ailing implant surface threads.

Osteoblastic differentiation of human bone marrow stromal cells in self-assembled BMP-2 receptor-binding peptide-amphiphiles.

Self-assembled nanostructures consisting of BMP receptor-binding peptides, termed osteopromotive domains (OPDs), and hydrophobic alkyl chains were fabricated with the aim of developing three-dimensional scaffolding materials for osteoblastic differentiation. OPD peptide was identified from BMP-2 and had an affinity for BMP receptors thereby inducing differentiation of human bone marrow stromal cells into osteoblastic cells. The peptide-hydrophobic alkyl chain amphiphiles were designed to mimic nanofibrous extracellular structures and to add osteogenic ligands to enhance osteoblastic cell function. The OPD peptide-amphiphiles (OPDAs) that end with the alkylation of the N-terminus of the OPD peptide were synthesized by standard solid phase chemistry. The self-assembly was triggered by mixing OPDA solution with calcium ions. Observation using scanning electron microscopy (SEM) revealed the formation of nanofibrous structures with extremely high aspect ratios and high surface areas. The FT-IR and circular dichroism (CD) spectrophotometry demonstrated that self-assembled nanofibers have a beta-sheet structure. The activation of Smad, an osteoblastic differentiation marker, was obtained in the cell culture gel of self-assembled OPDA; therefore, the intracellular signal transduction for osteogenesis was performed like an OPD peptide. Cell survival was supported in the OPDA gel for 10 days, and osteoblastic differentiation of human bone marrow stromal cells (hBMSCs) was evident as demonstrated by calcein staining and ALP activity measurement. These results revealed that self-assembled OPDA maintained osteogenic activity by the surface-exposed OPD peptide. Taken together, the self-assembled OPDA nanofibrous gel can be utilized as a cell culture scaffold in bone regeneration.