Injectable demineralized bone matrix particles and their hydrogel bone grafts loaded with ß-tricalcium phosphate powder and granules: A comparative study.

Demineralized bone matrix (DBM), has been used as a bone-graft material because of its osteoconductivity and osteoinductivity. However, the previous research report that supports the single use of DBM is limited by its rapid resorption caused by the lack of calcium and phosphate. ß-Tricalcium phosphate (TCP) is an enriched calcium phosphate material suitable for bone healing with osteoconductive properties. In this study, we have developed injectable bone graft by the loading two kinds of TCP in DBM particles and thermo-sensitive DBM-derived hydrogel (hDBM). TCP powder (pTCP) and TCP granules (gTCP) were loaded into hDBM and DBM, respectively. The bone formation effect was investigated according to the morphological features of TCP. Residual growth factor concentrations were investigated; microstructure and morphology were characterized by SEM. In-vitro studies showed that hDBM/DBM/pTCP and hDBM/DBM/gTCP bone grafts were biocompatible and could promote osteogenesis by up-regulating the expression of Runx2 and OPN, bone-related genes. In-vivo studies using the rabbit-femur defect model revealed that the implanted hDBM/DBM/pTCP bone graft showed similar histology to that of fibrous dysplasia with the expression of CD68, whereas hDBM/DBM/gTCP showed good bone formation. Loading of gTCP in place of pTCP was noticed as an effective way to improve bone regeneration in an injectable hDBM/DBM hydrogel-based bone graft.

Liver tissue-derived ECM loaded nanocellulose-alginate-TCP composite beads for accelerated bone regeneration.

Guided bone regeneration with osteoinductive scaffolds is a competitive edge of tissue engineering due to faster and more consistent healing. In the present study, we developed such composite beads with nanocellulose reinforced alginate hydrogel that carriedß-tricalcium phosphate (ß-TCP) nano-powder and liver-derived extracellular matrix (ECM) from porcine. Interestingly, it was observed that the beads' group containing ECM-ß-TCP-alginate-nanocellulose (ETAC) was more cytocompatible than the others comprised ofß-TCP-alginate-nanocellulose (TAC) and alginate-nanocellulose (AC). Cell attachment on ETAC beads was dramatically increased with time. In parallel within vitroresults, ETAC beads produced uniform cortical and cancellous bone in the femur defect model of rabbits within 2 months. Although the group TAC also produced noticeable bone in the defect site, the healing quality was improved and regeneration was faster after adding ECM. This conclusion was not only confirmed by micro-anatomical analysis but also demonstrated with x-ray microtomography. In addition, the characteristic moldable and injectable properties made ETAC a promising scaffold for clinical applications.

Physico-mechanical and biological evaluation of heparin/VEGF-loaded electrospun polycaprolactone/decellularized rat aorta extracellular matrix for small-diameter vascular grafts.

Although the continuous development of small-diameter vascular grafts (SDVGs) (D < 5 mm) continues, most vascular grafts are made from synthetic polymers, which lead to serious complications from arteriosclerosis, thrombosis, and vascular ischemia. Here, to address these shortcomings, we combine synthetic polymers with natural decellularized small-diameter vessels and loaded with growth factor. We fabricated vascular grafts by electrospinning polycaprolactone (PCL) to decellularized rat aorta matrix (ECM) followed by heparin and vascular endothelial growth factor (VEGF) loading. In- vitro studies showed that PCL/ECM/VEGF vascular grafts, showed excellent hemocompatibility and biocompatibility properties. The vascular grafts implanted into the rat aorta revealed that the PCL/ECM/VEGF grafts promotes endothelial cells and smooth-muscle cells infiltration with a rate of FLK-1, ICAM1, and a-SMA distribution higher than that of the PCL and PCL/ECM vascular grafts at 2 weeks and 4 weeks after implantation. The PCL/ECM/VEGF vascular graft should be considered for potential small-diameter vascular grafts in clinical fields.

Synthesis and characterization of biphasic calcium phosphate laden thiolated hyaluronic acid hydrogel based scaffold: physical and in-vitro biocompatibility evaluations.

The present study focused on the combination of biphasic calcium phosphate (BCP) nanoparticles into the modified hyaluronic acid based injectable hydrogels for bone tissue engineering. Self-cross-linked thiolated hyaluronic acid (HA-HS) injectable hydrogels loaded with biphasic calcium phosphate (BCP) nanoparticles were prepared by disulfide cross-linking to mimic the extracellular matrix as a potential material for bone treatment. Varying concentration of HA-HS ranging between 1 and 5w/v% was tested to optimize the optimum concentration and were further modified with varying BCP concentrations for final optimization. Physico-chemical characterizations of the prepared hydrogel such as SEM, EDS, FT-IR, and XRD confirmed that the BCP has effectively loaded and distributed homogeneously in the HA-HS hydrogel. The results showed that the 3% (w/v) HA-HS hydrogel exhibits the appropriate properties for injectable hydrogel system such as gelation times, swelling rate and in vitro degradation behavior among all tested concentrations. Cell viability and cell proliferation using osteoblast cells (MC3T3-E1) demonstrated that the BCP laden modified hydrogel are biocompatible in vitro. In light of the encouraging results obtained, BCP laden HA-HS hydrogels might offer the potential to be used as injectable hydrogel in bone tissue engineering.

Comparative study on biodegradation and biocompatibility of multichannel calcium phosphate based bone substitutes.

The objective of this study was to fabricate multichannel biphasic calcium phosphate (BCP) and ß-tricalcium phosphate (TCP) bone substitutes and compare their long-term biodegradation and bone regeneration potentials. Multi-channel BCP and TCP scaffolds were fabricated by multi-pass extrusion process. Both scaffolds were cylindrical with a diameter of 1-mm, a length of 1-mm, and seven interconnected channels. Morphology, chemical composition, phase, porosity, compressive strength, ion release behavior, and in-vitro biocompatibility of both scaffolds were studied. In-vivo biodegradation and bone regeneration efficacies of BCP and TCP were also evaluated using a rabbit model for 1 week, 1 month, and 6 months. BCP exhibited superior compressive strength compared to TCP scaffold. TCP showed higher release of both calcium ions and phosphorous ions than BCP in SBF solution. Both scaffolds showed excellent in-vitro biocompatibility and upregulated the expression of osteogenic markers of MC3T3-E1 cells. In-vivo studies revealed that both cylindrical TCP and BCP scaffolds were osteoconductive and supported new bone formation. Micro-CT data showed that the bone-regeneration efficacy of TCP was higher at one month and at six months after implantation. Histological examination confirmed that TCP degraded faster and had better bone regeneration than BCP after 6 months.

TEMPO oxidized nano-cellulose containing thermo-responsive injectable hydrogel for post-surgical peritoneal tissue adhesion prevention.

The objective of this study was to present an effective injectable adhesion barrier comprised of TEMPO-oxidized cellulose nanofiber (TOCN), methyl cellulose, carboxymethyl cellulose, and polyethylene glycol. Hydrogels with different concentrations (0.2, 0.5, 0.8, 1% w/v) of bio compatible TOCN were investigated to determine their abilities to prevent post-surgical peritoneal adhesion using a rat cecal wall abrasion model. Sol-gel transition at body temperature (37 °C) was optimized by adjusting concentration of sodium ions (Na+), with a gelation time of 45 ±â€¯7 s. These TOCN containing hydrogels showed non cytotoxicity to rat bone marrow mesenchymal stem cells (RBMSCs) and L929 fibroblast cells as cell models during in vitro assessment. Degradation studies revealed that, TOCN concentration in hydrogel was inversely proportional to hydrolytic degradation rate. From in vivo evaluations, TOCN 0.2 hydrogel significantly reduced peritoneal adhesion in rat (n = 8) compared to untreated controls based on gross observation, histological analysis, and expression analysis of marker proteins. By taking advantages of thermo gelling, high stability, non-invasive way of application and rapid recovery potential, TOCN containing bio compatible hydrogel could be used as a cost-effective barrier to efficiently inhibit post-surgical peritoneal adhesions.

Development and properties of duplex MgF2/PCL coatings on biodegradable magnesium alloy for biomedical applications.

The present work addresses the performance of polycaprolactone (PCL) coating on fluoride treated (MgF2) biodegradable ZK60 magnesium alloy (Mg) for biomedical application. MgF2 conversion layer was first produced by immersing Mg alloy substrate in hydrofluoric acid solution. The outer PCL coating was then prepared using dip coating technique. Morphology, elements profile, phase structure, roughness, mechanical properties, invitro corrosion, and biocompatibility of duplex MgF2/PCL coating were then characterized and compared to those of fluoride coated and uncoated Mg samples. The invivo degradation behavior and biocompatibility of duplex MgF2/PCL coating with respect to ZK60 Mg alloy were also studied using rabbit model for 2 weeks. SEM and TEM analysis showed that the duplex coating was uniform and comprised of porous PCL film (~3.3 µm) as upper layer with compact MgF2 (~2.2 µm) as inner layer. No significant change in microhardness was found on duplex coating compared with uncoated ZK60 Mg alloy. The duplex coating showed improved invitro corrosion resistance than single layered MgF2 or uncoated alloy samples. The duplex coating also resulted in better cell viability, cell adhesion, and cell proliferation compared to fluoride coated or uncoated alloy. Preliminary invivo studies indicated that duplex MgF2/PCL coating reduced the degradation rate of ZK60 Mg alloy and exhibited good biocompatibility. These results suggested that duplex MgF2/PCL coating on magnesium alloy might have great potential for orthopedic applications.

In vitro and in vivo evaluation of effectiveness of a novel TEMPO-oxidized cellulose nanofiber-silk fibroin scaffold in wound healing.

In this study, a novel TEMPO-oxidized cellulose nanofiber (TOCN)-silk fibroin scaffold was prepared using a cost effective freeze drying method. Fundamental physical characterizations were carried out by scanning electron microscopy (SEM), pore diameter determination, FT-IR. PBS uptake behavior of the scaffold showed that, silk fibroin can enhance the swelling capacity of TOCN. L929 primary fibroblast cell was selected for in vitro studies, which showed that the scaffolds facilitated growth of cells. In vivo evaluation of TOCN, TOCN-silk fibroin composites was examined using critical sized rat skin excisional model for one and two weeks. The results of rat wound model revealed that, compared to only TOCN scaffold, TOCN-silk fibroin scaffold successfully promoted wound healing by the expression of wound healing markers. TOCN-silk fibroin 2% has the fastest wound healing capacity. Thus, it appears that TOCN-silk fibroin composite scaffolds can be useful as wound healing material in clinical applications.

Sorting nexin-4 regulates ß-amyloid production by modulating ß-site-activating cleavage enzyme-1.

BACKGROUND: Amyloid precursor protein (APP) is cleaved by ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) to produce ß-amyloid (Aß), a critical pathogenic peptide in Alzheimer's disease (AD). Aß generation can be affected by the intracellular trafficking of APP or its related secretases, which is thus important to understanding its pathological alterations. Although sorting nexin (SNX) family proteins regulate this trafficking, the relevance and role of sorting nexin-4 (SNX4) regarding AD has not been studied yet. METHODS: In this study, human brain tissue and APP/PS1 mouse brain tissue were used to check the disease relevance of SNX4. To investigate the role of SNX4 in AD pathogenesis, several experiments were done, such as coimmunoprecipitation, Western blotting, immunohistochemistry, and gradient fractionation. RESULTS: We found that SNX4 protein levels changed in the brains of patients with AD and of AD model mice. Overexpression of SNX4 significantly increased the levels of BACE1 and Aß. Downregulation of SNX4 had the opposite effect. SNX4 interacts with BACE1 and prevents BACE1 trafficking to the lysosomal degradation system, resulting in an increased half-life of BACE1 and increased production of Aß. CONCLUSIONS: We show that SNX4 regulates BACE1 trafficking. Our findings suggest novel therapeutic implications of modulating SNX4 to regulate BACE1-mediated ß-processing of APP and subsequent Aß generation.

Autophagy in microglia degrades extracellular ß-amyloid fibrils and regulates the NLRP3 inflammasome.

Accumulation of ß-amyloid (Aß) and resultant inflammation are critical pathological features of Alzheimer disease (AD). Microglia, a primary immune cell in brain, ingests and degrades extracellular Aß fibrils via the lysosomal system. Autophagy is a catabolic process that degrades native cellular components, however, the role of autophagy in Aß degradation by microglia and its effects on AD are unknown. Here we demonstrate a novel role for autophagy in the clearance of extracellular Aß fibrils by microglia and in the regulation of the Aß-induced NLRP3 (NLR family, pyrin domain containing 3) inflammasome using microglia specific atg7 knockout mice and cell cultures. We found in microglial cultures that Aß interacts with MAP1LC3B-II via OPTN/optineurin and is degraded by an autophagic process mediated by the PRKAA1 pathway. We anticipate that enhancing microglial autophagy may be a promising new therapeutic strategy for AD.

CA-074Me, a cathepsin B inhibitor, decreases APP accumulation and protects primary rat cortical neurons treated with okadaic acid.

Upregulation of the lysosomal system has been suggested to contribute to the pathogenesis of Alzheimer's disease (AD). But the exact role of this system remains unknown. Okadaic acid (OA), a protein phosphatase-2A inhibitor, increases tau phosphorylation, ß-amyloid deposition, and neuronal cell death, which are the pathological hallmarks of AD. To investigate the role of lysosomal activation in AD brain cells, cultured neurons were treated with OA and assessed lysosomal morphology and enzyme activity and the protective effect of cathepsin B, D, or L inhibitors. It was found that although it induced lysosomal swelling and enzyme activation, OA did not induce lysosomal rupture. While inhibition of cathepsin D and L failed to protect neurons from OA-induced cell death, CA074-Me, a cathepsin B inhibitor, conferred a protective effect. Interestingly, CA-074Me reduced amyloid precursor protein (APP) accumulation and α-spectrin cleavage, similar to the effect of calpain inhibition.

Selective apoptotic effect of Zelkova serrata twig extract on mouth epidermoid carcinoma through p53 activation.

Apoptosis or programmed cell death plays an essential role in chemotherapy-induced tumor cell killing, and inducers of apoptosis are commonly used in cancer therapy. Treatment with Zelkova serrata extracts was performed in human gingival fibroblast (HGF), mouth epidermoid carcinoma cell (KB), lower gingival squamous cancer cell (YD38) and tongue mucoepidermoid carcinoma cells (YD15). We observed that extract prepared from Zelkova serrata twig selectively inhibited proliferation of various oral cancer cells, but not normal gingival fibroblasts, in a dose-dependent manner. Caspase-8-mediated apoptosis was induced by treatment with the extract only in mouth epidermoid carcinoma and not in other types of cancer cells, including lower gingival squamous cell carcinoma. The selective apoptotic effect of Zelkova serrata twig extract in mouth epidermoid carcinoma was dependent on normal p53 status. Apoptosis was not remarkably induced by treatment with the extract in either lower gingival squamous or tongue mucoepidermoid carcinoma cells, both of which contain abnormalities of p53. Upon treatment with Zelkova serrata twig extract, mouth epidermoid carcinoma cells accumulated in S phase by activation of p21. These data indicate that Zelkova serrata twig extract exerted a cancer type-specific, p53-dependent apoptotic effect and disturbed the cell cycle, which suggests that herbal medicine could be a treatment for specific types of cancers.

Purification and proteomic identification of putative upstream regulators of polo-like kinase-1 from mitotic cell extracts.

Polo-like kinase-1 (Plk1) is phosphorylated on Thr210 for activation during mitosis. Here, we investigated the question of which kinase(s) is the specific upstream kinase of mitotic Plk1. Upstream kinases of Plk1 were purified from mitotic cell extracts through column chromatography procedures, and identified by mass spectrometry. Candidates for Plk1 kinase included p21-activated kinase, aurora A, and mammalian Ste20-like kinases. Immunoprecipitates of these proteins from mitotic cell extracts phosphorylated Plk1 on Thr210. Even if the activity of Aurora A was blocked with a specific inhibitor, Plk1 phosphorylation still occurred, suggesting that function of Plk1 could be controlled by these kinases for proper mitotic progression, as well as by Aurora A in very late G2 phase for the beginning of mitosis.