3D-bioimplants mimicking the structure and function of spine units for the treatment of spinal tuberculosis.

Approximately 1-2% of the reported tuberculosis (TB) cases have skeletal system problems, particularly spinal TB. The complications of spinal TB involve the destruction of vertebral body (VB) and intervertebral disc (IVD) which consequently leads to kyphosis. This work aimed at utilizing different technologies to develop, for the first time, a functional spine unit (FSU) replacement to mimic the structure and function of the VB and IVD along with a good ability to treat spinal TB. 3D-printed scaffolds with different porous patterns (hexagonal or grid) were fabricated from biocompatible acrylonitrile butadiene styrene, and polylactic acid to replace damaged VB and IVD, respectively. The VB scaffold is filled with gelatine-based semi-IPN hydrogel containing mesoporous silica nanoparticles loaded with two antibiotics, rifampicin and levofloxacin, to act against TB. The IVD scaffold incorporates a gelatin hydrogel loaded with regenerative platelet-rich plasma and anti-inflammatory simvastatin-loaded mixed nanomicelles. The obtained results confirmed the superior mechanical strength of both 3D-printed scaffolds and loaded hydrogels as compared to normal bone and IVD with high in vitro (cell proliferation, anti-inflammation and anti-TB), and in vivo biocompatibility profiles. Moreover, the custom-designed replacements have achieved the expected prolonged release of antibiotics up to 60 days. Given the promising study findings, the utilization of the developed drug-eluting scaffold system can be extrapolated to treat not only spinal TB but also to resolve diverse backbone/spine problems that need a critical surgical process including degenerative IVD and its consequences like atherosclerosis, sliding or spondylolisthesis and severe traumatic bone fracture.

Dual antituberculosis drugs-loaded gelatin hydrogel bioimplant for treating spinal tuberculosis.

Spinal tuberculosis (TB) represents around 1% of the recorded TB with a high mortality rate due to neurological complications and kyphosis. The current work aimed to develop a bioimplant scaffold to treat spinal TB disease. The scaffold is composed of a biocompatible semi-interpenetrating (semi-IPN) gelatin-based hydrogel incorporating mesoporous silica nanoparticles (MPS-NPs) loaded with rifampicin (RIF) and levofloxacin (LEV) to treat TB. The elastic modulus of the hydrogel was 7.18 ± 0.78 MPa. Minimum inhibitory concentrations (MIC) value against Mycobacterium bovis for LEV-loaded and RIF-loaded MPS-NPs were 6.50 and 1.33 µm/ml, respectively.Sequential release of drugs was observed after 15 days. Loading of the MPS-NPs in the hydrogel matrix governed the amount of released drugs by prolonging the period of release up to 60 days. WST-1 test confirmed the biocompatibility and safety of the developed vertebral hydrogel bioimplant. Histological and immunohistochemistry micrographs showed the progress in healing process with the bioimplant. Besides, loading of LEV and RIF in the implants declined the presence of the giant macrophages clusters as compared to control groups. All the obtained results support the potential use of the developed vertebral hydrogel bioimplant as a scaffold with good mechanical and biocompatible properties along with a good ability to eradicate the TB pathogen.

Fortified gelatin-based hydrogel scaffold with simvastatin-mixed nanomicelles and platelet rich plasma as a promising bioimplant for tissue regeneration.

Treatment of intervertebral disc (IVD) degeneration includes conservative and surgical strategies that have a high risk of recurrence. Consequently, tissue engineering represents a promising alternative treatment. This study aimed at healing damaged IVD with a bioimplant that can maintain the function of defected IVD. The developed IVD scaffold is composed of a fortified biocompatible gelatin-based hydrogel to mimic the ECM mechanical properties of IVD and to allow a sustained release of loaded bioactive agents. The hydrogel is laden with platelet-rich plasma (PRP) and simvastatin (SIM)-loaded mixed pluronics nanomicelles because of their regenerative ability and anti-inflammatory effect, respectively. The gelatin-based hydrogel attained swelling of 508.9 ± 7.9 % to 543.1 ± 5.9 % after 24 h. Increasing crosslinking degree of the hydrogel improved its mechanical elasticity up to 0.3 ± 0.1 N/mm2, and retarded its degradation. The optimum mixed nanomicelles had particle size of 84 ± 0.5 nm, a surface charge of -10 ± 7.1 mv, EE% of 84.9 %, and released 88.4 % of SIM after 21 days. Cytotoxicity of IVD components was evaluated using human skin fibroblast for 3 days. WST-test results proved biocompatibility of IVD scaffold. Subcutaneous implantation of the IVD scaffold was performed for 28 days to test in-vivo biocompatibility. Histological and histochemical micrographs depicted normal healing signs such as macrophages, T-cells, angiogenesis and granulation reactions. Introducing PRP in IVD improved healing process and decreased inflammation reactions. The developed multicomponent implant could be used as potential IVD scaffold with desirable mechanical properties, biocompatibility and healing process.

Nanomicelles-in-coaxial nanofibers with exit channels as a transdermal delivery platform for smoking cessation.

Smoking is a life-threatening habit; that is why many nicotine-replacement therapies (NRTs), which include chewing gums, nicotine patches, lozenges, mouth sprays, inhalers and nasal sprays that are usually administered for 8-12 weeks, have been reported for smoking cessation. We report the fabrication of patches comprising nanomicelles-in-coaxial nanofibers (NFs) for the transdermal delivery of varenicline (VAR) tartrate, a partial agonist of the α4ß2 receptor subtype, for smoking cessation. The cores of the fabricated coaxial NF structures are composed of polyethylene oxide, VAR-loaded Pluronic F127 nanomicelles (NPs) and free VAR, while the shell consists of a blend of cellulose acetate (CA) and polycaprolactone (PCL) in a ratio of 1 : 9 (w/w) that incorporates 50% (wt%) free VAR. The morphology and the coaxial structure of the NFs were investigated using TEM, SEM and fluorescent microscopy. The physicochemical and mechanical properties of the scaffolds were analyzed using FTIR, DSC, DLS, TGA and a universal testing machine. SEM micrographs depict NFs with a size ranging from 793.7 ± 518.9 to 324.5 ± 144.1 nm. In vitro release of VAR reaches almost 100% after 3, 9 and 28 days for free VAR, VAR-loaded NPs and the NPs-in-NFs patches, respectively, while the ex vivo release tested using albino rat skin, over a period of 60 days, showed up to 94% sustained release of VAR. Besides, skin permeation, in vivo release and plasma concentrations of VAR from the NF transdermal patches were monitored via cyclic voltammetric measurements during the course of treatment. DFT calculations as well as mathematical release kinetic models were performed in order to study the release mechanism. The cell viability of human skin fibroblast (HSF) cells in the case of plain and VAR-loaded NFs was 75.09 and 32.11%, respectively. The in vivo results showed that VAR was being continuously released from the transdermal patch over a period of 14 days. Besides, the treatment with VAR-loaded patches did not cause any severe conditions in the studied animal model. The new fabricated NPs-in-NFs transdermal patch for VAR tartrate delivery is considered as an effective, economic, safe and long-acting NRT for smoking cessation.

Efficacy of biocompatible trilayers nanofibrous scaffold with/without allogeneic adipose-derived stem cells on class II furcation defects of dogs' model.

OBJECTIVE: This study aimed to evaluate the regenerative capacity of a newly-developed polycaprolactone (PCL)-based nanofibrous composite scaffold either alone or in combination with adipose-derived mesenchymal stem cells (ADSCs) as a treatment modality for class II furcation defects. MATERIALS AND METHODS: After ADSCs isolation and scaffold characterization, the mandibular premolars of adult male mongrel dogs were selected and randomly assigned into three equal groups. In group I, class II furcation defects were surgically induced to the inter-radicular bone. While class II furcation defects of group II were induced as in group I. In addition, the defects were filled with the prefabricated scaffold. Moreover, class II furcation defects of group III were induced as in group II and instead the defects were filled with the prefabricated scaffold seeded with ADSCs. The dogs were sacrificed at 30 days or at 60 days. Periodontal wound healing/regeneration was evaluated by radiological examination using cone beam computed tomography and histologically using ordinary, histochemical, and immunohistochemical staining. RESULTS: In the two examination periods, group II defects compared to group I, and group III compared to the other groups showed a decrease in defect dimensions radiographically. Histologically, histochemically, and immunohistochemically, they significantly demonstrated better periodontal wound healing/regeneration, predominant collagen type I of newly formed bone and periodontal ligament with a significant increase in the immunoreactivity of vascular endothelial growth factor and osteopontin. CONCLUSIONS: The newly fabricated nanofibrous scaffold has enhanced periodontal wound healing/regeneration of class II furcation defects with further enhancement achieved when ADSCs seeded onto the scaffold before implantation. CLINICAL RELEVANCE: The implementation of our newly-developed PCL-based nanofibrous composite scaffolds in class II furcation defect either alone or in conjunction with ADSCs can be considered as a suitable treatment modality to allow periodontal tissues regeneration.

Evaluation of the osteogenic potential of rat adipose-derived stem cells with different polycaprolactone/alginate-based nanofibrous scaffolds: an in vitro study.

BACKGROUND: Bone tissue engineering is a widely growing field that requires the combination of cells, scaffolds and signaling molecules. Adipose derived stem cells (ADSCs) are an accessible and abundant source of mesenchymal stem cells with high plasticity. Polycaprolactone/alginate (PCL/Alg) composite scaffolds have been used in bone regeneration and nano-hydroxyapatite (n-HA) is used as a reinforcing, osteoconductive component in scaffold fabrication. This study was conducted to assess the ability of three different PCL/Alg based scaffolds to induce osteogenic differentiation of ADSCs and to compare between them. METHODS: The study comprised 5 groups; negative control group with ADSCs cultured in complete culture media, positive control group with ADSCs cultured in osteogenic differentiation media, and 3 experimental groups with ADSCs seeded onto 3 scaffolds: S1 (PCL/Alg), S2 (PCL/Alg/Ca) and S3 (PCL/Alg/Ca/n-HA) respectively and cultured in osteogenic media. Mineralization and gene expression were assessed by Alizarin red S (ARS) staining and real time quantitative polymerase chain reaction (RT-qPCR). Evaluation was done at 7, 14 and 21 days. RESULTS: ARS staining reflected a time dependent increase through days 7, 14 and 21, with S3 (PCL/Alg/Ca/n-HA) group showing the highest mineralization levels. RT-qPCR detected upregulation of ALP gene expression at day 7 and decline thereafter. S2 (PCL/Alg/Ca) and S3 (PCL/Alg/Ca/n-HA) groups showed significantly higher gene expression levels than S1 (PCL/Alg). CONCLUSIONS: ADSCs and PCL/Alg-based scaffolds compose a good tissue engineering complex for bone regeneration. Addition of n-HA to PCL/Alg scaffolds and crosslinking with CaCl2 efficiently improve the osteogenic potential of ADSCs.

Niclosamide-loaded polymeric micelles ameliorate hepatocellular carcinoma in vivo through targeting Wnt and Notch pathways.

AIM: Niclosamide (NIC) is an anthelmintic agent repurposed as a potent anticancer agent. However, its use is hindered by its poor solubility. We investigated the underlying mechanisms of NIC anticancer activity employing a novel oral NIC pluronic-based nanoformulation and tested its effect in thioacetamide-induced hepatocellular carcinoma (HCC) in rats. We evaluated its antitumor effect through regulating Wnt/ß-catenin and Notch signaling pathways and apoptosis. MAIN METHODS: Niclosamide-loaded pluronic nanoparticles (NIC-NPs) were optimally developed and characterized with sustained release properties up to 7 days. Sixteen weeks after HCC induction, NIC (70 mg/kg) and an equivalent dose of NIC-NPs were administered orally for 3 consecutive weeks. Hepatocyte integrity was assessed by measuring serum levels of aminotransferases, ALP, GGT, bilirubin, albumin and total protein. HCC development was detected by measuring AFP expression. Necroinflammation and fibrosis were scored by histopathological examination. Wnt/ß-catenin and Notch signaling were evaluated by measuring hepatic mRNA levels of Wnt3A, Lrp5 and Lrp6 Co-receptors, Dvl-2, Notch1 and Hes1 and ß-catenin protein levels. Apoptosis was assessed by measuring mRNA and protein levels of cyclin D1 and caspase-3. KEY FINDING: The novel NIC-NPs restored liver integrity, reduced AFP levels and showed improved anticancer and proapoptotic activities compared to drug alone. The inhibitory effect of NIC on Wnt/ß-catenin and Notch signaling pathways was potentiated by the NIC-NPs formulation. SIGNIFICANCE: We conclude that NIC acts by inhibiting Wnt/ß-catenin and Notch signaling and inducing apoptosis in HCC. Developing pluronic-based nanoformulations may be a promising approach to improve NIC solubility and offer the possibility of controlled release.

Sandwich-Like Nanofibrous Scaffolds for Bone Tissue Regeneration.

Advanced bone healing approaches included a wide range of biomaterials that mainly mimic the composition, structure, and properties of bone extracellular matrix with osteogenic activity. The present study aimed to develop a sandwich-like structure of electrospun nanofibers (NFs) based on polycaprolactone (PCL) and chitosan/polyethylene oxide (CS/PEO) composite to stimulate bone fracture healing. The morphology of the fabricated scaffolds was examined using scanning electron microscopy (SEM). Apatite deposition was evaluated using simulated body fluid (SBF). The physicochemical and mechanical properties of samples were analyzed by Fourier transform infrared, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and universal testing machine. SEM images exhibited a porous three-dimensional structure with NF diameters of 514-4745 nm and 68-786 nm for PCL NFs layer and the sandwich-like NFs scaffolds, respectively. Deposition of apatite crystal on scaffolds started at week 2 followed by heavy deposition at week 8. This was confirmed by measuring the consumption of calcium and phosphorous ions from SBF. Thermal stability of scaffolds was confirmed using DSC and TGA. Moreover, the PCL NF layer in the middle of the developed sandwich structure reinforced the scaffolds with bear load up to 12.224 ± 1.12 MPa and Young's modulus of 17.53 ± 3.24 MPa. The scaffolds' porous structure enhanced both cell propagation and proliferation. Besides, the presence of CS in the outer NF layers of the scaffolds increased the hydrophilicity, as evidenced by the reduction of contact angle from 116.6 to 57.6°, which is essential for cell attachment. Cell viability study on mesenchymal stem cells proved the cytocompatibility of the fabricated scaffolds. Finally, in vivo mandibular bone defect rabbit model was used to confirm the regeneration of a new healthy bone within 28 days. In conclusion, the developed scaffolds could be a promising solution to stimulate bone regeneration.

In vivo evaluation of ß-CS/n-HA with different physical properties as a new bone graft material.

BACKGROUND: Natural polymer composite materials are becoming increasingly important as scaffolds for bone tissue engineering. Composite materials based on combinations of biodegradable polymers and bioactive ceramics, including CTS and Hap. PURPOSE: ß-Chitosan/n-HA composite with different percentages was prepared. Some of the physical and mechanical properties were examined by using (scanning electron microscope and transmission electron microscope). Histological evaluation of in vivo implantation of ß-Chitosan/n-HA composite as bone graft material was done. MATERIAL AND METHODS: ß-type chitosan was obtained through a modified procedure from squid pens (Loligo vulgaris). It was used in combination with different proportions of nano-hydroxyapatite (n-HA), to develop new series of ß-CS/n-HA nanocomposites. Sample were obtained in a powder form with the ratio of 30 CH to 70% nHA. The product was implanted in the femoral condyle of the animals (adult rabbits). RESULTS: Compact strength was 13.05 MPa for the weight ratio of 30/70. Histological examinations showed that the implant not only biological compatible but also its presence promotes and accelerate bone growth. CONCLUSIONS: The composite ß-CS/HPa (30/70) as biodegradable bone substitute that not only enhance bone generation but also accelerate the formation of Haversian system. We used the composite in a powder form and examined its suitability as artificial bone graft; yet the mechanical properties have shown that 30/70 ratio of ß-CS/HPa offer suitable mechanical strength to be employed as a solid-shaped implants.