In vivo neural tissue engineering using adipose-derived mesenchymal stem cells and fibrin matrix.

BACKGROUND: The multipotency of adipose-derived mesenchymal stem cells (ADMSC) could be an advantage to regenerate tissues with multiple cell types. However, due to the hostile nature, trauma sites like spinal cord injury can augment the ADMSC differentiation into undesirable lineages. Immersing pre-differentiated neural progenitors in a biomimetic niche during delivery could guard them against any undesired differentiation or death. OBJECTIVE: The study proposes using an insoluble cell-specific fibrin niche for in vitro differentiation of rat ADMSCs to neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs). Further, the study explores fibrin hydrogel for in vivo progenitor cell delivery, and that can aid post-transplant survival/differentiation. DESIGN: The in vitro experiments analyzed for differentiation-specific markers to establish derivation of rADMSCs to rNPCs and rOPCs. The derived progenitors, tagged with fluorescent tracker dye were delivered in rat T10 contusion SCI using fibrin hydrogel. After 28 days, imaged the experiment site to determine cell survival, immunostained the tissues to identify differentiation of transplanted cells, and evaluated the effect of fibrin and cells on regulating the injury-associated immune response. RESULTS: The study demonstrated fibrin niche aided stable differentiation of rat ADMSCs into neural progenitors. Fibrin matrix holds up the delivered progenitor cells in the SCI site. The H&E stained tissues revealed regulated cavitation, astrogliosis, and inflammation in test tissues. Progression of transplanted cells into oligodendrocytes upon delivering a mixture of rNPCs, rOPCs, and fibrin is evident. CONCLUSION: Fibrin niche-based derivation of neural progenitors from ADMSC seems valuable for transplantation using fibrin hydrogel. It is a promising strategy for extensive study towards further development of translational stem cell-based neural replacement therapy.

Silk Fibroin-Based Bioengineered Scaffold for Enabling Hemostasis and Skin Regeneration of Critical-Size Full-Thickness Heat-Induced Burn Wounds.

Millions of people around the globe are affected by full-thickness skin injuries. A delay in the healing of such injuries can lead to the formation of chronic wounds, posing several clinical and economic challenges. Current strategies for wound care aim for skin regeneration and not merely skin repair or faster wound closure. The present study aimed to develop a bioactive wound-healing matrix comprising natural biomaterial silk fibroin (SF), clinical-grade human fibrin (FIB), and human hyaluronic acid (HA), resulting in SFFIBHA for regeneration of full-thickness burn wounds. A porous, hemostatic, self-adhesive, moisture-retentive, and biomimetic scaffold that promotes healing was the expected outcome. The study validated a terminal sterilization method, suggesting the stability and translational potential of the novel scaffold. Also, the study demonstrated the regenerative abilities of scaffolds using in vitro cell culture experiments and in vivo full-thickness burn wounds of critical size (4 cm × 4 cm) in a rabbit model. Under in vitro conditions, the scaffold enhanced primary dermal fibroblast adhesion and cell proliferation with regulated extracellular matrix (ECM) synthesis. In vivo, the scaffolds promoted healing with mature epithelium coverage involving intact basal cells, superficial keratinocytes, multilayers of keratohyalin, dermal regeneration with angiogenesis, and deposition of remodeled ECM in 28 days. The relative gene expression of the IL6 marker indicated transitions from inflammation to proliferation stage. In addition, we observed skin appendages and rete peg development in the SFFIBHA-treated wound tissues. Although wound closure was observed, neither negative (untreated/sham) nor positive (commercially available product; NeuSkin) control wounds developed skin appendages/rete pegs or native skin architecture. After 56 days, healing with organized ECM production enabled the recovery of mechanical properties of skin with higher tissue maturity in SFFIBHA-treated wounds. Thus, in a single application, the SFFIBHA scaffold proved to be an efficient biomimetic matrix that can guide burn wound regeneration. The developed matrix is a suture-less, hemostatic, off-the-shelf product for potential wound regenerative applications.

Cellular and sub-chronic toxicity of hydroxyapatite porous beads loaded with antibiotic in rabbits, indented for chronic osteomyelitis.

Bioceramics have emerged as a hopeful remedy for site-specific drug delivery in orthopaedic complications, especially in chronic osteomyelitis. The bioresorbable nature of bioceramic materials shaped them into a versatile class of local antibiotic delivery systems in the treatment of chronic osteomyelitis. Hydroxyapatite (HA) based bioceramics with natural bone mimicking chemical composition are of particular interest due to their excellent biocompatibility, better osteoconductive and osteointegrative properties. Although HA has been widely recognized as an efficient tool for local delivery of antibiotics, information regarding its subchronic systemic toxicity have not been explored yet. Moreover, a detailed investigation of in vivo subchronic systemic toxicity of HA is critical for understanding its biocompatibility and futuristic clinical applications of these materials as novel therapeutic system in its long haul. Evaluation of biocompatibility and sub-chronic systemic toxicity are significant determinants in ensuring biomedical device's long-term functionality and success. Sub-chronic systemic toxicity allows assessing the potential adverse effects caused by leachable and nanosized wear particles from the device materials under permissible human exposure to the distant organs that are not in direct contact with the devices. In this context, the present study evaluates the sub-chronic systemic toxicity of in-house developed Hydroxyapatite porous beads (HAPB), gentamicin-loaded HAPB (HAPB + G) and vancomycin- loaded HAPB (HAPB + V) through 4 and 26-week muscle implantation in New Zealand white rabbits, as per ISO 10993-6 and ISO 10993-11. Analysis of cellular responses of HAPB towards Human Osteosarcoma (HOS) cell line through MTT assay, direct contact cytotoxicity, live/dead assay based on Imaging Flow Cytometry (IFC) showed its non-cytotoxic behaviour. Histopathological analysis of muscle tissue, organs like heart, lungs, liver, kidney, spleen, adrenals, intestine, testes, ovaries, and uterus did not reveal any abnormal biological responses. Our study concludes that the HAPB, gentamicin-loaded HAPB (HAPB + G) and vancomycin-loaded HAPB (HAPB + V) are biocompatible and did not induce sub-chronic systemic toxicity and hence satisfies the criteria for regulatory approval of HAs as a plausible candidate for clinical applications.

Asialoglycoprotein receptor targeted optical and magnetic resonance imaging and therapy of liver fibrosis using pullulan stabilized multi-functional iron oxide nanoprobe.

Early diagnosis and therapy of liver fibrosis is of utmost importance, especially considering the increased incidence of alcoholic and non-alcoholic liver syndromes. In this work, a systematic study is reported to develop a dual function and biocompatible nanoprobe for liver specific diagnostic and therapeutic applications. A polysaccharide polymer, pullulan stabilized iron oxide nanoparticle (P-SPIONs) enabled high liver specificity via asialogycoprotein receptor mediation. Longitudinal and transverse magnetic relaxation rates of 2.15 and 146.91 mM-1 s-1 respectively and a size of 12 nm, confirmed the T2 weighted magnetic resonance imaging (MRI) efficacy of P-SPIONs. A current of 400A on 5 mg/ml of P-SPIONs raised the temperature above 50 °C, to facilitate effective hyperthermia. Finally, a NIR dye conjugation facilitated targeted dual imaging in liver fibrosis models, in vivo, with favourable histopathological results and recommends its use in early stage diagnosis using MRI and optical imaging, and subsequent therapy using hyperthermia.

Similar regeneration of articular cartilage defects with autologous & allogenic chondrocytes in a rabbit model.

Background & objectives: Articular cartilage defects in the knee have a very poor capacity for repair due to avascularity. Autologous chondrocyte transplantation (ACT) is an established treatment for articular cartilage defects. Animal studies have shown promising results with allogenic chondrocyte transplantation since articular cartilage is non-immunogenic. In addition to being economical, allogenic transplantation has less morbidity compared to ACT. This study was undertaken to compare ACT with allogenic chondrocyte transplantation in the treatment of experimentally created articular cartilage defects in rabbit knee joints. Methods: Cartilage was harvested from the left knee joints of six New Zealand white rabbits (R1-R6). The harvested chondrocytes were cultured to confluence and transplanted onto a 3.5 mm chondral defect in the right knees of 12 rabbits [autologous in 6 rabbits (R1-R6) and allogenic in 6 rabbits (R7-R12)]. After 12 wk, the rabbits were euthanized and histological evaluation of the right knee joints were done with hematoxylin and eosin and safranin O staining. Quality of the repair tissue was assessed by the modified Wakitani histological grading scale. Results: Both autologous and allogenic chondrocyte transplantation resulted in the regeneration of hyaline/mixed hyaline cartilage. The total histological scores between the two groups showed no significant difference. Interpretation & conclusions: Allogenic chondrocyte transplantation seems to be as effective as ACT in cartilage regeneration, with the added advantages of increased cell availability and reduced morbidity of a single surgery.

Citrate coated iron oxide nanoparticles with enhanced relaxivity for in vivo magnetic resonance imaging of liver fibrosis.

Superparamagnetic iron oxide nanoparticles are widely used for the magnetic resonance imaging (MRI) applications. The surface characteristics, magnetic properties, size and targeting efficiency of the material are crucial factors for using the same as contrast agents. We report a simple synthesis method of citrate coated iron oxide nanoparticles and its systematic characterization. The developed system is highly water dispersible with an average particle size of 12 nm. The particles in water are monodisperse and are found to be stable over long periods. The efficiency of the material to de-phase water proton has been studied for various concentrations of iron using longitudinal (T1) and transverse (T2) weighted MRI. The coating thickness of the nanoparticle was optimized so that they exhibited a high transverse to longitudinal relaxivity (r2/r1) ratio of 37.92. A clear dose-dependent contrast enhancement was observed in T2 weighted in vivo MR imaging of liver fibrosis model in rodents. The labelling efficacy of the particle and the intracellular magnetic relaxivity were also investigated and presented. The particles were also tested for blood and cellular compatibility studies. Development of fibrosis and presence of iron in the liver was confirmed by histopathological analysis. From this study, we conclude that the citrate coated ultra small superparamagnetic iron oxide nanoparticles (C-USPION) with optimized parameters like particle size and magnetic property are capable of producing good MR contrast in imaging of liver diseases.

Determination of oxidative stress related toxicity on repeated dermal exposure of hydroxyapatite nanoparticles in rats.

Hydroxyapatite nanoparticles (HANPs) have numerous applications, such as substitute for bone grafting, bone fillers, bioceramic coating, and dental fillings. The toxicity of these nanomaterials is of growing concern despite their significant scientific interest and promising potential in many applications. In this study, an in-house synthesized, characterized HANP of size <50 nm was investigated for the dermal toxicity. A paste of HANPs was prepared in water and applied on the dorsal side of the rats for 28 days. At the end of 28 days, blood was subjected to haematological and biochemical analysis. Gross necropsy was conducted and major organs were collected for histopathological observations. Liver from the animals was evaluated for lipid peroxidation, reduced glutathione, and antioxidant enzymes activity. It was observed that none of the animals showed any abnormality during the experimental period. Gross examination of carcasses did not reveal any abnormality in the organs examined. The results also demonstrated that there was no significant fluctuation in the level of antioxidant defense mechanisms, lipid peroxidation, and haematological and biochemical parameters. There was no histopathological lesion observed in any of the organs. Hence, it can be concluded that the synthesized HANPs were nontoxic at cellular level, when exposed dermally to rats.

Synthesis and characterization of dextran stabilized superparamagnetic iron oxide nanoparticles for in vivo MR imaging of liver fibrosis.

The field of medical imaging has recently seen rapid advances in the development of novel agents for enhancing image contrast. In particular, superparamagnetic iron oxide nanoparticles (SPIONs) with a variety of surface properties have been tried as effective contrast agents for magnetic resonance imaging, but with major side effects. In this study, the surface chemistry of SPIONs of size 12 nm was modified with high molecular weight dextran to yield particles of size 50 nm, without compromising the magnetic properties. A systematic characterization of the material for its size, coating efficiency, magnetic properties and biocompatibility has been carried out. The magnetic relaxivity as evaluated on a 1.5 T clinical magnet showed r2/r1 ratio of 56.28 which is higher than that reported for any other dextran stabilized ironoxide nanoparticles. Liver uptake and magnetic resonance imaging potential of dextran stabilized SPIONs (D-SPIONs) has been evaluated on liver fibrosis induced animal model, which is further supported by histopathology results.

Pulsed electromagnetic field therapy results in healing of full thickness articular cartilage defect.

This study aimed to determine the efficacy of PEMF (pulsed electromagnetic field) treatment in experimental osteochondral defect healing in a rabbit model. The study was conducted on 12 New Zealand white rabbits. Six rabbits formed the study group and six rabbits the control group. The right knee joints of all 12 animals were exposed and a 3.5-mm diameter osteochondral defect was created in the trochlear groove. The defect was filled with calcium phosphate scaffold. Six animals from the study group were given PEMF of one hour duration once a day for six weeks with set parameters for frequency of 1 Hz, voltage 20 V, sine wave and current ±30 mA. At six weeks the animals were sacrificed and histological evaluation was done using H&E, Safranin O, Maissons trichrome staining and immunohistochemistry for type 2 collagen. The quality of the repair tissue was graded and compared between groups with the Wakitani histological grading scale and a statistical analysis was done. The total histological score was significantly better in the study group (p = 0.002) with regeneration similar to adjacent normal hyaline cartilage. Immunohistochemistry for collagen type II was positive in the study group. PEMF stimulation of osteochondral defects with calcium phosphate scaffold is effective in hyaline cartilage formation. PEMF is a non-invasive and cost effective adjuvant treatment with salvage procedures such as abrasion chondroplasty and subchondral drilling.

In vitro and in vivo performance of a dual drug-eluting stent (DDES).

This study reports on a dual drug-eluting stent (DDES) that has an anti-proliferative and an anti-thrombotic in a biodegradable polymer-coated onto a cobalt-chromium stent. The DDES was prepared by spray coating the bare metal stent with a biodegradable polymer loaded with sirolimus and triflusal, to treat against restenosis and thrombosis, respectively. The 2-layered dual-drug coated stent was characterized in vitro for surface properties before and after expansion, as well as for possible delamination by cross-sectioning the stent in vitro. The in vitro anti-platelet behavior of the triflusal-loaded films was investigated by using dynamic platelet adhesion measurements. Additionally, the in vitro degradation and release study of the films and the stents w/single sirolimus and dual sirolimus-triflusal in different formulations were examined. Finally, in vivo studies (in a porcine carotid artery model) were performed for acute thrombosis, inflammation and restenosis at 30 days. The in vitro results show DDES can sustain release both anti-proliferation drug (sirolimus) and anti-thrombosis drug (triflusal), two drugs were controlled in different rates to effectively reduce thrombosis and proliferation at the same time. In vivo results show a significant reduction in restenosis with dual-drug eluting stent compared with the controls (a bare metal stent, a sirolimus coated and a pure polymer-coated stent). The reduction in restenosis with a dual sirolimus-triflusal eluting stent is associated with an inhibition of inflammation, especially thrombus formation, suggesting that such dual-drug eluting stents have a role to play for the treatment of coronary artery disease.

The short-term effect on restenosis and thrombosis of a cobalt-chromium stent eluting two drugs in a porcine coronary artery model.

The aim of this article was to study the effect of dual drug-eluting stent (DES) on both restenosis and thrombosis in a porcine coronary artery model. This study reports on the use of two drugs coated on the stent to simultaneously minimize both restenosis and thrombosis. The DES was prepared by spray coating a bare metal stent with a biodegradable polymer loaded with sirolimus and triflusal, to treat against restenosis and thrombosis, respectively. The two-layered dual drug-coated stent was characterized in vitro for surface properties before and after expansion, as well as for possible delamination by cross-sectioning the stent in vitro. In vivo animal studies (in a pig model) were then performed for acute thrombosis, inflammation, and restenosis. The results show a significant reduction in restenosis with a stent coated with both drugs compared with the controls (a bare metal stent, a sirolimus-coated, and a pure polymer-coated stent). The reduction in restenosis with a sirolimus/triflusal-eluting stent is associated with an inhibition of inflammation and thrombus formation, suggesting that such dual DES have a role to play for the treatment of coronary artery diseases.