Electrochemical lipolysis of subcutaneous adipose tissue in a porcine animal model.

OBJECTIVES: There is a considerable demand for noninvasive low-cost fat reduction methods with fewer side effects and shorter recovery times. This study aims to develop a fat-reduction method through electrochemical lipolysis of subcutaneous adipocytes using needle-based electrodes, body tissue fluids, and electrical current application. METHODS: Electrochemical lipolysis was performed by inserting a 4-pin needle electrode connected to a DC power supply into the pig's abdomen. Applied electrical current (0.5 and 1 mA) and treatment time (5 or 10 minutes) were varied systematically. Ultrasound imaging was performed before and after treatment to determine changes in fat thickness. Tissue samples were collected at 0, 2, and 4 weeks posttreatment for histological evaluation to determine the mechanism of action and the procedure's efficacy. RESULTS: Electrochemical subcutaneous adipose tissue lipolysis in a porcine model was achieved through hydrolysis of physiologic fluid within the vicinity of the inserted electrode where an electric current is applied, leading to localized disruption of fat cell membranes and necrosis. Electric current configuration 1.0 mA showed more pronounced lipolysis effects applied for 10 minutes, significantly decreasing adipocyte content per treatment area. The electrochemical treatment method also stimulates collagen synthesis, which helps reduce fat. CONCLUSIONS: Electrochemical lipolysis is a potential new noninvasive localized technique to reduce fat. The treatment method induces fat cell necrosis via in situ reduction-oxidation reaction by the electrochemical activation of physiologic fluid in the surrounding tissue. Electrochemical lipolysis is a simple, low-cost, fat-reducing treatment method without harmful side effects.

Differentiation of embryonic stem cells into a putative hair cell-progenitor cells via co-culture with HEI-OC1 cells.

Several studies have shown how different cell lines can influence the differentiation of stem cells through co-culture systems. The House Ear Institute-Organ of Corti 1 (HEI-OC1) is considered an important cell line for in vitro auditory research. However, it is unknown if HEI-OC1 cells can promote the differentiation of embryonic stem cells (ESCs). In this study, we investigated whether co-culture of ESCs with HEI-OC1 cells promotes differentiation. To this end, we developed a co-culture system of mouse ESCs with HEI-OC1 cells. Dissociated or embryonic bodies (EBs) of ESCs were introduced to a conditioned and inactivated confluent layer of HEI-OC1 cells for 14 days. The dissociated ESCs coalesced into an EB-like form that was smaller than the co-cultured EBs. Contact co-culture generated cells expressing several otic progenitor markers as well as hair cell specific markers. ESCs and EBs were also cultured in non-contact setup but using conditioned medium from HEI-OC1 cells, indicating that soluble factors alone could have a similar effect. The ESCs did not form into aggregates but were still Myo7a-positive, while the EBs degenerated. However, in the fully differentiated EBs, evidence to prove mature differentiation of inner ear hair cell was still rudimentary. Nevertheless, these results suggest that cellular interactions between ESCs and HEI-OC1 cells may both stimulate ESC differentiation.

Collagen and bone morphogenetic protein-2 functionalized hydroxyapatite scaffolds induce osteogenic differentiation in human adipose-derived stem cells.

Surface modification is one important way to fabricate successful biocompatible materials in bone tissue engineering. Hydroxyapatite (HAp) materials have received considerable attention as suitable bioceramics for manufacturing osseous implants because of their similarity to bone mineral in terms of chemical composition. In this study, the surface of porous HAp scaffold was modified by collagen treatment and bone morphogenetic protein-2 (BMP-2) conjugation. The surface modification did not affect the HAp scaffold's bulk properties. No significant difference in compressive strength was found among different scaffolds, with HAp, collagen modified HAp, and collagen-BMP-2-functionalized HAp having compressive strengths of 45.8 ± 3.12, 51.2 ± 4.09, and 50.7 ± 3.98 MPa, respectively. In vitro studies were performed to compare adhesion and osteogenic differentiation between human adipose-derived stem cells (hADSCs) with modified surfaces and those unmodified HAp surfaces. Collagen or BMP-2 alone was insufficient and that both collagen and BMP-2 are necessary to get the desired results. The findings suggest the possibility of using three-dimensional HAp scaffold treated with gold-standard collagen coating and highly researched BMP-2 growth factor as a platform to deliver hADSCs. Results of this study could be used to develop treatment strategy for regenerating completely transected models using more synergistic approaches.

Enzymatic in situ formed hydrogel from gelatin-tyramine and chitosan-4-hydroxylphenyl acetamide for the co-delivery of human adipose-derived stem cells and platelet-derived growth factor towards vascularization.

An injectable, in situ forming hydrogel system capable of co-delivering human adipose-derived stem cells (hADSC) and platelet-derived growth factor (PDGF) was investigated as a new system for tissue engineering, envisaged to support vascularization. The system consists of tyramine-conjugated gelatin and hydroxyphenyl acetamide chitosan derivative. Both are soluble and stable at physiologic conditions, which is a key factor for retaining viable cells and active growth factor. In situ gelation involved enzymatic crosslinking using horseradish peroxidase as a catalyst and hydrogen peroxide as an oxidant. Gel formation occurred within 30-90 s by controlling the concentration of polymers. PDGF release showed adequate release kinetics within the intended period of time and hADSC showed good compatibility with the hydrogel formulation based on the in vitro assay and subcutaneous implantation into BALB/c-nu/nu nude female mice. Immunohistochemical analysis confirmed viability of delivered hADSC. Histological analysis showed no immune reaction and confirmed blood vessel formation. The results implicate the hydrogel as a promising delivery vehicle or carrier of both cell and growth factor, which support vascularization for tissue engineering applications.

Evaluation of egg white ovomucin-based porous scaffold as an implantable biomaterial for tissue engineering.

Studies have shown the technological and functional properties of ovomucin (OVN) in the food-agricultural industry. But research has yet to explore its potential as an implantable biomaterial for tissue engineering and regenerative medicine. In this study we isolated OVN from egg white by isoelectric precipitation and fabricated scaffolds with tunable porosity by utilizing its foaming property. Gelatin a known biocompatible material was introduced to stabilize the foams, wherein different ratios of OVN and gelatin had a significant effect on the degree of porosity, pore size and stability of the formed hydrogels. The porous scaffolds were crosslinked with EDC resulting in stable scaffolds with prolonged degradation. Improved cell proliferation and adhesion of rat bone marrow-derived mesenchymal stem cells were observed for OVN containing scaffolds. Although, scaffolds with 75% OVN showed decrease in cell proliferation for L929 fibroblast type of cells. Further biocompatibility assessment as implant material was determined by subcutaneous implantation in rats of selected scaffold. H&E staining showed reasonable vascularization over time and little evidence of severe fibrosis at the implant site. Persistent polarization of classically activated macrophage was not observed, potentially reducing inflammatory response, and showed increased expression of alternatively activated macrophage cells that is favorable for tissue repair. Analysis of IgE levels in rat serum after implantation indicated minimal and resolvable allergic response to the OVN implants. The results demonstrate OVN as an acceptable implant scaffold that could provide new opportunities as an alternative natural biocompatible and functional biomaterial in various biomedical applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2107-2117, 2017.

Phosphonate-chitosan functionalization of a multi-channel hydroxyapatite scaffold for interfacial implant-bone tissue integration.

Nonunion associated with long bone defects continues to be highly researched both experimentally and clinically. A porous hydroxyapatite (HAp) scaffold has been recognized as a bone repair and substitute material clinically, but its use in segmental bone defects has been limited by poor integration and stability, as a consequence of scaffold strength unmatched with the native bone. Herein, we designed a multi-channel HAp-based scaffold for application in segmental bone defects, with a specific geometry and design. It possesses the required porosity for bone tissue regeneration with sufficient mechanical properties. We also developed a surface functionalization/modification method with the goal of early scaffold integration and stability. Initial functionalization with poly(vinyl phosphonic acid), PVPA, allowed simple attachment of a chitosan polymeric layer. The modification improved the biocompatibility of the scaffold and attachment of rat bone marrow-derived mesenchymal stem cells (rBMSC) in vitro. The modification also served as a buffer between the implant scaffold and bone tissue. Significant improvement in the integration behavior with better interlocking of the scaffold to bone tissue was observed for the modified scaffolds implanted in rabbit tibiae. The modified HAp scaffolds exhibited early interfacial implant-bone tissue integration with enhanced new bone formation and high potential for use in segmental bone defects.

Preformed chitosan cryogel-biphasic calcium phosphate: a potential injectable biocomposite for pathologic fracture.

The increasing interest in chitosan-based biomaterials stems from its desirable physicochemical properties. Although calcium phosphates have been mixed with chitosan to form injectable scaffolds, its application for bone tissue engineering has been limited and is still being explored to improve its clinical translatability. We report a biocomposite comprised of preformed chitosan cryogel with dispersed biphasic calcium phosphate that can flow under moderate pressure allowing passage through a small gauge needle, while maintaining sufficient integrity and strength during injection for gel recovery. The formed samples were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, X-ray diffraction analysis and protein adsorption measurements. Composite with 1% w/v biphasic calcium phosphate (CSG1) resulted in a homogeneous and rigid final structure. Injectable composite cryogel CSG1 (2.5 ± 0.2 N, 23G needle) exhibited good protein adsorption and biocompatibility. Results of subcutaneous implantation in rats reveal relatively high presence of polymorphonuclear cells but with no fibrous encapsulation with the composites, allowing further infiltration of cells within the sample implants. The biocomposite system presents a less-invasive delivery of bone filling material for stabilizing pathologic fractures.

Poly(vinylphosphonic acid) immobilized on chitosan: a glycosaminoglycan-inspired matrix for bone regeneration.

Glycosaminoglycans modulate the attraction of bone precursor cells including the actions of proteins essential for bone regeneration. In this study, poly(vinylphosphonic acid) was entrapped and immobilized in chitosan prior to formation of a porous three-dimensional matrix. Mimicking glycosaminoglycan interactions occurring in vivo, we evaluate its bone regeneration potential. Immobilized phosphonate was characterized by the absence of covalent linkage in Fourier transform infrared spectroscopy and polyanion effect in X-ray diffraction analysis. Higher surface and bulk protein adsorption was observed for the matrices containing phosphonate groups, consequently improving the proliferation and attachment of MC3T3-E1 cells. The porous poly(vinylphosphonic acid)-chitosan matrix was able to promote significant bone formation after in vivo implantation in rat calvarial defect as observed from the reconstructed images and histological analysis of tissue sections taken after 4 and 8 weeks. The unique porous matrix showed potential for bone tissue engineering applications.