Design of gelatin cryogel scaffolds with the ability to release simvastatin for potential bone tissue engineering applications.

Tissue engineering aims to improve or restore damaged tissues by using scaffolds, cells and bioactive agents. In tissue engineering, one of the most important concepts is the scaffold because it has a key role in keeping up and promoting the growth of the cells. It is also desirable to be able to load these scaffolds with drugs that induce tissue regeneration/formation. Based on this, in our study, gelatin cryogel scaffolds were developed for potential bone tissue engineering applications and simvastatin loading and release studies were performed. Simvastatin is lipoliphic in nature and this form is called inactive simvastatin (SV). It is modified to be in hydrophilic form and converted to the active form (SVA). For our study's drug loading and release process, simvastatin was used in both inactive and active forms. The blank cryogels and drug-loaded cryogels were prepared at different glutaraldehyde concentrations (1, 2, and 3%). The effect of the crosslinking agent and the amount of drug loaded were discussed with morphological and physicochemical analysis. As the glutaraldehyde concentration increased gradually, the pores size of the cryogels decreased and the swelling ratio decreased. For the release profile of simvastatin in both forms, we can say that it depended on the form (lipophilic and hydrophilic) of the loaded simvastatin.

Electrospun nanofiber mats caged the mammalian macrophages on their surfaces and prevented their inflammatory responses independent of the fiber diameter.

Poly-ε-caprolactone (PCL) has been widely used as biocompatible materials in tissue engineering. They have been used in mammalian cell proliferation to polarization and differentiation. Their modified versions had regulatory activities on mammalian macrophages in vitro. There are also studies suggesting different nanofiber diameters might alter the biological activities of these materials. Based on these cues, we examined the inflammatory activities and adherence properties of mammalian macrophages on electrospun PCL nanofibrous scaffolds formed with PCL having different nanofiber diameters. Our results suggest that macrophages could easily attach and get dispersed on the scaffolds. Macrophages lost their inflammatory cytokine TNF and IL6 production capacity in the presence of LPS when they were incubated on nanofibers. These effects were independent of the mean fiber diameters. Overall, the scaffolds have potential to be used as biocompatible materials to suppress excessive inflammatory reactions during tissue and organ transplantation by caging and suppressing the inflammatory cells.

Electrospun Nanofibers for Biomedical, Sensing, and Energy Harvesting Functions.

The process of electrospinning is over a century old, yet novel material and method achievements, and later the addition of nanomaterials in polymeric solutions, have spurred a significant increase in research innovations with several unique applications. Significant improvements have been achieved in the development of electrospun nanofibrous matrices, which include tailoring compositions of polymers with active agents, surface functionalization with nanoparticles, and encapsulation of functional materials within the nanofibers. Recently, sequentially combining fabrication of nanofibers with 3D printing was reported by our group and the synergistic process offers fiber membrane functionalities having the mechanical strength offered by 3D printed scaffolds. Recent developments in electrospun nanofibers are enumerated here with special emphasis on biomedical technologies, chemical and biological sensing, and energy harvesting aspects in the context of e-textile and tactile sensing. Energy harvesting offers significant advantages in many applications, such as biomedical technologies and critical infrastructure protection by using the concept of finite state machines and edge computing. Many other uses of devices using electrospun nanofibers, either as standalone or conjoined with 3D printed materials, are envisaged. The focus of this review is to highlight selected novel applications in biomedical technologies, chem.-bio sensing, and broadly in energy harvesting for use in internet of things (IoT) devices. The article concludes with a brief projection of the future direction of electrospun nanofibers, limitations, and how synergetic combination of the two processes will open pathways for future discoveries.

Impact of injectable chitosan cryogel microspherescaffolds on differentiation and proliferation of adiposederived mesenchymal stem cells into fat cells.

Difficulty in the clinical practice of stem cell therapy is often experienced in achieving desired target tissue cell differentiation and migration of stem cells to other tissue compartments where they are destroyed or die. This study was performed to evaluate if mesenchymal stem cells (MSCs) may differentiate into desired cell types when injected after combined with an injectable cryogel scaffold and to investigate if this scaffold may help in preventing cells from passing into different tissue compartments. MSCs were obtained from fat tissue of the rabbits as autografts and nuclei and cytoplasms of these cells were labeled with BrdU and PKH26. In Group 1, only-scaffold; in Group 2, only-MSCs; and in Group 3, combined stem cell/scaffold were injected to the right malar area of the rabbits. At postoperative 3 weeks, volumes of the injected areas were calculated by computer-tomography scans and histopathological evaluation was performed. The increase in the volume of the right malar areas was more in Group 3. In histopathological evaluation, chitosan cryogel microspheres were observed microscopically within the tissue and the scaffold was only partially degraded. Normal tissue form was seen in Group 2. Cells differentiated morphologically into fat cells were detected in Groups 2 and 3. Injectable chitosan cryogel microspheres were used in vivo for the first time in this study. As it was demonstrated to be useful in carrying MSCs to the reconstructed area, help cell differentiation to desired cells and prevent migration to other tissue compartments, it may be used for reconstructive purposes in the future.

Differential anti-inflammatory properties of chitosan-based cryogel scaffolds depending on chitosan/gelatin ratio.

Chitosan/gelatine-based materials have been widely used as biocompatible scaffolds in the tissue engineering field. Chitosan suppresses the inflammatory activities of macrophages whereas gelatine induces inflammatory cytokine production by these cells. Cryogel form of the scaffolds created an effect that was mostly dominated by chitosan activity. Since independent of chitosan to gelatine ratio, the cryogels eliminated the inflammatory cytokine production by the activated macrophages. This will enable suppression of inflammatory reactions by macrophages during implant procedure while enabling a nest of the matrix for the macrophages to reside. Determining the immunomodulatory effect of these materials during the decay is crucial to assess their biocompatibility and safety. Our results suggest that when the chitosan ratio was higher than that of gelatine the materials had anti-inflammatory activity in their powder forms based on TNFα production levels by LPS activated macrophages, whereas higher gelatine to chitosan ratio eliminated this effect. To our knowledge, this is the first study to assess the powder vs. gel forms of the chitosan/gelatine-based materials for their immunomodulatory potentials as well as how the ratio of chitosan to gelatine might affect these materials immunomodulatory effects on the activated macrophages.HIGHLIGHTSChitosan/gelatin composite cryogels have anti-inflammatory activities.Different ratios of chitosan to gelatin content altered the immunomodulatory activities.They can be safely and effectively used as implant materials for tissue engineering applications.They will also reduce the use of anti-inflammatory drugs during implantation.

Development of Hypericum perforatum oil incorporated antimicrobial and antioxidant chitosan cryogel as a wound dressing material.

In this study, a herbal infused oil (Hypericum perforatum, HP) incorporated chitosan (CS) cryogel as a wound dressing material was produced in order to be used in wound healing process. The main strategy is to combine the traditional perspective of using medicinal oils with polymeric scaffolds manufactured by an engineering approach to fabricate a potential tissue engineering product that provides both new tissue formation and wound healing. The scaffolds manufactured by cryogelation were soft, spongy, highly porous, physically stable, elastic and could be easily cut in any desired shape. Physicochemical, mechanical and morphological analyzes were used to characterize the produced cryogels. Young modulus of the plain chitosan cryogel was about 21 kPa whereas it increased with increasing HP oil content and became 61 kPa for 20% HP oil ratio. Further, the antimicrobial studies, antioxidant and DNA cleavage effects were investigated. Samples including the highest ratio of oil (CS4) showed the highest DPPH scavenging activity as 69.9%. In addition, 20% HP oil loaded chitosan cryogel demonstrated single strain DNA cleavage activitiy at 500 µg/mL concentration. Antimicrobial studies were applied against seven strains. The lowest activities were obtained against E. hirae and B. cereus, the highest against E. coli and L. pneumophila. This study concluded that the newly developed HP oil loaded chitosan cryogel scaffolds with unique antimicrobial and antioxidant properties are promising candidates to be used in tissue engineering applications as wound dressing for exudative and long-term healing wounds.

Stem cells combined 3D electrospun nanofibrous and macrochannelled matrices: a preliminary approach in repair of rat cranial bones.

Repair of cranial bone defects is an important problem in the clinical area. The use of scaffolds combined with stem cells has become a focus in the reconstruction of critical-sized bone defects. Electrospinning became a very attracting method in the preparation of tissue engineering scaffolds in the last decade, due to the unique nanofibrous structure of the electrospun matrices. However, they have a limitation for three dimensional (3D) applications, due to their two-dimensional structure and pore size which is smaller than a cellular diameter which cannot allow cell migration within the structure. In this study, electrospun poly(ε-caprolactone) (PCL) membranes were spirally wounded to prepare 3D matrices composed of nanofibers and macrochannels. Mesenchymal stromal/stem cells were injected inside the scaffolds after the constructs were implanted in the cranial bone defects in rats. New bone formation, vascularisation and intramembranous ossification of the critical size calvarial defect were accelerated by using mesenchymal stem cells combined 3D spiral-wounded electrospun matrices.

Gelatin- and hydroxyapatite-based cryogels for bone tissue engineering: synthesis, characterization, in vitro and in vivo biocompatibility.

The aim of this study was the synthesis and characterization of gelatin- and hydroxyapatite (osteoconductive component of bone)-based cryogels for tissue-engineering applications. Preliminary in vitro and in vivo biocompatibility tests were conducted. Gelatin- and hydroxyapatite-based cryogels of varying concentrations were synthesized using glutaraldehyde as the crosslinking agent. Chemical structure, pore morphology, pore size distribution, mechanical properties, swelling characteristics and degradation profiles of the synthesized cryogels were demonstrated by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), mercury porosimetry, a mechanical test device, swelling ratio tests and weight loss measurements, respectively. In vitro cell viability and in vivo biocompatility tests were performed in order to show the performance of the cryogels in the biological environment. Changing the concentrations of gelatin, hydroxyapatite and crosslinker changed the chemical structure, pore size and pore size distribution of the cryogels, which in turn resulted in the ultimate behaviour (mechanical properties, swelling ratio, degradation profile). In vitro cell culture tests showed the viability of the cells. The cryogels did not show any cytotoxic effects on the cells. Clinical outcomes and the gross pathological results demonstrated that there was no necrosis noted in the abdominal and thoracic regions at the end of implantation and the implanted cryogel was found to be non-irritant and non-toxic at 12 weeks of implantation. Copyright © 2013 John Wiley & Sons, Ltd.

Effect of crosslinking methods on the structure and biocompatibility of polyvinyl alcohol/gelatin cryogels.

In this study, polyvinyl alcohol (PVA) and gelatin based cryogels were prepared by crosslinking chemically or physically for tissue engineering applications. Different PVA/Gelatin ratios (100:0, 90:10, 70:30, 50:50) and crosslinking methods have been used to prepare cryogels; chemical and physical structure of the prepared matrices were analysed by FTIR and SEM; swelling and degradation profiles were followed. Chemical and physical crosslinking was obtained by using glutaraldehyde as crosslinker and by applying freeze thawing cycle, respectively. Gelatin concentration and crosslinking method had significant effect on the pore size, swelling ratio and degradation profiles of the cryogels. Biocompatibility of the cryogels were also investigated by MTT assay. SEM was used to investigate the cell morphology on the scaffolds. The MTT assay findings prove that physically crosslinked PVA/Gelatin scaffolds are more biocompatible and enhance more the adhesion and proliferation of mouse embryonic fibroblast cells (MEF) than chemically crosslinked PVA/Gelatin scaffolds. The overall results demonstrated that, the PVA/Gelatin cyrogels as a suitable biomaterial for tissue engineering applications and crosslinking methods affect the architecture and characteristic properties of the cryogels.

Thermoresponsive biodegradable HEMA-lactate-Dextran-co-NIPA cryogels for controlled release of simvastatin.

Abstract NIPA and HEMA-lactate-Dextran-based biodegradable and thermoresponsive cryogels were synthesized at different compositions by cryogelation. Chemical and morphological properties of the HEMA-lactate-Dextran-co-NIPA cryogel matrices were demonstrated by FTIR, SEM, and ESEM. Thermoresponsivity of the prepared cryogels was investigated by DSC, imaging NMR, and swelling studies. For possible use of the cryogels in potential bone tissue engineering applications, either hydrophobic simvastatin was embedded, or hydrophilic simvastatin was incorporated in the cryogels. Release profiles of simvastatin delivering cryogel scaffolds depending on their composition, hydrophobicity or hydrophilicity of loaded simvastatin and the medium temperature were demonstrated.

Stem cell suspension injected HEMA-lactate-dextran cryogels for regeneration of critical sized bone defects.

HEMA-Lactate-Dextran cryogel scaffolds were produced by cryogelation. Mesencyhmal stem cells (MSC) were isolated from rat bone marrow. Critical sized cranial bone defects were created in rat cranium. Stem cells were injected inside the macropores of the cryogel scaffolds prepared from HEMA-Lactate-Dextran possessing the same dimensions with the defect and placed in the cranial bone. The cryogels placed in the defect without stem cells served as control. After selected time intervals the experimental sites were removed from the animals and new bone formation and tissue integration were investigated by histological analysis. The in vivo results exhibited osseous tissue integration within the implant and mineralized functionally stable bone restoration of the cranial defects. Tissue formation started in the macrospores of the scaffold starting from periphery to the center. A significant ingrowth of connective tissue cells and new blood vessels allowed new bone formation. Histological data demonstrated that new bone per total defect area ratio, were not significantly different in "scaffold-stem cells" group compared to that of "scaffold only" group on all time points. However, the blood vessel density was significantly higher in "scaffold-stem cells" group comparing to that of the "scaffold only" group on day 30. "Scaffold-stem cells" given group gave better tissue response score when compared to "scaffold only" group on day 180.

In vivo performance of simvastatin-loaded electrospun spiral-wound polycaprolactone scaffolds in reconstruction of cranial bone defects in the rat model.

Reconstruction of large bone defects is still a major problem. Tissue-engineering approaches have become a focus in regeneration of bone. In particular, critical-sized defects do not ossify spontaneously. The use of electrospinning is attracting increasing attention in the preparation of tissue-engineering scaffolds. Recently, acellular scaffolds carrying bioactive agents have been used as scaffolds in "in situ" tissue engineering for soft and hard tissue repair. Poly(epsilon-caprolactone) (PCL) with two different molecular weights were synthesized, and the blends of these two were electrospun into nonwoven membranes composed of nanofibers/micropores. To stimulate bone formation, an active drug, "simvastatin" was loaded either after the membranes were formed or during electrospinning. The matrices were then spiral-wound to produce scaffolds with 3D-structures having both macro- and microchannels. Eight-millimeter diameter critical size cranial defects were created in rats. Scaffolds with or without simvastatin were then implanted into these defects. Samples from the implant sites were removed after 1, 3, and 6 months postimplantation. Bone regeneration and tissue response were followed by X-ray microcomputed tomography and histological analysis. These in vivo results exhibited osseous tissue integration within the implant and mineralized bone restoration of the calvarium. Both microCT and histological data clearly demonstrated that the more successful results were observed with the "simvastatin-containing PCL scaffolds," in which simvastatin was incorporated into the PCL scaffolds during electrospinning. For these samples, bone mineralization was quite significant when compared with the other groups.

Tissue responses to novel tissue engineering biodegradable cryogel scaffolds: an animal model.

Biodegradable macroporous cryogels with highly open and interconnected pore structures were produced from dextran modified with oligo L-lactide bearing hydroxyethylmethacrylate (HEMA) end groups in moderately frozen solutions. Tissue responses to these novel scaffolds were evaluated in rats after dorsal subcutaneous implantation, iliac submuscular implantation, auricular implantation, or in calvarial defect model. In no case, either necrosis or foreign body reaction was observed during histological studies. The cryogel scaffolds integrated with the surrounding tissue and the formation of a new tissue were accompanied with significant ingrowth of connective tissue cells and new blood vessels into the cryogel. The tissue responses were significantly lower in auricular and calvarial implantations when compared with the subcutanous and the submuscular implantations. The degradation of the scaffold was slower in bone comparing to soft tissues. The biodegradable cryogels are highly biocompatible and combine extraordinary properties including having soft and elastic nature, open porous structure, and very rapid and controllable swelling. Therefore, the cryogels could be promising candidates for further clinical applications in tissue regeneration.

Three-dimensional ingrowth of bone cells within biodegradable cryogel scaffolds in bioreactors at different regimes.

Three-dimensional cell ingrowth within biodegradable cryogel scaffolds made of cross-linked 2-hydroxyethyl methacrylate (HEMA)-lactate-dextran with interconnected macropores was studied in bioreactors at different regimes (static, perfusion, and compression-perfusion). An osteoblast-like cell line (MG63) was used in these studies. The samples taken after selected times from the bioreactors were examined by microscopy techniques (light, SEM, TEM, and laser scanning confocal). The cell culture conditions were found to have a significant impact not only on the cell morphology, such as the extent of cell attachment and ingrowth, but also on cellular activities. Dynamic conditions (perfusion and/or compression) greatly improved cell ingrowth and extracellular matrix (ECM) synthesis. Alkaline phosphatase activity results confirmed the positive effect of dynamic conditions on bone cells.

Cryogelation for preparation of novel biodegradable tissue-engineering scaffolds.

2-Hydroxyethyl methacrylate-L-lactate (HEMA-LLA) and HEMA-L-lactate-dextran (HEMA-LLA-D) were synthesized. 1H-NMR confirmed the formation of these oligomers and macromers. Cryogels with different pore structures were prepared using different amounts of HEMA, HEMA-LLA and HEMA-LLA-D by a cryogelation technique. SEM micrographs exhibited pore morphologies. Cryogels were highly porous with interconnected pore structures, opaque, spongy and highly elastic. It was possible to compress them to remove the water in the pores and to return to their original form just by immersing them in water in few minutes, which was quite reproducible. Their swelling abilities, compressive strengths and degradation in buffer solutions were found to be related with their structural properties which was controlled by changing the cryogelation recipe.

Electrospun matrices made of poly(alpha-hydroxy acids) for medical use.

Biomaterials are widely used in diverse applications as substances, materials or important elements of biomedical devices. Biodegradable polymers, both natural and synthetic, have been utilized in applications in which they act as temporary substitutes. Poly(alpha-hydroxy acids), especially lactic acids and glycolic acid and their copolymers with epsilon-caprolactone, are the most widely known and used among all biodegradable polymers. They degrade in vivo into safe end products mainly by hydrolysis in a few weeks to several months, depending on several factors, including molecular structure/morphology, average molecular weight, size and shape. They are processed into tailor-made materials for diverse applications, although mainly for soft and hard tissue repair. Electrospinning is a method of producing nanofibers and nonwoven matrices from their solutions and melts. Several factors affect fiber diameter and resulting nonwoven structures/morphologies. Recently, electrospun matrices made of lactic acids, glycolic acid and epsilon-caprolactone homo- and co-polymers have been attracting increasing attention for fabrication of novel materials for medical use. This review briefly describes poly(alpha-hydroxy acids) and the elecrospinning process, and gives some selected recent applications of electrospun matrices made from these polymers.