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Biomaterials (v.31, #24)

Editorial board (pp. ifc).

A review on stereolithography and its applications in biomedical engineering by Ferry P.W. Melchels; Jan Feijen; Dirk W. Grijpma (pp. 6121-6130).
Stereolithography is a solid freeform technique (SFF) that was introduced in the late 1980s. Although many other techniques have been developed since then, stereolithography remains one of the most powerful and versatile of all SFF techniques. It has the highest fabrication accuracy and an increasing number of materials that can be processed is becoming available. In this paper we discuss the characteristic features of the stereolithography technique and compare it to other SFF techniques. The biomedical applications of stereolithography are reviewed, as well as the biodegradable resin materials that have been developed for use with stereolithography. Finally, an overview of the application of stereolithography in preparing porous structures for tissue engineering is given.

Keywords: Rapid prototyping; Stereolithography; Microstructure; Tissue engineering scaffold; Three-dimensional printing

The dependence of MG63 osteoblast responses to (meth)acrylate-based networks on chemical structure and stiffness by Kathryn E. Smith; Sharon L. Hyzy; MoonHae Sunwoo; Ken A. Gall; Zvi Schwartz; Barbara D. Boyan (pp. 6131-6141).
The cell response to an implant is regulated by the implant’s surface properties including topography and chemistry, but less is known about how the mechanical properties affect cell behavior. The objective of this study was to evaluate how the surface stiffness and chemistry of acrylate-based copolymer networks affect the in vitro response of human MG63 pre-osteoblast cells. Networks comprised of poly(ethylene glycol) dimethacrylate (PEGDMA; Mn ∼750) and diethylene glycol dimethacrylate (DEGDMA) were photopolymerized at different concentrations to produce three compositions with moduli ranging from 850 to 60 MPa. To further decouple chemistry and stiffness, three networks comprised of 2-hydroxyethyl methacrylate (2HEMA) and PEGDMA or DEGDMA were also designed that exhibited a range of moduli similar to the PEGDMA–DEGDMA networks. MG63 cells were cultured on each surface and tissue culture polystyrene (TCPS), and the effect of copolymer composition on cell number, osteogenic markers (alkaline phosphatase specific activity and osteocalcin), and local growth factor production (OPG, TGF-β1, and VEGF-A) were assessed. Cells exhibited a more differentiated phenotype on the PEGDMA–DEGDMA copolymers compared to the 2HEMA–PEGDMA copolymers. On the PEGDMA–DEGDMA system, cells exhibited a more differentiated phenotype on the stiffest surface indicated by elevated osteocalcin compared with TCPS. Conversely, cells on 2HEMA–PEGDMA copolymers became more differentiated on the less stiff 2HEMA surface. Growth factors were regulated in a differential manner. These results indicate that copolymer chemistry is the primary regulator of osteoblast differentiation, and the effect of stiffness is secondary to the surface chemistry.

Keywords: Surface stiffness; Osteoblasts; Hydroxyethyl methacrylate; Polyethylene glycol dimethacrylate; In vitro; Mineralized and demineralized bone

The promotion of human malignant melanoma growth by mesoporous silica nanoparticles through decreased reactive oxygen species by Xinglu Huang; Jie Zhuang; Xu Teng; Linlin Li; Dong Chen; Xiyun Yan; Fangqiong Tang (pp. 6142-6153).
The concept that mesoporous silica nanoparticles (MSNs) are regarded as ideal novel drug delivery carriers in tumor therapy has been introduced extensively, but the effects of MSNs on tumor growth have received little attention. Here a model of nude mice xenografted with human malignant melanoma cells (A375) was used to investigate the effect of MSNs on tumor growth. Surprisingly, we found that MSNs have no toxicity to human malignant melanoma but increasing tumor growth in vivo. It was also confirmed that MSNs significantly promoted A375 cell proliferation and accelerated cell cycle progression in vitro. Cellular uptake mechanism showed that MSNs may affect molecular behavior of A375 cells when they entered into cytoplasm. Then, a detailed mechanism indicated that the promotion effect induced by MSNs was due to the decreasing of endogenous reactive oxygen species (ROS) in cells. Further results demonstrated that the upregulation of anti-apoptotic molecules Bcl-2 expression and the inhibition of NF-κB activation by MSNs may promote cell proliferation in a redox-sensitive signal pathway. These results show that tumor growth can be regulated by nanocarriers themselves in a ROS-dependent manner and imply that nanocarriers are not necessarily suitable for all kinds of tumor therapy in development drug delivery system.

Keywords: Mesoporous silica nanoparticles; Promotion tumor growth; Reactive oxygen species; Molecular mechanism

Self-assembling nanostructures to deliver angiogenic factors to pancreatic islets by Lesley W. Chow; Ling-jia Wang; Dixon B. Kaufman; Samuel I. Stupp (pp. 6154-6161).
Supramolecular self-assembly of nanoscale filaments offers a vehicle to signal cells within dense cell aggregates such as pancreatic islets. We previously developed a heparin-binding peptide amphiphile (HBPA) that self-assembles into nanofiber gels at concentrations of 1% by weight when mixed with heparin and activates heparin-binding, angiogenic growth factors. We report here on the use of these molecules at concentrations 100 times lower to drive delivery of the nanofibers into the dense islet interior. Using fluorescent markers, HBPA molecules, heparin, and FGF2 were shown to be present in and on the surface of murine islets. The intraislet nanofibers were found to be necessary to retain FGF2 within the islet for 48h and to increase cell viability significantly for at least 7 days in culture. Furthermore, enhanced insulin secretion was observed with the nanofibers for 3 days in culture. Delivery of FGF2 and VEGF in conjunction with the HBPA/heparin nanofibers also induced a significant amount of islet endothelial cell sprouting from the islets into a peptide amphiphile 3-D matrix. We believe the infiltration of bioactive nanofibers in the interior of islets as an artificial ECM can improve cell viability and function in vitro and enhance their vascularization in the presence of growth factors such as FGF2 and VEGF. The approach described here may have significant impact on islet transplantation to treat type 1 diabetes.

Keywords: Angiogenesis; Cell viability; Diabetes; Heparin; Peptide; Self-assembly

Relationships between degradability of silk scaffolds and osteogenesis by Sang-Hyug Park; Eun Seok Gil; Hai Shi; Hyeon Joo Kim; Kyongbum Lee; David L. Kaplan (pp. 6162-6172).
Bone repairs represent a major focus in orthopedic medicine with biomaterials as a critical aspect of the regenerative process. However, only a limited set of biomaterials are utilized today and few studies relate biomaterial scaffold design to degradation rate and new bone formation. Matching biomaterial remodeling rate towards new bone formation is important in terms of the overall rate and quality of bone regeneration outcomes. We report on the osteogenesis and metabolism of human bone marrow derived mesenchymal stem cells (hMSCs) in 3D silk scaffolds. The scaffolds were prepared with two different degradation rates in order to study relationships between matrix degradation, cell metabolism and bone tissue formation in vitro. SEM, histology, chemical assays, real-time PCR and metabolic analyses were assessed to investigate these relationships. More extensively mineralized ECM formed in the scaffolds designed to degrade more rapidly, based on SEM, von Kossa and type I collagen staining and calcium content. Measures of osteogenic ECM were significantly higher in the more rapidly degrading scaffolds than in the more slowly degrading scaffolds over 56 days of study in vitro. Metabolic analysis, including glucose and lactate levels, confirmed the degradation rate differences with the two types of scaffolds, with the more rapidly degrading scaffolds supporting higher levels of glucose consumption and lactate synthesis by the hMSCs upon osteogenesis, in comparison to the more slowly degrading scaffolds. The results demonstrate that scaffold degradation rates directly impact the metabolism of hMSCs, and in turn the rate of osteogenesis. An understanding of the interplay between cellular metabolism and scaffold degradability should aid in the more rational design of scaffolds for bone regeneration needs both in vitro and in vivo.

Keywords: Silk; Scaffold degradation; Osteogenesis; Stem cell metabolism; Tissue Regeneration

Bioprinting vessel-like constructs using hyaluronan hydrogels crosslinked with tetrahedral polyethylene glycol tetracrylates by Aleksander Skardal; Jianxing Zhang; Glenn D. Prestwich (pp. 6173-6181).
Bioprinting enables deposition of cells and biomaterials into spatial orientations and complexities that mirror physiologically relevant geometries. To facilitate the development of bioartificial vessel-like grafts, two four-armed polyethylene glycol (PEG) derivatives with different PEG chain lengths, TetraPEG8 and TetraPEG13, were synthesized from tetrahedral pentaerythritol derivatives. The TetraPEGs are unique multi-armed PEGs with a compact and symmetrical core. The TetraPEGs were converted to tetra-acrylate derivatives (TetraPAcs) which were used in turn to co-crosslink thiolated hyaluronic acid and gelatin derivatives into extrudable hydrogels for printing tissue constructs. First, the hydrogels produced by TetraPAc crosslinking showed significantly higher shear storage moduli when compared to PEG diacrylate (PEGDA)-crosslinked synthetic extracellular matrices (sECMs) of similar composition. These stiffer hydrogels have rheological properties more suited to bioprinting high-density cell suspensions. Second, TetraPAc-crosslinked sECMs were equivalent or superior to PEGDA-crosslinked gels in supporting cell growth and proliferation. Third, the TetraPac sECMs were employed in a proof-of-concept experiment by encapsulation of NIH 3T3 cells in sausage-like hydrogel macrofilaments. These macrofilaments were then printed into tubular tissue constructs by layer-by-layer deposition using the Fab@Home printing system. LIVE/DEAD viability/cytotoxicity-stained cross-sectional images showed the bioprinted cell structures to be viable in culture for up to 4 weeks with little evidence of cell death. Thus, biofabrication of cell suspensions in TetraPAc sECMs demonstrates the feasibility of building bioartificial blood vessel-like constructs for research and potentially clinical uses.

Keywords: Hyaluronic acid; Tissue engineering; Hydrogel; Polyethylene glycol; Pentaerythritol; Bioartificial vascular graft

Effect of mechanical factors on the function of engineered human blood microvessels in microfluidic collagen gels by Gavrielle M. Price; Keith H.K. Wong; James G. Truslow; Alexander D. Leung; Chitrangada Acharya; Joe Tien (pp. 6182-6189).
This work examines how mechanical signals affect the barrier function and stability of engineered human microvessels in microfluidic type I collagen gels. Constructs that were exposed to chronic low flow displayed high permeabilities to bovine serum albumin and 10 kDa dextran, numerous focal leaks, low size selectivity, and short lifespan of less than one week. Higher flows promoted barrier function and increased longevity; at the highest flows, the barrier function rivaled that observed in vivo, and all vessels survived to day 14. By studying the physiology of microvessels of different geometries, we established that shear stress and transmural pressure were the dominant mechanical signals that regulated barrier function and vascular stability, respectively. In microvessels that were exposed to high flow, elevation of intracellular cyclic AMP further increased the selectivity of the barrier and strongly suppressed cell proliferation. Computational models that incorporated stress dependence successfully predicted vascular phenotype. Our results indicate that the mechanical microenvironment plays a major role in the functionality and stability of engineered human microvessels in microfluidic collagen gels.

Keywords: Mechanical microenvironment; Barrier function; Microvascular tissue engineering; Shear stress; Transmural pressure

The influence of an aligned nanofibrous topography on human mesenchymal stem cell fibrochondrogenesis by Brendon M. Baker; Ashwin S. Nathan; Albert O. Gee; Robert L. Mauck (pp. 6190-6200).
Fibrocartilaginous tissues serve critical load-bearing functions in numerous joints throughout the body. As these structures are often injured, there exists great demand for engineered tissue for repair or replacement. This study assessed the ability of human marrow-derived mesenchymal stem cells (MSCs) to elaborate a mechanically functional, fibrocartilaginous matrix in a nanofibrous microenvironment. Nanofibrous scaffolds, composed of ultra-fine biodegradable polymer fibers, replicate the structural and mechanical anisotropy of native fibrous tissues and serve as a 3D micro-pattern for directing cell orientation and ordered matrix formation. MSCs were isolated from four osteoarthritic (OA) patients, along with meniscal fibrochondrocytes (FC) which have proven to be a potent cell source for engineering fibrocartilage. Cell-seeded nanofibrous scaffolds were cultured in a chemically-defined medium formulation and mechanical, biochemical, and histological features were evaluated over 9 weeks. Surprisingly, and contrary to previous studies with juvenile bovine cells, matrix assembly by adult human MSCs was dramatically hindered compared to donor-matched FCs cultured similarly. Unlike FCs, MSCs did not proliferate, resulting in sparsely colonized constructs. Increases in matrix content, and therefore changes in tensile properties, were modest in MSC-seeded constructs compared to FC counterparts, even when normalized to the lower cell number in these constructs. To rule out the influence of OA sourcing on MSC functional potential, constructs from healthy young donors were generated; these constructs matured no differently than those formed with OA MSCs. Importantly, there was no difference in matrix production of MSCs and FCs when cultured in pellet form, highlighting the sensitivity of human MSCs to their 3D microenvironment.

Keywords: Mesenchymal stem cells; Microenvironment; Tissue engineering; Fibrocartilage; Nanofibers

Effects of fractal surface on C6 glioma cell morphogenesis and differentiation in vitro by Ping Wang; Lei Li; Cheng Zhang; Qunfang Lei; Wenjun Fang (pp. 6201-6206).
Neurons and glial cells in the brain are surrounded by a fractal environment. A fractal alkylketene dimmer (AKD) surface was shown to provide such a biomimetic environment for glial cell culture. However, little is known about the effects of fractal surface on the complexity of cell morphology. In particular, whether fractal surface induces glial cell differentiation remains to be elucidated. The present work, thus determined the fractal dimension (FD) of cell complexity with a geometrically calculational parameter, the expressions of GFAP gene and protein in C6 glioma cells on fractal AKD, non-fractal AKD and PLL-coated surfaces. Fractal surface suppressed the proliferation of glioma cell, and significantly increased the length and number of cell process. Furthermore, the enhanced values of FD were accompanied with the expressions of GFAP gene and protein, especially that of gene. However, cells on non-fractal and PLL surface proliferated gradually along with the culture time, showing the fibroblast-like morphology, and accompanied with the consistent expressions of GFAP gene and protein. These results suggested that C6 glioma cell differentiation can be induced by fractal AKD surface.

Keywords: Fractal; Fractal dimension; C6 glioma cell; Cell differention

Urinary bladder smooth muscle regeneration utilizing bone marrow derived mesenchymal stem cell seeded elastomeric poly(1,8-octanediol-co-citrate) based thin films by Arun K. Sharma; Partha V. Hota; Derek J. Matoka; Natalie J. Fuller; Danny Jandali; Hatim Thaker; Guillermo A. Ameer; Earl Y. Cheng (pp. 6207-6217).
Bladder regeneration studies have yielded inconclusive results possibly due to the use of unfavorable cells and primitive scaffold design. We hypothesized that human mesenchymal stem cells seeded onto poly(1,8-octanediol-co-citrate) elastomeric thin films would provide a suitable milieu for partial bladder regeneration. POCfs were created by reacting citric acid with 1,8-octanediol and seeded on opposing faces with human MSCs and urothelial cells; normal bladder smooth muscle cells and UCs, or unseeded POCfs. Partial cystectomized nude rats were augmented with the aforementioned POCfs, enveloped with omentum and sacrificed at 4 and 10 weeks. Isolated bladders were subjected to Trichrome and anti-human γ-tubulin, calponin, caldesmon, smooth muscle γ-actin, and elastin stainings. Mechanical testing of POCfs revealed a Young’s modulus of 138 kPa with elongation 137% its initial length without permanent deformation demonstrating its high uniaxial elastic potential. Trichrome and immunofluorescent staining of MSC/UC POCf augmented bladders exhibited typical bladder architecture with muscle bundle formation and the expression and retention of bladder smooth muscle contractile proteins of human derivation. Quantitative morphometry of MSC/UC samples revealed muscle/collagen ratios approximately 1.75× greater than SMC/UC controls at 10 weeks. Data demonstrate MSC seeded POCfs support partial regeneration of bladder tissue in vivo.

Keywords: Bladder tissue engineering; Bone marrow; Elastomer; Mesenchymal stem cell; Smooth muscle cell

A strategy for fabrication of a three-dimensional tissue construct containing uniformly distributed embryoid body-derived cells as a cardiac patch by Chieh-Cheng Huang; Chen-Kang Liao; Mei-Ju Yang; Chun-Hung Chen; Shiaw-Min Hwang; Yi-Wen Hung; Yen Chang; Hsing-Wen Sung (pp. 6218-6227).
Growing three-dimensional (3D) scaffolds that contain more than a few layers of seeded cells in vitro is crucial for the creation of thick and viable cardiac tissues in vivo. Embryonic stem cells (ESCs) have been used as an alternative cell source for cardiac repair; however, dissociated ESCs show poor viability in the scaffold and do not form the embryoid body (EB)-like structures. In this study, a strategy intended for cultivating EB-derived cells (EBDCs) uniformly in a porous 3D tissue scaffold was developed. This strategy employed techniques of formation of spherically symmetric EBs in a thermo-responsive hydrogel system, production of cell sheets of EBDCs in a similar hydrogel system coated with collagen and fabrication of sliced porous tissue scaffolds. The prepared EBs were collected and plated evenly in the cell-sheet culture system. After 8 days in culture, a continuous sheet of EBDCs with cell beating was obtained; our qPCR and flow cytometric analyses showed that the collagen-coated on the cell-sheet culture system can significantly enhance the population of cardiac-lineage cells. The produced EBDC sheets were then sandwiched into the sliced porous tissue scaffold. After reculture, the seeded EBDCs were redistributed uniformly throughout the scaffold, with a significant increase in mechanical strength. Cardiac-specific myosin heavy chain and α-actinin were expressed for some cells grown in the scaffold, while connexin 43 was clearly expressed at the cell borders. Additional studies such as employing purification techniques to enrich the population of cardiomyocytes are needed to further improve the developed tissue constructs as a bioengineered cardiac patch.

Keywords: Embryonic stem cell; Cell-sheet; Thermo-responsive hydrogel; Myocardial infarction; Bioengineered scaffold

Regulating orientation and phenotype of primary vascular smooth muscle cells by biodegradable films patterned with arrays of microchannels and discontinuous microwalls by Ye Cao; Yin Fun Poon; Jie Feng; Shahrzad Rayatpisheh; Vincent Chan; Mary B. Chan-Park (pp. 6228-6238).
Vascular smooth muscle cells (vSMCs) cultured in vitro are known to exhibit phenotype hyperplasticity. This plasticity is potentially very useful in tissue engineering of blood vessels. The synthetic phenotype is necessary for cell proliferation on the tissue scaffold but the cells must ultimately assume a quiescent, contractile phenotype for normal vascular function. In vitro control of vSMC phenotype has been challenging. This study shows that microchannel scaffolds with discontinuous walls can support primary vSMC proliferation and, when the cells reach confluence inside the channels, transform the cell phenotype towards greater contractility and promote cell alignment. A thorough time-resolved study was undertaken to characterize the expression of the contractile proteins alpha-actin, calponin, myosin heavy chain (MHC) and smoothelin as a function of time and initial cell density on microchannel scaffolds. The results consistently indicate that primary vSMCs cultured on the microchannel substrate substantially align parallel to the microwalls, become more elongated and significantly increase their expression of contractile proteins only when the cells reach confluence. MHC immunostaining was visible in the micropatterned cells after confluence but not in flat substrate cells or non-confluent micropatterned cells, which further verifies the increased contractility of the confluent channel-constrained vSMCs. The higher total amount of deposited elastin and collagen in confluent flat cultures than in confluent micropatterned cultures also provides confirmation of the higher contractility of the channel-constrained cells. These results establish that our microchanneled film can trigger the switch of primary vSMCs from a proliferative state to a more contractile phenotype at confluence.

Keywords: Phenotype; Smooth muscle cells; Micropattern; Confluence; Contractile proteins

Osteogenic differentiation of human mesenchymal stem cells using RGD-modified BMP-2 coated microspheres by Ji S. Park; Han N. Yang; Su Y. Jeon; Dae G. Woo; Kun Na; Keun-Hong Park (pp. 6239-6248).
Micro-structured scaffolds formed with poly(lactic- co -glycolic acid) (PLGA) microspheres were composed of adhesion molecules and growth factors. PLGA microspheres, constructed with Arg-Gly-Asp (RGD) peptide and bone morphogenic protein 2 (BMP-2) were created as a stem cell delivery vehicle. In this study, a high potential for cell adhesion and differentiation of human mesenchymal stem cells (hMSCs) was achieved by constructing the scaffolds with different compositions of coating materials. Specific gene and protein detection by RT-PCR and western blot analysis of the embedded hMSCs revealed that a combination of RGD peptide and BMP-2 induced differentiation of bone cells. Histology and immunohistochemistry results confirmed that bone cell-differentiated transplanted hMSCs were present in the micro-structured scaffolds. The results of this study demonstrate that the regulation of stem cell differentiation by adhesion molecules and growth factors has the potential to enable formation of therapeutic vehicles for the delivery of stem cells that are easily fabricated, less expensive, and more easily controlled than currently available delivery systems. The micro-structure typed PLGA microspheres used in this study possessed unique properties of ideal scaffolds. The embedded hMSCs easily adhered onto the PLGA microspheres mediated by RGD peptide, proliferated well onto the scaffolds, and differentiated to perform the distinct functions of bone tissues.

Keywords: RGD; BMP-2; hMSC; Differentiation; Scaffold

Iodinated blood pool contrast media for preclinical X-ray imaging applications – A review by François Hallouard; Nicolas Anton; Philippe Choquet; André Constantinesco; Thierry Vandamme (pp. 6249-6268).
The in vivo X-ray micro-computed tomography (micro-CT) is a very powerful and non-invasive tool used to establish high-resolution images with isotropic voxels in typical scan times ranging from minutes to tenths of minutes. This preclinical imaging technology is primarily adapted to visualize bones. X-ray imaging of soft tissues is made possible by using opaque compounds, providing contrast through tissue vascularization. Thus, using control agents with a long-lasting time in the blood, active or passive targeting of soft tissue is made possible in small animals. In this respect, the use of hydrophilic iodinated X-ray contrast media remains limited due to their rapid blood clearance, albeit at a slightly slower pace in humans as compared with rodents. The development of an iodinated contrast medium with increased vascular residence time is thus necessary. This is precisely the scope of the present paper, which will review and compare in detail the different vectors used as long-circulating iodinated contrast agents for micro-CT, i.e. liposomes, nano-emulsions, micelles, dendrimers and other polymeric particles. The discussion is focused, for each of these nanoparticulate systems, on their method of formulation and production, their stability properties, encapsulation properties, release properties, pharmacokinetics, and toxicology. The different aspects relative to the adaptation of these properties and physico-chemical characteristics for blood pool contrast agents aimed at angiographic micro-CT applications are also discussed. The aim of this review is to propose an overview into the formulation and properties of iodinated micro-CT contrast agents for preclinical applications.

Keywords: Molecular imaging; Nanoparticle; Liposome; Micelle; Dendrimer

Dual therapeutic action of antibiotic-loaded nanosheets for the treatment of gastrointestinal tissue defects by Toshinori Fujie; Akihiro Saito; Manabu Kinoshita; Hiromi Miyazaki; Shinya Ohtsubo; Daizoh Saitoh; Shinji Takeoka (pp. 6269-6278).
An ultra-thin polymer film (nanosheet) composed of polysaccharides (i.e., polysaccharide nanosheet) provides sufficient adhesiveness, flexibility and robustness to act as an effective wound dressing. We have recently demonstrated the sealing effect of a nanosheet on a murine cecal puncture. Nevertheless, a small percentage of bacteria penetrated the nanosheet because of its ultra-thin structure. Here, we have developed an antibiotic-loaded nanosheet to inhibit bacterial penetration and investigated its therapeutic efficacy using a model of a murine cecal puncture. Tetracycline (TC) was sandwiched between a poly(vinylacetate) (PVAc) layer and the polysaccharide nanosheet (named PVAc-TC-nanosheet). Under physiological conditions, TC was released from the nanosheet for 6 h. Microscopic observation between the interface of the PVAc-TC-nanosheet and bacteria demonstrated how its potent anti-microbial effect was achieved. In vivo studies show that overlapping therapy with the PVAc-TC-nanosheet (thickness: 177 nm) significantly increases mouse survival rate after cecal puncture as well as suppressing an increase in the intraperitoneal bacterial count and leukocyte count.

Keywords: anti-microbial; In vivo test; Inflammation; Nanocomposite; Polysaccharide; Wound dressing

Toward delivery of multiple growth factors in tissue engineering by Fa-Ming Chen; Min Zhang; Zhi-Fen Wu (pp. 6279-6308).
Inspired by physiological events that accompany the “wound healing cascade”, the concept of developing a tissue either in vitro or in vivo has led to the integration of a wide variety of growth factors (GFs) in tissue engineering strategies in an effort to mimic the natural microenvironments of tissue formation and repair. Localised delivery of exogenous GFs is believed to be therapeutically effective for replication of cellular components involved in tissue development and the healing process, thus making them important factors for tissue regeneration. However, any treatment aiming to mimic the critical aspects of the natural biological process should not be limited to the provision of a single GF, but rather should release multiple therapeutic agents at an optimised ratio, each at a physiological dose, in a specific spatiotemporal pattern. Despite several obstacles, delivery of more than one GF at rates mimicking an in vivo situation has promising potential for the clinical management of severely diseased tissues. This article summarises the concept of and early approaches toward the delivery of dual or multiple GFs, as well as current efforts to develop sophisticated delivery platforms for this ambitious purpose, with an emphasis on the application of biomaterials-based deployment technologies that allow for controlled spatial presentation and release kinetics of key biological cues. Additionally, the use of platelet-rich plasma or gene therapy is addressed as alternative, easy, cost-effective and controllable strategies for the release of high concentrations of multiple endogenous GFs, followed by an update of the current progress and future directions of research utilising release technologies in tissue engineering and regenerative medicine.

Keywords: Biomaterials; Wound healing; Controlled delivery; Drug delivery system; Gene therapy; Platelet-rich plasma (PRP)

A pH/Enzyme-responsive tumor-specific delivery system for doxorubicin by Lei Dong; Suhua Xia; Ke Wu; Zhen Huang; Huan Chen; Jiangning Chen; Junfeng Zhang (pp. 6309-6316).
To overcome the cardiotoxicity of doxorubicin, a self-assembled pH/enzyme-responsive system was developed. Cationic gelatin combined polyGC–DOX intercalation tightly to form compact nanoscale complexes (CPX1) which then combined by a pH-sensitive pegylated alginate to form CPX2. CPX2 could be digested and release DOX under the co-digestion of gelatinase (GA) and Dnase I when pH < 6.9. More importantly, tumor homogenate supernatant (THS) could effectively release DOX from CPX2 while the plasma and liver homogenate supernatant (LHS) could not, which was confirmed by an in vivo test. The results indicated that this formulation had the tumor-specific drug-release effect. This effect resulted in an increased drug concentration in tumor tissue and decreased content in heart and liver. The changed bio-distribution of DOX delivered by CPX2 greatly enhanced the anti-cancer activity and reduced the cardiotoxicity of the drug. The anti-cancer efficiency of DOX delivered by CPX2 is more than 2 times of the free doxorubicin, and the mortality caused by the high-dose DOX was completely prevented by CPX2. All results suggested that this easy-manufactured, cost-effective nanocomplex holds great promise to be developed into a formulation of doxorubicin and the other anthracyclines with high anti-cancer activity and low cardiotoxicity.

Keywords: Doxorubicin; pH/enzyme-responsive; Cardiotoxicity; Gelatinase

Polyethyleneimine-modified iron oxide nanoparticles for brain tumor drug delivery using magnetic targeting and intra-carotid administration by Beata Chertok; Allan E. David; Victor C. Yang (pp. 6317-6324).
This study aimed to examine the applicability of polyethyleneimine (PEI)-modified magnetic nanoparticles (GPEI) as a potential vascular drug/gene carrier to brain tumors. In vitro, GPEI exhibited high cell association and low cell toxicity – properties which are highly desirable for intracellular drug/gene delivery. In addition, a high saturation magnetization of 93emu/gFe was expected to facilitate magnetic targeting of GPEI to brain tumor lesions. However, following intravenous administration, GPEI could not be magnetically accumulated in tumors of rats harboring orthotopic 9L-gliosarcomas due to its poor pharmacokinetic properties, reflected by a negligibly low plasma AUC of 12±3μgFe/mlmin. To improve “passive” GPEI presentation to brain tumor vasculature for subsequent “active” magnetic capture, we examined the intra-carotid route as an alternative for nanoparticle administration. Intra-carotid administration in conjunction with magnetic targeting resulted in 30-fold ( p=0.002) increase in tumor entrapment of GPEI compared to that seen with intravenous administration. In addition, magnetic accumulation of cationic GPEI (ζ-potential=+ 37.2mV) in tumor lesions was 5.2-fold higher ( p=0.004) than that achieved with slightly anionic G100 (ζ-potential=−12mV) following intra-carotid administration, while no significant accumulation difference was detected between the two types of nanoparticles in the contra-lateral brain ( p=0.187). These promising results warrant further investigation of GPEI as a potential cell-permeable, magnetically-responsive platform for brain tumor delivery of drugs and genes.

Keywords: Nanoparticle; Magnetic targeting; Drug delivery; Brain tumor; Iron oxide

Self-quenching polysaccharide-based nanogels of pullulan/folate-photosensitizer conjugates for photodynamic therapy by Byoung-chan Bae; Kun Na (pp. 6325-6335).
Self-quenching polysaccharide-based nanogels synthesized from pullulan/folate-pheophorbide-a (Pheo-A) conjugates were investigated for their potential to reduce photosensitizer (PS) phototoxicity in normal tissue and to enhance the efficacy of tumor treatment. While the nanogels showed photoactive properties including fluorescence and singlet oxygen generation in organic solvent (DMF), these properties were suppressed in PBS due to the self-quenching of photosensitizer moieties similar to the fluorescence resonance energy transfer (FRET) effect. When the PFP2 nanogel was co-incubated with esterase or HeLa cancer cells, its photoactivity was restored. These results demonstrate that the nanogel was internalized in cancer cells by folate receptor-mediated endocytosis and was then disintegrated by various enzymes in the lysosome, leading to restoration of photoactivity. In an in vivo study, free Pheo-A showed fluorescence immediately after injection; however, nanogel fluorescence was detected 30 min after injection, increased significantly over 12 h, and was maintained beyond 3 weeks. The phototoxic properties of the nanogel were similar to those of free Pheo-A, resulting in an IC50 < 0.25 and apoptic cell death. Based on these results, we suggest that self-quenching PFP nanogels can be used to design new photodynamic therapies with minimal unfavorable phototoxicity.

Keywords: Nanogel; Self-quenching; Pullulan; Pheophorbide-a; Photoactivity

Polymer–xerogel composites for controlled release wound dressings by Marius C. Costache; Haibo Qu; Paul Ducheyne; David I. Devore (pp. 6336-6343).
Many polymers and composites have been used to prepare active wound dressings. These materials have typically exhibited potentially toxic burst release of the drugs within the first few hours followed by a much slower, potentially ineffective drug release rate thereafter. Many of these materials also degraded to produce inflammatory and cytotoxic products. To overcome these limitations, composite active wound dressings were prepared here from two fully biodegradable and tissue compatible components, silicon oxide sol–gel (xerogel) microparticles that were embedded in tyrosine-poly(ethylene glycol)-derived poly(ether carbonate) copolymer matrices. Sustained, controlled release of drugs from these composites was demonstrated in vitro using bupivacaine and mepivacaine, two water-soluble local anesthetics commonly used in clinical applications. By systematically varying independent compositional parameters of the composites, including the hydrophilic:hydrophobic balance of the tyrosine-derived monomers and poly(ethylene glycol) in the copolymers and the porosity, weight ratio and drug content of the xerogels, drug release kinetics approaching zero-order were obtained. Composites with xerogel mass fractions up to 75% and drug payloads as high as 13% by weight in the final material were fabricated without compromising the physical integrity or the controlled release kinetics. The copolymer–xerogel composites thus provided a unique solution for the sustained delivery of therapeutic agents from tissue compatible wound dressings.

Keywords: Composite; Wound dressing; Sol–gel; Polymer; Controlled drug release

Bioreducible BPEI-SS-PEG-cNGR polymer as a tumor targeted nonviral gene carrier by Sejin Son; Kaushik Singha; Won Jong Kim (pp. 6344-6354).
The work demonstrated development of multifunctional gene carrier which has incorporated reducible moiety, tumor targeting ligands as well as PEG to achieve efficient release of pDNA, enhanced tumor-specificity and long circulation, respectively. In our successful one-pot synthesis of multifunctional polymer, low molecular weight branched polyethylenimine (BPEI) was thiolated with propylene sulfide, and mixed with α-Maleimide-ω-N-hydroxysuccinimide ester polyethylene glycol (MAL–PEG–NHS, MW: 5000), and cyclic NGR peptide. The structural elucidation of the cNGR conjugated reducible BPEI containing disulfide bond (BPEI-SS-PEG-cNGR), was done by NMR and GPC study. Complex formation as well as reducible property of the polymer was confirmed by gel retardation assay. In order to achieve efficient tumor targeting, we have used cNGR peptide which is known to bind to CD13 overexpressed in neovasculature endothelial cells. Tumor target-specificity of polymer was established by carrying out competitive inhibition assay with free cNGR peptide. Cellular uptake of polymers was evaluated by confocal laser scanning microscope (CLSM). Finally, addition of free cNGR and buthionine sulfoximine (BSO) reduced transfection efficiency synergistically, which implied that multifunctional polymer-mediated gene transfection took place tumor-specifically and via GSH-dependent pathway.

Keywords: NGR peptide; Polyethylenimine; Reducible; Tumor targeting; Gene delivery

2-Methacryloyloxyethyl phosphorylcholine polymer (MPC)-coating improves the transfection activity of GALA-modified lipid nanoparticles by assisting the cellular uptake and intracellular dissociation of plasmid DNA in primary hepatocytes by Masami Ukawa; Hidetaka Akita; Tomoya Masuda; Yasuhiro Hayashi; Tomohiro Konno; Kazuhiko Ishihara; Hideyoshi Harashima (pp. 6355-6362).
We previously reported that modification of GALA peptide on the surface of liposomes enhanced fusion with endosomal membrane, and cytoplasmic release of encapsulated macromolecules. We report herein that an additional coating of GALA-modified liposomes with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer resulted in a two order of magnitude enhancement in the transfection activity of encapsulating plasmid DNA (pDNA). Quantification of the delivered gene copies in whole cells and isolated nuclei revealed that the increase of transfection activity can be attributed to improved efficiencies in cellular uptake and post-nuclear delivery processes. Imaging studies revealed that the intracellular dissociation of pDNA from the lipid envelope is enhanced by GALA modification and further coating with MPC polymer in a stepwise manner. The MPC polymer-coating decreased the ζ-potential of GALA-modified liposomes, suggesting that it assisted in the functional display of negatively charged GALA on the cationic liposomes by providing shielding from mutual electrostatic interactions. Collectively, these data indicate that MPC polymer-coating induced the fusogenic activity of the GALA-modified envelope with endosomes, leading to a more effective cytoplasmic release pDNA. The extensive fusion of the lipid envelope may also reduce electrostatic interactions between mRNA and cationic lipid components, thereby resulting in an enhancement in the translation process.

Keywords: Isolated hepatocyte; Gene delivery; Intracellular trafficking; Endosomal escape; GALA; MPC polymer

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