Biomaterials (v.32, #32)

The effect of plasma chemical oxidation of titanium alloy on bone-implant contact in rats by Michael Diefenbeck; Thomas Mückley; Christian Schrader; Jürgen Schmidt; Sergiy Zankovych; Jörg Bossert; Klaus D. Jandt; Mathilde Faucon; Ulrich Finger (8041-8047).
Many different technologies have been used to enhance osseointegration in orthopaedic and dental implant surgery. Hydroxyapatite coatings, pure or in combination with growth factors or bisphosphonates, showed improved osseointegration of titanium alloy implants. We choose a different approach to enhance osseointegration: plasma chemical oxidation was used to modify the surface of titanium alloy implants. This technique converts the nm-thin natural occurring titanium oxide layer on an implant to a 4 μm thick ceramic coating (TiOB surface). Bioactive TiOB surfaces have a macroporous structure and were loaded with calcium and phosphorus, while bioinert TiOB surfaces are smooth. A rat tibial model with bilateral placement of titanium alloy implants was employed to analyze the bone response to TiOB surfaces in vivo. 64 rats were randomly assigned to four groups of implants: (1) titanium alloy (control), (2) titanium alloy, type III anodization, (3) bioinert TiOB surface and (4) bioactive TiOB surface. Mechanical fixation, peri-implant-bone area and bone contact were evaluated by pull-out tests and histology at three and eight weeks. Shear strength and bone contact at eight weeks were significantly increased in the bioactive TiOB group compared to all other groups. The results of plasma chemical oxidation in a rat model showed that the bioactive TiOB surface has a positive effect on implant anchorage by enhancing the bone-implant contact in normal bone.
Keywords: Titanium alloy; Titanium oxide; Surface modification; Osseointegration; Animal model;

Micropatterns of arginine–glycine–aspartic acid (RGD) on poly(ethylene glycol) (PEG) hydrogels were fabricated. Under an appropriate size of microislands on this strong and persistent non-fouling background, single mesenchymal stem cells (MSCs) from rats were well localized, keeping the same adhesive area but different shapes. The cell shapes influenced the differentiation of MSCs, and the osteogenic and adipogenic differentiations exhibited different trends. According to comparison between square and rectangular cells, optimal adipogenic differentiation occurred at aspect ratio (AR) 1, but the optimal osteogenic differentiation was found when AR was about 2. We further interpreted the optimal ratios as reflecting the inherent global anisotropy of free adipoblasts and osteoblasts on unpatterned culture plates. According to comparison between globally isotropic circular, square, triangular, and star cells, the optimal adipogenic and osteogenic differentiations happened in circular and star cells, respectively. In this case we found that extents of both adipogenic and osteogenic differentiations were linearly related to cell perimeter, which reflects the non-roundness or local anisotropy of cells. Hence, the present study makes semi-quantitative investigations of the effects of cell shape on differentiation of stem cells based on a material technique, and reveals that the shape anisotropy is very important in directing the lineage commitments of stem cells.
Keywords: Micropatterning; Stem cells; Hydrogels; Surface modification; Polyethylene oxide; RGD peptide;

A synthetic substrate to support early mesodermal differentiation of human embryonic stem cells by Yang Liu; Xintong Wang; Dan S. Kaufman; Wei Shen (8058-8066).
Our ability to guide differentiation of human pluripotent stem cells (hPSCs) toward desired lineages efficiently and reproducibly in xeno-free conditions is the key to advancing hPSC technology from the laboratory to clinical use. Here we report an engineered biomimetic substrate functionalized with both peptide ligands for α5β1 and α6β1 integrins to support efficient early mesodermal differentiation of human embryonic stem cells (hESCs) when cultured in a differentiation medium containing BMP4. In contrast, mesodermal differentiation is not induced on substrates functionalized with either ligand alone even though the culture medium is identical. Mesodermal differentiation was characterized by immunofluorescent staining, flow cytometric analysis, and RT-PCR analysis of early mesodermal markers Brachyury, Mixl1, and Wnt3. The early mesodermal progenitors derived on the substrate functionalized with both integrin ligands have the normal developmental potential to further differentiate along the hemato-endothelial and cardiac lineages. Immobilized ligands for α5β1 and α6β1 integrins both are permissive, necessary, and sufficient insoluble ligands in this engineered system to support early mesodermal differentiation of hESCs. This synthetic substrate, in conjunction with defined soluble factors, constructs a well-controlled and xeno-free early mesodermal differentiation niche that offers advantages over the previously reported niche constructed with the Matrigel-coated substrate.
Keywords: Stem cell; Biomimetic material; Surface modification; Peptide; Integrin;

Engineering extracellular matrix structure in 3D multiphase tissues by Brian M. Gillette; Ninna S. Rossen; Nikkan Das; Debra Leong; Meixin Wang; Arushi Dugar; Samuel K. Sia (8067-8076).
In native tissues, microscale variations in the extracellular matrix (ECM) structure can drive different cellular behaviors. Although control over ECM structure could prove useful in tissue engineering and in studies of cellular behavior, isotropic 3D matrices poorly replicate variations in local microenvironments. In this paper, we demonstrate a method to engineer local variations in the density and size of collagen fibers throughout 3D tissues. The results showed that, in engineered multiphase tissues, the structures of collagen fibers in both the bulk ECM phases (as measured by mesh size and width of fibers) as well as at tissue interfaces (as measured by density of fibers and thickness of tissue interfaces) could be modulated by varying the collagen concentrations and gelling temperatures. As the method makes use of a previously published technique for tissue bonding, we also confirmed that significant adhesion strength at tissue interfaces was achieved under all conditions tested. Hence, this study demonstrates how collagen fiber structures can be engineered within all regions of a multiphase tissue scaffold by exploiting knowledge of collagen assembly, and presents an approach to engineer local collagen structure that complements methods such as flow alignment and electrospinning.
Keywords: Adhesion; Alginate; Collagen; Hydrogel; Interface; Micropatterning;

Comparison of polymer scaffolds in rat spinal cord: A step toward quantitative assessment of combinatorial approaches to spinal cord repair by Bingkun K. Chen; Andrew M. Knight; Nicolas N. Madigan; LouAnn Gross; Mahrokh Dadsetan; Jarred J. Nesbitt; Gemma E. Rooney; Bradford L. Currier; Michael J. Yaszemski; Robert J. Spinner; Anthony J. Windebank (8077-8086).
The transected rat thoracic (T9/10) spinal cord model is a platform for quantitatively comparing biodegradable polymer scaffolds. Schwann cell-loaded scaffolds constructed from poly (lactic co-glycolic acid) (PLGA), poly(ɛ-caprolactone fumarate) (PCLF), oligo(polyethylene glycol) fumarate (OPF) hydrogel or positively charged OPF (OPF+) hydrogel were implanted into the model. We demonstrated that the mechanical properties (3-point bending and stiffness) of OPF and OPF + hydrogels closely resembled rat spinal cord. After one month, tissues were harvested and analyzed by morphometry of neurofilament-stained sections at rostral, midlevel, and caudal scaffold. All polymers supported axonal growth. Significantly higher numbers of axons were found in PCLF (P < 0.01) and OPF+ (P < 0.05) groups, compared to that of the PLGA group. OPF + polymers showed more centrally distributed axonal regeneration within the channels while other polymers (PLGA, PCLF and OPF) tended to show more evenly dispersed axons within the channels. The centralized distribution was associated with significantly more axons regenerating (P < 0.05). Volume of scar and cyst rostral and caudal to the implanted scaffold was measured and compared. There were significantly smaller cyst volumes in PLGA compared to PCLF groups. The model provides a quantitative basis for assessing individual and combined tissue engineering strategies.
Keywords: OPF; PLGA; PCLF; Axon regeneration; Spinal cord injury; Schwann cell;

Concave microwell based size-controllable hepatosphere as a three-dimensional liver tissue model by Sau Fung Wong; Da Yoon No; Yoon Young Choi; Dong Sik Kim; Bong Geun Chung; Sang-Hoon Lee (8087-8096).
We have developed a size-controllable spheroidal hepatosphere and heterosphere model by mono-culturing of primary hepatocytes and by co-culturing primary hepatocytes and hepatic stellate cells (HSCs). We demonstrated that uniform-sized heterospheres, which self-aggregated from primary hepatocytes and HSCs, formed within concave microwell arrays in a rapid and homogeneous manner. The effect of HSCs was quantitatively and qualitatively investigated during spheroid formation, and HSC played an important role in controlling the organization of the spheroidal aggregates and formation of tight cell–cell contacts. An analysis of the metabolic function showed that heterospheres secreted 30% more albumin than hepatospheres on day 8. In contrast, the urea secretion from heterospheres was similar to that of hepatospheres. A quantitative cytochrome P450 assay showed that the enzymatic activity of heterospheres cultured for 9 days was higher as compared with primary hepatospheres. These size-controllable heterospheres could be mass-produced using concave plate and be useful for creating artificial three-dimensional hepatic tissue constructs and regeneration of failed liver.
Keywords: Co-culture; Hepatocytes; Hepatic stellate cells; Concave microwell array; Hepatospheres; Heterospheres;

Bioprinting of growth factors onto aligned sub-micron fibrous scaffolds for simultaneous control of cell differentiation and alignment by Elmer D.F. Ker; Amrinder S. Nain; Lee E. Weiss; Ji Wang; Joseph Suhan; Cristina H. Amon; Phil G. Campbell (8097-8107).
The capability to spatially control stem cell orientation and differentiation simultaneously using a combination of geometric cues that mimic structural aspects of native extracellular matrix (ECM) and biochemical cues such as ECM-bound growth factors (GFs) is important for understanding the organization and function of musculoskeletal tissues. Herein, oriented sub-micron fibers, which are morphologically similar to musculoskeletal ECM, were spatially patterned with GFs using an inkjet-based bioprinter to create geometric and biochemical cues that direct musculoskeletal cell alignment and differentiation in vitro in registration with fiber orientation and printed patterns, respectively. Sub-micron polystyrene fibers (diameter ∼ 655 nm) were fabricated using a Spinneret-based Tunable Engineered Parameters (STEP) technique and coated with serum or fibrin. The fibers were subsequently patterned with tendon-promoting fibroblast growth factor-2 (FGF-2) or bone-promoting bone morphogenetic protein-2 (BMP-2) prior to seeding with mouse C2C12 myoblasts or C3H10T1/2 mesenchymal fibroblasts. Unprinted regions of STEP fibers showed myocyte differentiation while printed FGF-2 and BMP-2 patterns promoted tenocyte and osteoblast fates, respectively, and inhibited myocyte differentiation. Additionally, cells aligned along the fiber length. Functionalizing oriented sub-micron fibers with printed GFs provides instructive cues to spatially control cell fate and alignment to mimic native tissue organization and may have applications in regenerative medicine.
Keywords: Bone; Muscle; Tendon; Growth factors;

A significant stumbling block in the creation of functional three-dimensional (3D) engineered tissues is the proper vascularization of the constructs. Furthermore, in the context of electrospinning, the development of 3D constructs using this technique has been hindered by the limited infiltration of cells into their structure. In an attempt to address these issues, a hybrid mesh of poly (ɛ-caprolactone)-collagen blend (PCL/Col) and hyaluronic acid (HA) hydrogel, Heprasil™ was created via a dual electrodeposition system. Simultaneous deposition of HA and PCL/Col allowed the dual loading and controlled release of two potent angiogenic growth factors VEGF165 and PDGF-BB over a period of five weeks in vitro. Furthermore, this manner of loading sustained the bioactivity of the two growth factors. Utilizing an in-house developed 3D co-culture assay model of human umbilical vein endothelial cells and lung fibroblasts, the growth factor-loaded hybrid meshes was shown to not only support cellular attachment, but also their infiltration and the recapitulation of primitive capillary network in the scaffold’s architecture. Thus, the creation of a PCL/Col-Heprasil hybrid scaffold is a step forward toward the attainment of a 3D bio-functionalized, vascularized tissue engineering construct.
Keywords: Angiogenesis; Hyaluronic acid/hyaluronan; Polycaprolactone; Collagen; Scaffold; Endothelial cell;

Natural biological conduits containing seed cells have been widely used as an alternative strategy for nerve gap reconstruction to replace traditional nerve autograft techniques. The purpose of this study was to investigate the effects of a decellularized allogeneic artery conduit containing autologous transdifferentiated adipose-derived stem cells (dADSCs) on an 8-mm facial nerve branch lesion in a rat model. After 8 weeks, functional evaluation of vibrissae movements and electrophysiological assessment, retrograde labeling of facial motoneurons and morphological analysis of regenerated nerves were performed to assess nerve regeneration. The transected nerves reconstructed with dADSC-seeded artery conduits achieved satisfying regenerative outcomes associated with morphological and functional improvements which approached those achieved with Schwann cell (SC)-seeded artery conduits, and superior to those achieved with artery conduits alone or ADSC-seeded artery conduits, but inferior to those achieved with nerve autografts. Besides, numerous transplanted PKH26-labeled dADSCs maintained their acquired SC-phenotype and myelin sheath-forming capacity inside decellularized artery conduits and were involved in the process of axonal regeneration and remyelination. Collectively, our combined use of decellularized allogeneic artery conduits with autologous dADSCs certainly showed beneficial effects on nerve regeneration and functional restoration, and thus represents an alternative approach for the reconstruction of peripheral facial nerve defects.
Keywords: Facial nerve; Nerve regeneration; Decellularized artery conduit; Adipose-derived stem cells; Transdifferentiation;

The role of protein solubilization in antigen removal from xenogeneic tissue for heart valve tissue engineering by Maelene L. Wong; J. Kent Leach; Kyriacos A. Athanasiou; Leigh G. Griffiths (8129-8138).
Decellularization techniques have been developed in an attempt to reduce the antigenicity of xenogeneic biomaterials, a critical barrier in their use as tissue engineering scaffolds. However, numerous studies have demonstrated inadequate removal and subsequent persistence of antigens in the biomaterial following decellularization, resulting in an immune response upon implantation. Thus, methods to enhance antigen removal (AR) are critical for the use of xenogeneic biomaterials in tissue engineering and regenerative medicine. In the present study, AR methods incorporating protein solubilization principles were investigated for their ability to reduce antigenicity of bovine pericardium (BP) for heart valve tissue engineering. Bovine pericardium following AR (BP-AR) was assessed for residual antigenicity, tensile properties, and extracellular matrix composition. Increasing protein solubility during AR significantly decreased the residual antigenicity of BP-AR—by an additional 80% compared to hypotonic solution or 60% compared to 0.1% (w/v) SDS decellularization methods. Moreover, solubilizing agents have a dominant effect on reducing the level of residual antigenicity of BP-AR beyond that achieved by AR additives alone. Tested AR methods did not compromise the tensile properties of BP-AR compared to native BP. Furthermore, residual cell nuclei did not correlate to residual antigenicity, demonstrating that residual nuclei counts may not be an appropriate indicator of successful AR. In conclusion, AR strategies promoting protein solubilization significantly reduced residual antigens compared to decellularization methods without compromising biomaterial functional properties. This study demonstrates the importance of solubilizing protein antigens for their removal in the generation of xenogeneic scaffolds.
Keywords: Antigen removal; Xenogeneic scaffold; Decellularization; Extracellular matrix; Heart valve tissue engineering;

Chondrogenic potential of stem cells derived from amniotic fluid, adipose tissue, or bone marrow encapsulated in fibrin gels containing TGF-β3 by Ji Sun Park; Myung-Sun Shim; Sung Han Shim; Han Na Yang; Su Yeon Jeon; Dae Gyun Woo; Dong Ryul Lee; Tae Ki Yoon; Keun-Hong Park (8139-8149).
In this study, several types of hMSCs, derived from bone marrow, adipose tissue, or amniotic fluid, were encapsulated in a fibrin hydrogel mixed with TGF-β3 and then evaluated for their capacity for differentiation in vitro and in vivo. For determination of stem cell differentiation, RT-PCR, real time quantitative PCR (qPCR), histology, and immunohistochemical assays were used for analysis of chondrogenesis. Using these analysis methods, several of the cultured hMSCS were found to highly express genes and proteins specific to cartilage forming tissues. Additionally, similar trends in expression were found in tissue recovered from nude mice transplanted with several types of hMSCs encapsulated in a fibrin hydrogel containing TGF-β3. The results of both in vitro and in vivo analyses showed that cultured or transplanted hMSCs mixed with TGF-β3 in a fibrin hydrogel differentiated into chondrocytes, suggesting that these cells would be suitable for reconstruction of hyaline articular cartilage.
Keywords: Fibrin; hMSC; TGF-β3; Chondrogenesis; Cartilage;

Scaffold vascularization in vivo driven by primary human osteoblasts in concert with host inflammatory cells by Shahram Ghanaati; Ronald E. Unger; Matthew J. Webber; Mike Barbeck; Carina Orth; Jenny A. Kirkpatrick; Patrick Booms; Antonella Motta; Claudio Migliaresi; Robert A. Sader; C. James Kirkpatrick (8150-8160).
Successful cell-based tissue engineering requires a rapid and thorough vascularization in order to ensure long-term implant survival and tissue integration. The vascularization of a scaffold is a complex process, and is modulated by the presence of transplanted cells, exogenous and endogenous signaling proteins, and the host tissue reaction, among other influencing factors. This paper presents evidence for the significance of pre-seeded osteoblasts for the in vivo vascularization of a biodegradable scaffold. Human osteoblasts, cultured on silk fibroin micronets in vitro, migrated throughout the interconnected pores of the scaffold and produced extensive bone matrix. When these constructs were implanted in SCID mice, a rapid and thorough vascularization of the scaffold by the host blood capillaries occurred. This profound response was not seen for the silk fibroin scaffold alone. Moreover, when the pre-cultivation time of human osteoblasts was reduced from 14 days to only 24 h, the significant effect these cells exerted on vascularization rate in vivo was still detectable. From these studies, we conclude that matrix and soluble factors produced by osteoblasts can serve to instruct host endothelial cells to migrate, proliferate, and initiate the process of scaffold vascularization. This finding represents a potential paradigm shift for the field of tissue engineering, especially in bone, as traditional strategies to enhance scaffold vascularization have focused on endovascular cells and regarded osteoblasts primarily as cell targets for mineralization. In addition, the migration of host macrophages and multinucleated giant cells into the scaffold was also found to influence the vascularization of the biomaterial. Therefore, the robust effect on scaffold vascularization seen by pre-culturing with osteoblasts appears to occur in concert with the pro-angiogenic stimuli arising from host immune cells.
Keywords: Angiogenesis; Bone tissue engineering; Osteoblasts; Multinucleated giant cells; Silk fibroin;

Photo-cured hyaluronic acid-based hydrogels containing simvastatin as a bone tissue regeneration scaffold by Min Soo Bae; Dae Hyeok Yang; Jung Bok Lee; Dong Nyoung Heo; Yong-Dae Kwon; In Chan Youn; Kuiwon Choi; Jong Hyun Hong; Gye Tae Kim; Yong Suk Choi; Eui Hwan Hwang; Il Keun Kwon (8161-8171).
We describe in this study the positive influences on in vitro and in vivo osteogenesis of photo-cured hyaluronic acid (HA) hydrogels loaded with simvastatin (SIM). Prior to loading SIM, we first characterized the HA hydrogels for their mechanical properties and swelling ratios. The results from this testing indicated that these two factors improved as the substitution degree of 2-aminoethyl methacrylate (AEMA) increased. MTT and live/dead assays showed that the HA hydrogels have good biocompatibility for use as scaffolds for bone tissue regeneration. Moreover, another MTT assay showed that the photo-cured HA hydrogels III fabricated with 30% AEMA (300 mg) conjugated HA (HA-AEMA iii) loaded with between 0.1 and 1 mg of SIM had a similar cytotoxicity as compared to the HA hydrogel III itself. The sustained release of SIM was observed to occur in the HA hydrogel III loaded with 1 mg of SIM. In vitro and in vivo experiments showed that the HA hydrogel III loaded with 1 mg of SIM had a significant influence on osteogenesis.
Keywords: Photopolymerization; Hyaluronic acid; Hydrogel; Simvastatin; Bone tissue regeneration;

Regeneration of uterine horns in rats by collagen scaffolds loaded with collagen-binding human basic fibroblast growth factor by Xin’an Li; Haixiang Sun; Nacheng Lin; Xianglin Hou; Jingmei Wang; Bai Zhou; Peizhen Xu; Zhifeng Xiao; Bing Chen; Jianwu Dai; Yali Hu (8172-8181).
Severe damages of uterine endometrium which prevent embryos from implantation and placentation finally often result in infertility or pregnant complications. There is lack of effective treatments due to the limitation of native materials available and complexity of the function and internal environment of uterus. In the present study, a collagen targeting basic fibroblast growth factor (bFGF) delivery system was constructed by a collagen membrane loaded with bFGF fused a collagen-binding domain (CBD) to the N-terminal which limits the diffusion of bFGF from collagen. We tested the bFGF delivery system in rats under the severe uterine damage model (partial rat uterine horn excision/reconstruction), and found this delivery system improved regeneration abilities of uterine endometrium and muscular cells, improved vascularization, as well as better pregnancy outcomes in rats. Therefore, this targeting delivery system may be an effective strategy for uterine tissue regeneration.
Keywords: Collagen; Basic fibroblast growth factor; Uterine horn regeneration; Tissue engineering;

Effects of hypoxias and scaffold architecture on rabbit mesenchymal stem cell differentiation towards a nucleus pulposus-like phenotype by Ganjun Feng; Xiaobing Jin; Jiang Hu; Haiyun Ma; Melanie J. Gupte; Hao Liu; Peter X. Ma (8182-8189).
Nucleus pulposus grafts are needed for patients requiring replacement of their degenerated intervertebral discs. Bone marrow-derived mesenchymal stem cells (MSCs) are a potential autologous stem cell source for the nucleus pulposus regeneration. One of the key issues of constructing functional nucleus pulposus using MSCs, however, is to differentiate MSCs into nucleus pulposus phenotype in vitro and to maintain their phenotypic stability in vivo. In this study, three-dimensional (3D) nanofibrous poly(l-lactide) (PLLA) scaffolds were seeded with multi-potent rabbit MSCs and the constructs were induced along nucleus pulposus development routes in a hypoxia chamber (2% O2) in the presence of TGF-β1. It was found that nanofibrous scaffold could support the differentiation of rabbit MSCs towards a nucleus pulposus-like phenotype in vitro, as evidenced by upregulated expression of a few important nucleus pulposus-associated genes (aggrecan, type II collagen and Sox-9), abundant deposition of extracellular matrix (glycosaminoglycan (GAG) and type II collagen), and the continuous expression of the nucleus pulposus-specific marker, hypoxia-inducible factor (HIF)-1α. The subcutaneous implantation results confirmed that hypoxic induction before implantation could help the constructs to retain their phenotype and resist calcification in vivo. Therefore, the above data showed the promise of using 3D nanofibrous scaffolds in combination with TGF-β1 and hypoxic induction to regenerate functional nucleus pulposus grafts for intervertebral disc replacement.
Keywords: Rabbit bone marrow-derived mesenchymal stem cell; Nanofiber; Porous scaffold; Hypoxia; Nucleus pulposus graft;

The stimulation of an osteogenic response by classical monocyte activation by Omar M. Omar; Cecilia Granéli; Karin Ekström; Camilla Karlsson; Anna Johansson; Jukka Lausmaa; Cecilia Larsson Wexell; Peter Thomsen (8190-8204).
The monocyte/macrophage system plays a central role in host defense, wound healing and immune regulation at biomaterial surfaces. Monocytes can be classically and alternatively activated, and can be stimulated differently in response to variations in biomaterial surface properties. In this study, human monocytes, cultured on polystyrene surfaces (Ps), were activated either classically, by lipopolysaccharide (LPS), or alternatively, by interleukin-4 (IL-4). Monocytes were also cultured on anodically oxidized (Ox) and machined (Ma) titanium surfaces, with and without LPS stimulation. Cells were cultured for 1 and 3 days and their conditioned media (CM) were collected. The osteogenic response of hMSCs to the monocyte CM was determined by analyzing the gene expression of key osteogenic markers. The CM from classically activated monocytes increased the hMSCs expression of runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP). Furthermore, CM from monocytes cultured on Ox surface resulted in a modest increase of the expression of bone morphogenetic protein-2 (BMP-2). LPS stimulation of the surface-seeded monocytes overwhelmed the effect of the surface properties and resulted in significant upregulation of BMP-2 and Runx2 for all samples. The results show that human monocytes, cultured on different surfaces and/or under different activation pathways, communicate pro-osteogenic signals to hMSCs. The signals involve regulation of autologous BMP-2 in the hMSCs. The classical activation results in profound and prolonged osteogenic effect compared to the effect of the investigated surface properties.
Keywords: Monocytes; Inflammation; Mesenchymal stem cells; Cell signalling; Cytokines; Osseointegration;

Mechanical and biological properties of keratose biomaterials by Roche C. de Guzman; Michelle R. Merrill; Jillian R. Richter; Rawad I. Hamzi; Olga K. Greengauz-Roberts; Mark E. Van Dyke (8205-8217).
The oxidized form of extractable human hair keratin proteins, commonly referred to as keratose, is gaining interest as a biomaterial for multiple tissue engineering studies including those directed toward peripheral nerve, spinal cord, skin, and bone regeneration. Unlike its disulfide cross-linked counterpart, kerateine, keratose does not possess a covalently cross-linked network structure and consequently displays substantially different characteristics. In order to understand its mode(s) of action and potential for clinical translatability, detailed characterization of the composition, physical properties, and biological responses of keratose biomaterials are needed. Keratose was obtained from end-cut human hair fibers by peracetic acid treatment, followed by base extraction, and subsequent dialysis. Analysis of lyophilized keratose powder determined that it contains 99% proteins by mass with amino acid content similar to human hair cortex. Metallic elements were also found in minute quantities. Protein oxidation led to disulfide bond cleavage and drastic reduction of free thiols due to conversion of sulfhydryl to sulfonic acid, chain fragmentation, and amino acid modifications. Mass spectrometry identified the major protein constituents as a heterogeneous mixture of 15 hair keratins (type I: K31–35 and K37–39, and type II: K81–86) with small amounts of epithelial keratins which exist in monomeric, dimeric, multimeric, and even degraded forms. Re-hydration with PBS enabled molecular assembly into an elastic solid-like hydrogel. Highly-porous scaffolds formed by lyophilization of the gel had the compression behavior of a cellular foam material and reverted back to gel upon wetting. Cytotoxicity assays showed that the EC50 for various cell lines were attained at 8–10 mg/mL keratose, indicating the non-toxic nature of the material. Implantation in mouse subcutaneous tissue pockets demonstrated that keratose resorption follows a rectangular hyperbolic regression with 92% degradation by an 8-week time point. Keratose was shown to integrate with the host tissue as evidenced by infiltration of leukocytes and fibroblasts, bulk material angiogenesis, and minimal fibrous encapsulation. Tissue response benchmarks were superior in keratose compared to the control PLGA 90:10 mesh. Finally, the degraded keratose was observed to remodel with the natural collagen extracellular matrix, verifying the benefit of using keratose as a temporary matrix for regenerative medicine applications.
Keywords: Keratin; Biomaterial; Protein oxidation; Compatibility; Tissue engineering; Regenerative medicine;

The role of the tumor suppressor p53 pathway in the cellular DNA damage response to zinc oxide nanoparticles by Kee Woei Ng; Stella P.K. Khoo; Boon Chin Heng; Magdiel I. Setyawati; Eng Chok Tan; Xinxin Zhao; Sijing Xiong; Wanru Fang; David T. Leong; Joachim S.C. Loo (8218-8225).
In this paper, we explored how ZnO nanoparticles cross-interact with a critical tumor suppressive pathway centered around p53, which is one of the most important known tumor suppressors that protects cells from developing cancer phenotypes through its control over major pathways like apoptosis, senescence and cell cycle progression. We showed that the p53 pathway was activated in BJ cells (skin fibroblasts) upon ZnO nanoparticles treatment with a concomitant decrease in cell numbers. This suggests that cellular responses like apoptosis in the presence of ZnO nanoparticles require p53 as the molecular master switch towards programmed cell death. This also suggests that in cells without robust p53, protective response can be tipped towards carcinogenesis when stimulated by DNA damage inducing agents like ZnO nanoparticles. We observed this precarious tendency in the same BJ cells with p53 knocked down using endogeneous expressing shRNA. These p53 knocked down BJ cells became more resistant to ZnO nanoparticles induced cell death and increased cell progression. Collectively, our results suggest that cellular response towards specific nanoparticle induced cell toxicity and carcinogenesis is not only dependent on specific nanoparticle properties but also (perhaps more importantly) the endogenous genetic, transcriptomic and proteomic landscape of the target cells.
Keywords: Nanotoxicology; Genotoxicology; Zinc oxide; Nanoparticles; DNA damage; p53;

Sorting and detection of circulating tumor cells (CTC) in peripheral blood as an efficient and non-invasive method to diagnose cancer have recently attracted much attention. In this article, we developed a multiply-engineered nanoparticle system for CTC sorting and detection, which consists of (1) conjugated oligomer (CO) as fluorescence signal source, (2) polyhedral oligomeric silsesquioxanes (POSS) scaffold for CO localization for better fluorescent effects, (3) silica nanoparticles (SiNPs) as formulation matrix of the POSS containing CO, (4) iron oxide (IO) layer on the silica nanoparticles (IO-SiNPs) for magnetic collection, and (5) herceptin surface functionalization of the IO-SiNPs to target cancer cells of HER2 overexpression. Such a multiply-engineered structure can be used for either traditional immunomagnetic methods or microfluidic devices for CTC sorting and detection.
Keywords: CTC; Cancer nanotechnology; Conjugated oligomers; Iron oxides; Silica nanoparticles; Trastuzumab;

Three reversible and controllable discrete steps of channel gating of a viral DNA packaging motor by Jia Geng; Huaming Fang; Farzin Haque; Le Zhang; Peixuan Guo (8234-8242).
The channel of the viral DNA packaging motor allows dsDNA to enter the protein procapsid shell during maturation and to exit during infection. We recently showed that the bacteriophage phi29 DNA packaging motor exercises a one-way traffic property using a channel as a valve for dsDNA translocation. This raises a question of how dsDNA is ejected during infection if the channel only allows the dsDNA to travel inward. We proposed that DNA forward or reverse travel is controlled by conformational changes of the channel. Here we reported our direct observation that the channel indeed exercises conformational changes by single channel recording at a single-molecule level. The changes were induced by high electrical voltage, or by affinity binding to the C-terminal wider end located within the capsid. Novel enough, the conformational change of the purified connector channel exhibited three discrete gating steps, with a size reduction of 32% for each step. We investigated the role of the terminal and internal loop of the channel in gating by different mutants. The step-wise conformational change of the channel was also reversible and controllable, making it an ideal nano-valve for constructing a nanomachine with potential applications in nanobiotechnology and nanomedicine.
Keywords: Nanotechnology; Bionanotechnology; DNA packaging; Viral motor; Nanopore; Single-molecule sensing;

Multifunctional rare-earth self-assembled nanosystem for tri-modal upconversion luminescence /fluorescence /positron emission tomography imaging by Qian Liu; Min Chen; Yun Sun; Guoying Chen; Tianshe Yang; Yuan Gao; Xianzhong Zhang; Fuyou Li (8243-8253).
Rare-earth upconversion nanoparticles (UCNP) which can absorb low-energy photons and emit high energy photons have attracted great interest not only because of their unique application in upconversion luminescence imaging, but also because they can be used as ideal building blocks for multimodal bioimaging probes. Improving the water-solubility of UCNP and functionalizing them are as yet unresolved problems. In this present study, a general strategy was developed to achieve these two aims by converting hydrophobic upconversion nanoparticles into hydrophilic ones. This was based on the self-assembly between oleic acid, which is a capping ligand, as the guest molecule, and alpha-cyclodextrin, as the host molecule, no matter what the particle size was (10–400 nm) or what synthesis method (thermal decomposition, hydrothermal, solvothermal) was used. The synthesized hydrophilic nanoparticles can further load hydrophobic molecule, e.g. Os(II) complex. The process of self-assembly and loading was confirmed by transmission electron microscopy, X-ray powder diffraction, 1H-nuclear magnetic resonance, Fourier transform-infrared and thermogravimetric analysis, upconversion luminescence and fluorescence spectra. Further bioapplication has also been investigated, including cell-labeling, in vivo lymphatic imaging, upconversion luminescence and positron emission tomography imaging of whole-body Kunming mice. The results indicate that this method is a potential candidate for the preparation of hydrophilic UCNP as a multimodal nanoprobe.
Keywords: Upconversion nanoparticles; Alpha-cyclodextrin; Positron emission tomography (PET); Upconversion luminescence (UCL) imaging;

Polycaprolactone (PCL)/Pluronic F127 cylindrical scaffolds with gradually increasing growth factor concentrations were fabricated by the centrifugation of fibril-like PCLs and the subsequent fibril surface immobilization of growth factors. The cylindrical scaffolds exhibited gradually increasing surface areas along the longitudinal direction [from 3.17 ± 0.05 m2/g (top position) to 5.42 ± 0.01 m2/g (bottom position)]. The growth factors (BMP-7, TGF-β2 and VEGF165) as model bioactive molecules were immobilized onto the fibril surfaces of the scaffolds via heparin binding to produce scaffolds with gradually increasing concentrations of growth factors from the top position (BMP-7, 60.89 ± 2.51; TGF-β2, 42.85 ± 2.00; VEGF165, 42.52 ± 3.22 ng/scaffold section) to the bottom position (BMP-7, 181.07 ± 3.21; TGF-β2, 142.08 ± 2.91; VEGF165, 112.00 ± 4.00 ng/scaffold section). The released amount of growth factor (VEGF165) from the cylindrical scaffold gradually decreased along the longitudinal direction in a sustained manner for up to 35 days, which can allow for a minutely controlled spatial distribution of growth factors in a 3D environment. The 3D porous scaffold with a concentration gradient of growth factors may become a useful tool for basic studies, including in vitro investigations of 3D chemotaxis/haptotaxis for the control of specific biological process. It may also be used as a tissue engineering scaffolding system for a variety of tissues/organs requiring the spatial regulation of growth factors for effective regeneration.
Keywords: Growth factor gradient; Scaffold; Polycaprolactone; Centrifugation; Surface immobilization;

In order to design a water soluble polymeric photosensitizer (WPS) with controllable photoactivity, a nano-photosensitizer (NPS) was prepared from a polyelectrolyte complex between polyethylene glycol-polyethylenimine-chlorine e6 conjugate (PEG-PEI-Ce6) and Black Hole Quencher-3 chondroitin sulfate conjugate (BHQ-3-CS). NPSs have a unimodal size distribution below 100 nm. Photoquenching of the NPS was dependent on the weight ratio of BHQ-3-CS/WPS. This phenomenon was maintained in a salt condition up to 300 mm, indicating that the photoactivity of the NPS disappears in the normal blood stream of the body. The quenched photoactivity was restored by the enzyme degradation of BHQ-3-CS after esterase treatment. In a HCT-116 (human colon cancer) cell test, the rapid cellular internalization of the NPS without any other ligands was observed by confocal imaging. Upon light irradiation after internalization, phototoxicity was detected via MTT colorimetric assay. Also, when the NPS was subcutaneously injected in both tumoral and normal regions of HCT-116 tumor-bearing mice, the fluorescence signal in the tumors rapidly increased compared to the normal region due to the enzymatic-triggered dissociation of the NPS in vivo. These results suggest that the NPS can provide both tumor diagnosis and therapy simultaneously, and has great potential for biological studies and clinical treatments of various tumors.
Keywords: Water soluble polymeric photosensitizer; Photoactivity; Nano-photosensitizer; Photoquenching;

We have endeavored to develop injectable, thermosensitive, biodegradable hydrogels that prolong human growth hormone (hGH) release, improving bioavailability through introducing balanced ionic interactions. Cationic poly(organophosphazene)-polyethylenimine (PEI, 1.8 kDa) conjugate hydrogels were synthesized as those hydrogels for sustained delivery of anionic hGH with proper ionic strength of association/dissociation. We have additionally prepared different chain lengths of α-amino-ω-methoxy-poly(ethylene glycol) (AMPEG550 and AMPEG750) for the synthesis of conjugates as a means to control hydrogel degradation rates. All Aqueous solutions of PEI-conjugates became hydrogels hydrolyzable in proportion to AMPEG molecular weight at body temperature; these PEI-conjugates complexed with hGH and extended hGH release in vitro. In pharmacokinetic studies of hGH behavior in SD rats, hydrogels of PEI-conjugate/hGH complexes could suppress the initial burst-phase, and extend the duration, of release, as well as increasing of area under the curve (AUC) compared to controls including hGH solution or non-ionic hydrogel. In a hypophysectomized rat model, the biological efficacy of hGH delivered from PEI-conjugate/hGH complex hydrogels was equivalent to that from daily administration over four days based on body weight gain and width of the tibial growth plate. These results suggest that ionic, thermosensitive, poly(organophosphazene)-PEI-conjugate hydrogel demonstrates potential as an injectable depot for sustained delivery of bioavailable hGH.
Keywords: Human growth hormone (hGH); Polyethylenimine (PEI); Thermosensitive hydrogel; Ionic interaction; Sustained delivery;

Enhanced anti-tumor efficacy by co-delivery of doxorubicin and paclitaxel with amphiphilic methoxy PEG-PLGA copolymer nanoparticles by Hai Wang; Ying Zhao; Yan Wu; Yu-lin Hu; Kaihui Nan; Guangjun Nie; Hao Chen (8281-8290).
The use of single chemotherapeutic drug has shown some limitations in anti-tumor treatment, such as development of drug resistance, high toxicity and limited regime of clinical uses. The combination of two or more therapeutic drugs is feasible means to overcome the limitations. Co-delivery strategy has been proposed to minimize the amount of each drug and to achieve the synergistic effect for cancer therapies. Attempts have been made to deliver chemotherapeutic drugs simultaneously using drug carriers, such as micelles, liposomes, and inorganic nanoparticles (NPs). Here we reported core-shell NPs that were doubly emulsified from an amphiphilic copolymer methoxy poly(ethylene glycol)-poly(lactide-co-glycolide) (mPEG-PLGA). These NPs offered advantages over other nanocarriers, as they were easy to fabricate by improved double emulsion method, biocompatible, and showed high loading efficacy. More importantly, these NPs could co-deliver hydrophilic doxorubicin (DOX) and hydrophobic paclitaxel (TAX). The drug-loaded NPs possessed a better polydispersity, indicating that they are more readily subject to controlled size distribution. Studies on drug release and cellular uptake of the co-delivery system demonstrated that both drugs were effectively taken up by the cells and released simultaneously. Furthermore, the co-delivery nanocarrier suppressed tumor cells growth more efficiently than the delivery of either DOX or TAX at the same concentrations, indicating a synergistic effect. Moreover, the NPs loading drugs with a DOX/TAX concentration ratio of 2:1 showed the highest anti-tumor activity to three different types of tumor cells. This nanocarrier might have important potential in clinical implications for co-delivery of multiple anti-tumor drugs with different properties.
Keywords: Nanotechnology; Co-delivery of drugs; Multifunctional nanoparticles; Double emulsion; Controlled drug release;

Intracellular dynamics of cationic and anionic polystyrene nanoparticles without direct interaction with mitotic spindle and chromosomes by Yuexian Liu; Wei Li; Fang Lao; Ying Liu; Liming Wang; Ru Bai; Yuliang Zhao; Chunying Chen (8291-8303).
The fate of nanomaterials with different sizes and charges in mitotic cells is of great importance but seldom explored. Herein we investigate the intracellular fate of negatively charged carboxylated polystyrene (COOH–PS) and positively charged amino-modified polystyrene (NH2–PS) nanoparticles of three different diameters (50, 100 and 500 nm) on cancer HeLa cells and normal NIH 3T3 cells during the cell cycles. The results showed that all the fluorescent PS nanoparticles differing in size and/or charge did not interact with chromosome reorganization and cytoskeleton assembly during the mitotic process in live cells. They neither disturbed chromosome reorganization nor affected the cytoskeleton reassembly in both normal and cancer cells. However, NH2–PS at the size of 50 nm caused G1 phase delay and a decrease of cyclin (D, E) expression, respectively. Moreover, NH2–PS displayed higher cellular toxicity and NH2–PS of 50 nm disturbed the integrity of cell membranes. Both cationic and anionic PS nanoparticles had a more pronounced effect on normal NIH 3T3 cells than cancer HeLa cell. Our research provides insight into the dynamic fate, intracellular behavior, and the effects of nanoparticles on spindle and chromosomes during cell division, which will enable the optimization of design and selection of much safer nanoparticles for lower risk to human health and widely medical applications.
Keywords: Polystyrene nanoparticles; Mitosis; Live cell imaging; Surface charge; Size effect;

A nanobiohybrid complex of recombinant baculovirus and Tat/DNA nanoparticles for delivery of Ang-1 transgene in myocardial infarction therapy by Arghya Paul; Zyad M. Binsalamah; Afshan A. Khan; Sana Abbasia; Cynthia B. Elias; Dominique Shum-Tim; Satya Prakash (8304-8318).
The study aims to design a new gene delivery method utilizing the complementary strengths of baculovirus, such as relatively high transduction efficiency and easy scale-up, and non-viral nanodelivery systems, such as low immunogenicity. This formulation was developed by generating a self assembled binary complex of negatively charged baculovirus (Bac) and positively charged endosomolytic histidine rich Tat peptide/DNA nanoparticles (NP). The synergistic effect of this hybrid (Bac-NP) system to induce myocardial angiogenesis in acute myocardial infarction (AMI) model has been explored in this study, using Angiopoietin-1 (Ang-1) as the transgene carried by both vector components. Under optimal transduction conditions, Bac-NPAng1 showed 1.75 times higher and sustained Ang-1 expression in cardiomyocytes than BacAng1, with significantly high angiogenic potential as confirmed by functional assays. For in vivo analysis, we intramyocardially delivered Bac-NPAng1 to AMI rat model. 3 weeks post AMI, data showed increase in capillary density (p < 0.01) and reduction in infarct sizes (p < 0.05) in Bac-NPAng1 compared to BacAng1, NPAng1 and control groups due to enhanced myocardial Ang-1 expression at peri-infarct regions (1.65 times higher than BacAng1). Furthermore, the Bac-NPAng1 group showed significantly higher cardiac performance in echocardiography than BacAng1 (44.2 ± 4.77% vs 37.46 ± 5.2%, p < 0.01), NPAng1 and the control group (32.26 ± 2.49% and 31.58 ± 2.26%). Collectively, this data demonstrates hybrid Bac-NP as a new and improved gene delivery system for therapeutic applications.
Keywords: Gene therapy; Baculovirus; Nanoparticle; Encapsulation; Microencapsulation; Myocardial angiogenesis;

Dual use of amphiphilic macromolecules as cholesterol efflux triggers and inhibitors of macrophage athero-inflammation by Nicole M. Iverson; Nicole M. Plourde; Sarah M. Sparks; Jinzhong Wang; Ekta N. Patel; Pratik S. Shah; Daniel R. Lewis; Kyle R. Zablocki; Gary B. Nackman; Kathryn E. Uhrich; Prabhas V. Moghe (8319-8327).
Activated vascular wall macrophages can rapidly internalize modified lipoproteins and escalate the growth of atherosclerotic plaques. This article proposes a biomaterials-based therapeutic intervention for depletion of non-regulated cholesterol accumulation and inhibition of inflammation of macrophages. Macromolecules with high scavenger receptor (SR)-binding activity were investigated for SR-mediated delivery of agonists to cholesterol-trafficking nuclear liver-X receptors. From a diverse feature space of a family of amphiphilic macromolecules of linear and aromatic mucic acid backbones modified with varied aliphatic chains and conjugated with differentially branched poly(ethylene glycol), a key molecule (carboxyl-terminated, C12-derivatized, linear mucic acid backbone) was selected for its ability to preferentially bind scavenger receptor A (SR-A) as the key target. At a basal level, this macromolecule suppressed the pro-inflammatory signaling of activated THP-1 macrophages while competitively lowering oxLDL uptake in vitro through scavenger receptor SRA-1 targeting. To further deplete intracellular cholesterol, the core macromolecule structure was exploited to solubilize a hydrophobic small molecule agonist for nuclear Liver-X Receptors, which regulate the efflux of intracellular cholesterol. The macromolecule-encapsulated agonist system was found to reduce oxLDL accumulation by 88% in vitro in comparison to controls. in vivo studies were designed to release the macromolecules (with or without encapsulated agonist) to injured carotid arteries within Sprague Dawley rats fed a high fat diet, conditions that yield enhanced cholesterol accumulation and macrophage recruitment. The macromolecules lowered intimal levels of accumulated cholesterol (50% for macromolecule alone; 70% for macromolecule-encapsulated agonist) and inhibited macrophage retention (92% for macromolecule; 96% for macromolecule-encapsulated agonist; 4 days) relative to non-treated controls. Thus, this study highlights the promise of designing bioactive macromolecule therapeutics based on scavenger receptor targeting, for potential management of vascular arterial disease.
Keywords: Macrophage; Inflammation; Atherosclerosis; Low density lipoproteins; Drug delivery; Copolymers;

Chitosan-graft-(PEI-β-cyclodextrin) copolymers and their supramolecular PEGylation for DNA and siRNA delivery by Yuan Ping; Chengde Liu; Zhongxing Zhang; Kerh Li Liu; Jianhai Chen; Jun Li (8328-8341).
Two water-soluble chitosan-graft-(polyethylenimine-β-cyclodextrin) (CPC) cationic copolymers were synthesized via reductive amination between oxidized chitosan (CTS) and low molecular weight polyethylenimine-modified β-cyclodextrin (β-CD-PEI). The two polycations, termed as CPC1 and CPC2, were characterized by proton nuclear magnetic resonance spectroscopy, gel permeation chromatography, and elemental analysis. These polycations exhibited good ability to condense both plasmid DNA (pDNA) and small interfering RNA (siRNA) into compact and spherical nanoparticles. Gene transfection activity of both polymers showed improved performance in comparison with native CTS in HEK293, L929, and COS7 cell lines. Further investigation of the gene transfection mediated by CPC2/DNA complexes showed both time-dependent and dose-dependent in the tested cell lines, where the polymer showed higher level luciferase expression than commercially available branched PEI (25 kDa) under the condition of high dose or extended time. Gene silencing activity mediated by CPC2/siRNA against luciferase expression showed superior knockdown effect in HEK293 and L929 cell lines. In addition, both polymers exhibited much lower cytotoxicity than PEI (25 kDa) in HEK293, L929, and COS7 cell lines. More interestingly, the pendent β-CD moieties of CPC copolymers allowed the supramolecular PEGylation though self-assembly of adamantyl-modified poly(ethylene glycol) with the β-CD moieties. The supramolecular PEGylation of the polyplexes significantly improved their stability under physiological conditions. The supramolecular PEGylated polyplexes of CPC with pDNA showed decreased transfection efficiency in all tested cell lines. However, remarkably, the supramolecular PEGylated polyplexes with siRNA exhibited even higher silencing efficiency in HEK293 and L929 cells (up to 84%), comparable to commercial DharmaFECT. The interesting mechanism for the enhanced silencing efficiency was discussed. With the pendent β-CD moieties on CTS chains, the system is expected to be further modified via inclusion complexation between β-CD unit and guest molecules to serve as a multifunctional delivery system.
Keywords: Chitosan; Cyclodextrin; Supramolecular self-assembly; PEGylation; Gene delivery; Polyplexes;