Biomaterials (v.29, #7)

Bioencapsulation of living bacteria (Escherichia coli) with poly(silicate) after transformation with silicatein-α gene by Werner E.G. Müller; Sylvia Engel; Xiaohong Wang; Stephan E. Wolf; Wolfgang Tremel; Narsinh L. Thakur; Anatoli Krasko; Mugdha Divekar; Heinz C. Schröder (771-779).
Bioencapsulation is an intriguing way to immobilize biological materials, including cells, in silica, metal-oxides or hybrid sol–gel polymers. Until now only the sol–gel precursor technology was utilized to immobilize bacteria or yeast cells in silica. With the discovery of silicatein, an enzyme from demosponges that catalyzes the formation of poly(silicate), it became possible to synthesize poly(silicate) under physiological (ambient) conditions. Here we show that Escherichia coli can be transformed with the silicatein gene, its expression level in the presence of isopropyl β-d-thiogalactopyranoside (IPTG) can be efficiently intensified by co-incubation with silicic acid. This effect could be demonstrated on the level of recombinant protein synthesis as well as by immunostaining analysis. The heterologously produced silicatein is enzymatically active, as confirmed by staining with Rhodamine 123 (formation for poly[silicate] from silicic acid) and by reacting free silicic acid with the β-silicomolybdato color system. Electron microscopic analysis revealed that the bacteria that express silicatein form a viscous cover around them when growing in the presence of silicic acid. Finally, we demonstrate that the growth kinetics of E. coli remains unaffected whether or not the bacteria had been transformed with silicatein or grown in medium, supplemented with silicic acid. It is concluded that silicatein-mediated encapsulation of bacteria with silica might improve, extend and optimize the range of application of bacteria for the production of recombinant protein.
Keywords: Bioencapsulation; Silicatein; Bacteria; Escherichia coli; Poly(silicate);

Hyaluronic acid containing hydrogels for the reduction of protein adsorption by Mark Van Beek; Lyndon Jones; Heather Sheardown (780-789).
Recently, new contact lens materials have been introduced which are reported to improve comfort by incorporating wetting agents either in a releasable or nonreleasable form. In the present work, model lens materials based on poly(2-hydroxyethyl methacrylate) (pHEMA) were developed which incorporate releasable or crosslinked and therefore physically entrapped hyaluronic acid (HA) of various molecular weights as a wetting agent.Crosslinked HA, despite being only present in very small amounts, resulted in consistently lower water contact angles over 4 h in comparison to controls, indicating that HA is present at the interface and was not being released over time. The presence of HA in the material was further confirmed by increases in the glass transition temperature measured by differential scanning calorimetry (DSC), and small increases in the stiffness as measured by Instron testing. This crosslinking procedure appeared to have no effect on optical transparency using 35 kDa HA, whereas small decreases in optical transparency at higher wavelengths were noted for the 169 kDa HA crosslinked material, as measured by UV spectrophotometry. Most importantly, protein adsorption results indicated that the adsorption of all proteins studied was considerably decreased by the presence of the small amount of crosslinked HA. The results provide insight into the mechanisms of comfort improvement with commercially available lens materials and suggest that HA containing materials may have significant potential for use in contact lens applications.
Keywords: Hyaluronic acid; Protein adsorption; Contact lens; Comfort; Hydrogel;

Alendronate–hydroxyapatite nanocomposites and their interaction with osteoclasts and osteoblast-like cells by Elisa Boanini; Paola Torricelli; Massimo Gazzano; Roberto Giardino; Adriana Bigi (790-796).
The direct synthesis of hydroxyapatite in the presence of bisphosphonates is quite difficult due to the great affinity for calcium of these compounds, which are widely used in the treatment of pathologies related to bone loss. We recently developed a new method which allowed to synthesize alendronate–hydroxyapatite composite nanocrystals with a bisphosphonate content up to about 7 wt%.Herein we report the results of an in vitro study aimed to investigate the effects of alendronate incorporation into hydroxyapatite on bone cells response.Osteoblast-like MG63 cells and human osteoclasts were cultured on nanocrystals at different alendronate content (3.9, 6.2, 7.1 wt%). MG63 cells cultured on the composite nanocrystals display normal morphology, good proliferation and increased values of the differentiation parameters. In particular, when cultured on composites at relatively high alendronate contents, osteoblasts display increased values of alkaline phosphatase activity (ALP), collagen type I, and osteocalcin production, as well as significant decrease of matrix metalloproteinases (MMP-1 and MMP-13) production, with respect both to the control and to pure hydroxyapatite nanocrystals. It follows that the presence of alendronate enhances osteoblast activation and extracellular matrix mineralization processes, without any abnormal collagen degradation. The osteoclast number on the composite nanocrystals decrease indicating that the bisphosphonate exerts its inhibitory effect on osteoclast proliferation even when incorporated into hydroxyapatite.
Keywords: Hydroxyapatite; Bisphosphonate; Osteoblast; Osteoclast;

The recruitment of primitive Lin Sca-1+, CD34+, c-kit+ and CD271+ cells during the early intraperitoneal foreign body reaction by Ilse Vranken; Geofrey De Visscher; An Lebacq; Erik Verbeken; Willem Flameng (797-808).
Implanted materials, such as medical devices, provoke the body to initiate an inflammatory reaction, known as the foreign body reaction (FBR), which causes several complications for example in hip prostheses, silicone implants, peritoneal dialysis catheters and left ventricular assist devices. FBR is initiated by macrophage adherence and results in granulation tissue formation. The early immunobiology and development of this tissue is not completely understood, but there are indications from related myofibroblast-forming diseases such as vascular repair and fibrosis that primitive stem cells also play a role in the formation of FBR-tissue. To investigate this, acellular photo-oxidized bovine pericardium patches were implanted intraperitoneally in rats and retrieved at time-points ranging from 6 h to 7 days. A significant fraction of Sca-1+ (6 h–2 days), c-kit+, CD34+ and CD271+ (2–3 days) stem/progenitor cells were detected. Colony-forming and differentiation capacity of the primitive stem cells into adipo-, osteo-, and myofibroblasts were shown. The presence of these primitive cells and their myofibroblastic differentiation potential were also confirmed at RNA level. The identification of specific primitive cells during FBR may have important implications for the inflammatory responses to inert materials and their use in tissue prostheses.
Keywords: Foreign body response; Inflammation; Cell adhesion; Primitive cell;

In vitro differentiation of human mesenchymal stem cells on Ti6Al4V surfaces by Isabella Tognarini; Sabina Sorace; Roberto Zonefrati; Gianna Galli; Alessia Gozzini; Silvia Carbonell Sala; Giorgia D. Zappoli Thyrion; Anna Maria Carossino; Annalisa Tanini; Carmelo Mavilia; Chiara Azzari; Fausto Sbaiz; Alessandro Facchini; Rodolfo Capanna; Maria Luisa Brandi (809-824).
Long-term stability of arthroplasty prosthesis depends on the integration between the bone tissue and the implanted biomaterials, which requires the contribution of osteoblastic precursors and their continuous differentiation into the osteoblastic phenotype. Classically, these interactions are tested in vitro using mesenchymal stem cells (MSCs) isolated and ex vivo expanded from bone marrow aspirates. Human adipose tissue-derived stromal cells (AMSCs) may be a more convenient source of MSCs, according to their abundance and accessibility, but no data are available on their in vitro interactions with hard biomaterials. The aim of this work is to compare the osteogenic potential of human AMSCs and bone marrow-derived MSCs (BMMSCs) and to evaluate their response to Ti6Al4V alloy in terms of adhesion, proliferation and differentiation features, using the human osteosarcoma cell line SaOS-2 for comparison. The overall results showed that AMSCs have the same ability to produce bone matrix as BMMSCs and that Ti6Al4V surfaces exhibit an osteoinductive action on AMSCs, promoting their differentiation into functional osteoblasts and increasing bone formation. In conclusion, adipose tissue is a promising autologous source of osteoblastic cells with important clinical implications for bone tissue engineering.
Keywords: Titanium alloy; Mesenchymal stem cells; Bone marrow; Adipose tissue; Osteoblast; Osteogenic differentiation;

Development of a tissue-engineered vascular graft combining a biodegradable scaffold, muscle-derived stem cells and a rotational vacuum seeding technique by Alejandro Nieponice; Lorenzo Soletti; Jianjun Guan; Bridget M. Deasy; Johnny Huard; William R. Wagner; David A. Vorp (825-833).
There is a clinical need for a tissue-engineered vascular graft (TEVG), and combining stem cells with biodegradable tubular scaffolds appears to be a promising approach. The goal of this study was to characterize the incorporation of muscle-derived stem cells (MDSCs) within tubular poly(ester urethane) urea (PEUU) scaffolds in vitro to understand their interaction, and to evaluate the mechanical properties of the constructs for vascular applications. Porous PEUU scaffolds were seeded with MDSCs using our recently described rotational vacuum seeding device, and cultured inside a spinner flask for 3 or 7 days. Cell viability, number, distribution and phenotype were assessed along with the suture retention strength and uniaxial mechanical behavior of the TEVGs. The seeding device allowed rapid even distribution of cells within the scaffolds. After 3 days, the constructs appeared completely populated with cells that were spread within the polymer. Cells underwent a population doubling of 2.1-fold, with a population doubling time of 35 h. Stem cell antigen-1 (Sca-1) expression by the cells remained high after 7 days in culture (77±20% vs. 66±6% at day 0) while CD34 expression was reduced (19±12% vs. 61±10% at day 0) and myosin heavy chain expression was scarce (not quantified). The estimated burst strength of the TEVG constructs was 2127±900 mmHg and suture retention strength was 1.3±0.3 N. We conclude from this study that MDSCs can be rapidly seeded within porous biodegradable tubular scaffolds while maintaining cell viability and high proliferation rates and without losing stem cell phenotype for up to 7 days of in-vitro culture. The successful integration of these steps is thought necessary to provide rapid availability of TEVGs, which is essential for clinical translation.
Keywords: Vascular tissue engineering; Muscle-derived stem cells; Scaffolds; Vascular grafts; Cell seeding; Dynamic culture;

Influence of electrospun collagen on wound contraction of engineered skin substitutes by Heather M. Powell; Dorothy M. Supp; Steven T. Boyce (834-843).
The treatment of massive full-thickness burns with engineered skin substitutes has shown promise in clinical trials. The majority of skin substitutes are comprised of fibroblasts and/or keratinocytes on collagen scaffolds, commonly generated by freeze drying which can generate significant structural heterogeneity. Electrospinning may generate collagen scaffolds with greater homogeneity. Skin substitutes were fabricated using either freeze-dried (FD) or electrospun (ES) collagen scaffolds. Cell distribution, proliferation, organization, and maturation were assessed on each scaffold type in vitro, and engraftment and healing of full thickness wounds in athymic mice were tested. In vitro evaluation of freeze-dried collagen skin substitutes (FCSS) and electrospun collagen skin substitutes (ECSS) revealed no significant differences in cell proliferation, surface hydration, or cellular organization between the ECSS and FCSS groups. Both groups exhibited excellent stratification with a continuous layer of basal keratinocytes present at the dermal–epidermal junction. After grafting to full thickness wounds in athymic mice, both skin substitutes had high rates of engraftment: 87.5% in the FCSS group and 100% in the ECSS group. Histological evaluation of wounds revealed that bovine collagen persisted in the wound at week 8 in the FCSS group while no bovine collagen was seen in the ECSS group. At 8 weeks post-grafting, the ECSS grafts were 61.3±7.9% original graft area whereas the FCSS grafts were 39.2±8.8% original area (p<0.01). These results indicate that ES scaffolds can be used to fabricate skin substitutes with optimal cellular organization and can potentially reduce wound contraction compared to FD scaffolds. These advantages may lead to reduced morbidity in patients treated with skin substitutes fabricated from ES collagen.
Keywords: Wound healing; Collagen; Scaffold; Wound closure;

The effect of cyclic strain on embryonic stem cell-derived cardiomyocytes by So-Jung Gwak; Suk Ho Bhang; Il-Kwon Kim; Sang-Soo Kim; Seung-Woo Cho; Oju Jeon; Kyung Jong Yoo; Andrew J. Putnam; Byung-Soo Kim (844-856).
Cardiomyocytes in the body are subjected to cyclic mechanical strain induced by the rhythmic heart beating. In this study, we tested the hypothesis that cyclic strain promotes cardiomyogenesis of embryonic stem cell-derived cardiomyocytes (ESCs). ESCs cultured on elastic polymer [poly(lactide-co-caprolactone), PLCL] scaffolds subjected to cyclic strain in vitro displayed elevated cardiac gene expression compared to unstrained controls. Six weeks after implantation into infarcted rat myocardium, the elastic cardiac patches (ESC-seeded PLCL scaffolds) showed reduced fibrotic tissue formation, likely due to a combination of lower apoptotic activity, higher vascular endothelial growth factor (VEGF) expression, and more extensive angiogenesis in the strained versus unstrained control [ESC-seeded, non-elastic poly(lactide-co-glycolide) scaffolds] patches. Importantly, cardiac gene expression was upregulated in the elastic patches compared to control, with evidence for cardiomyocyte-specific microstructures including myofibrillar bundles and Z-lines. This study shows that the use of an elastic polymer scaffold designed to permit mechanical strain transduction as a cell transplantation vehicle significantly increases cardiomyogenesis of the implanted ESCs.
Keywords: Cardiac patch; Cardiomyogenesis; Cyclic strain; Embryonic stem cell-derived cardiomyocytes;

Towards development of a dermal rudiment for enhanced wound healing response by Yolanda Garcia; Brendan Wilkins; Russell J. Collighan; Martin Griffin; Abhay Pandit (857-868).
Enhancement of collagen's physical characteristics has been traditionally approached using various physico-chemical methods frequently compromising cell viability. Microbial transglutaminase (mTGase), a transamidating enzyme obtained from Streptomyces mobaraensis, was used in the cross-linking of collagen-based scaffolds. The introduction of these covalent bonds has previously indicated increased proteolytic and mechanical stability and the promotion of cell colonisation. The hypothesis behind this research is that an enzymatically stabilised collagen scaffold will provide a dermal precursor with enhanced wound healing properties. Freeze-dried scaffolds, with and without the loading of a site-directed mammalian transglutaminase inhibitor to modulate matrix deposition, were applied to full thickness wounds surgically performed on rats’ dorsum and explanted at three different time points (3, 7 and 21 days). Wound healing parameters such as wound closure, epithelialisation, angiogenesis, inflammatory and fibroblastic cellular infiltration and scarring were analysed and quantified using stereological methods. The introduction of this enzymatic cross-linking agent stimulated neovascularisation and epithelialisation resisting wound contraction. Hence, these characteristics make this scaffold a potential candidate to be considered as a dermal precursor.
Keywords: Cross-linking collagen; Microbial transglutaminase; Wound healing; Angiogenesis; Stereology;

Alginate-based microencapsulation of retinal pigment epithelial cell line for cell therapy by Jonna Wikström; Matti Elomaa; Heli Syväjärvi; Johanna Kuokkanen; Marjo Yliperttula; Paavo Honkakoski; Arto Urtti (869-876).
The goals of this study were to evaluate human retinal pigment epithelial cell line (ARPE-19) for cell encapsulation and to optimize the alginate-based microencapsulation. We used immortalized ARPE-19 cells and the transfected sub-line that expresses secreted alkaline phosphatase (SEAP) reporter enzyme. Alginate was cross-linked with different divalent cations (Ca2+, Ba2+, Sr2+ and combination of Ca2+ and Ba2+), coated first with poly-l-lysine (PLL), and then with alginate. Microcapsules with different pore sizes and stability were generated. The pore size of the microcapsules was assessed by the release of encapsulated fluorescein isothiocyanate (FITC)-dextrans. The viability of the cells in the microcapsules was studied in vitro by assessing the secretion rates of SEAP and oxygen consumption by the cells. The best microcapsule morphology, durability and cellular viability were obtained with alginate microcapsules that were cross-linked with Ca2+ and Ba2+ ions and then coated with PLL and alginate. Based on FITC-dextran release these microcapsules have porous wall that enables the rapid contents release. The ARPE-19 cells maintained viability in the Ca2+ and Ba2+ cross-linked microcapsules for at least 110 days. The alginate microcapsules cross-linked with Ca2+ and Ba2+ have sufficiently large pore size for prolonged cell viability and for the release of secreted SEAP model protein (Mw 50 kDa; radius of gyration of 3 nm). ARPE-19 cells show long-term viability and protein secretion within alginate microcapsules cross-linked with Ca2+ and Ba2+. This combination may be useful in cell therapy.
Keywords: Cell encapsulation; Alginate; Protein release; Retinal pigment epithelial cells; ARPE-19;

Enhancement of bone-bonding ability of bioactive titanium by prostaglandin E2 receptor selective agonist by Eijiro Onishi; Shunsuke Fujibayashi; Mitsuru Takemoto; Masashi Neo; Takayuki Maruyama; Tadashi Kokubo; Takashi Nakamura (877-883).
Systemic administration of prostaglandin E2 receptor (EP4) selective agonist increases both bone formation and resorption, and consequently leads to an increase in bone mass. Although previous studies have reported that EP4 agonist enhanced bone remodeling and fracture healing, it was not known if EP4 agonist activates the bone–biomaterial interface. Bioactive titanium prepared by chemical and thermal treatment can bond to living bone and is suitable for use in clinical applications in cementless fixation devices. Therefore, we examined whether the administration of EP4 agonist enhances the bonding strength between bone and bioactive titanium. Bioactive titanium plates were inserted into the tibia bone of rabbits and examined histologically and biomechanically at 4, 8, and 16 weeks. EP4 agonist was administrated systemically every 2 weeks after surgery. A non-administrated control group, a low-dose group (10 μg/kg body weight (BW)), and a high-dose group (100 μg/kg BW) were compared. The bonding strength of bioactive titanium in the EP4 agonist groups was significantly higher than that in the control group at both 4 and 8 weeks, and enhanced bone remodeling and direct bonding around the bioactive titanium plates was observed only in the EP4 agonist groups at 4 weeks. EP4 agonist enhanced bone formation around the bioactive titanium plate, and achieved early direct bone bonding.
Keywords: Prostaglandin; Titanium; Biomaterial; Animal experiment;

Localized drug delivery from drug-eluting stents has been accepted as one of the most promising treatment methods for preventing restenosis after stenting. However, thrombosis, inflammation, and restenosis are still major problems for the utility of cardiovascular prostheses such as vascular grafts and stents. Epigallocatechin-3-O-gallate (EGCG), a major polyphenolic constituent of green tea, has been shown to have anti-thrombotic, anti-inflammatory and anti-proliferative activities. It was hypothesized that controlled release of EGCG from biodegradable poly(lactide-co-ε-caprolactone, PLCL) stent coatings would suppress migration and invasion of vascular smooth muscle cells (VSMCs) as well as platelet-mediated thrombosis. EGCG-releasing PLCL (E-PLCL) was prepared by blending PLCL with 5% EGCG. The surface morphology, roughness and melting temperature of PLCL were not changed despite EGCG addition. EGCG did, however, EGCG appreciably increase the hydrophilicity of PLCL. EGCG was found to be uniformly dispersed throughout E-PLCL without direct chemical interactions with PLCL. E-PLCL displayed diffusion controlled release of EGCG release for periods up to 34 days. E-PLCL significantly suppressed the migration and invasion of VSMCs as well as the adhesion and activation of platelets. E-PLCL coatings were able to smooth the surface of bare stents with neither cracks nor webbings after balloon expansion. The structural integrity of coatings was sufficient to resist delamination or destruction during 90% dilatation. These results suggest that EGCG-releasing polymers can be effectively applied for fabricating an EGCG-eluting vascular stent to prevent in-stent restenosis and thrombosis.
Keywords: Stent; Restenosis; Polylactic acid; Polycaprolactone; Drug release; Smooth muscle cells;

Controlled release from multilayer silk biomaterial coatings to modulate vascular cell responses by Xianyan Wang; Xiaohui Zhang; John Castellot; Ira Herman; Mark Iafrati; David L. Kaplan (894-903).
A multilayered silk fibroin protein coating system was employed as a drug carrier and delivery system to evaluate vascular cell responses to heparin, paclitaxel, and clopidogrel. The results demonstrated that the silk coating system was an effective system for drug-eluting coatings, such as for stent applications, based on its useful micromechanical properties and biological outcomes. Cell attachment and viability studies with human aortic endothelial cells (HAECs) and human coronary artery smooth muscle cells (HCASMCs) on the drug-incorporated silk coatings demonstrated that paclitaxel and clopidogrel inhibited smooth muscle cell (SMC) proliferation and retarded endothelial cell proliferation. Heparin-loaded silk multilayers promoted HAEC proliferation while inhibiting HCASMC proliferation, desired outcomes for the prevention of restenosis. The preservation of the phenotype of endothelial cells on silk and heparin-loaded silk coatings was confirmed with the presence of endothelial markers CD-31, CD-146, vWF and VE-Cadherin using immunocytochemistry assays. A preliminary in-vivo study in a porcine aorta showed integrity of the silk coatings after implantation and the reduction of platelet adhesion on the heparin-loaded silk coatings.
Keywords: Silk; Fibroin; Stent; Heparin; Paclitaxel; Clopidogrel;

In situ IGF-1 gene delivery to cells emerging from the injured anterior cruciate ligament by Andre F. Steinert; Meike Weber; Manuela Kunz; Glyn D. Palmer; Ulrich Nöth; Christopher H. Evans; Martha M. Murray (904-916).
Ruptures of the anterior cruciate ligament (ACL) are common knee injuries that do not heal, even with surgical repair. Our research is directed towards developing novel, biological approaches that enable suture repair of this ligament. One promising strategy involves the insertion of a collagen hydrogel between the severed ends of the ACL. Cells migrate from the damaged ligament into the hydrogel and produce repair tissue. Here we have investigated the potential for augmenting this process by the transfer of insulin like growth factor (IGF) 1 cDNA to the repair cells using an adenovirus vector. The goal is to achieve direct, in situ gene delivery by loading the hydrogel with vector prior to its insertion into the defect. In a step-wise approach towards evaluating this process, we confirmed that monolayers of ACL fibroblasts were efficiently transduced by adenovirus vectors and continued to express transgenes when subsequently incorporated into the hydrogel; indeed, transgene expression persisted longer within collagen gels than in monolayer culture. Transfer of IGF-1 cDNA increased the cellularity of the gels and led to the synthesis and deposition of increased amounts of types I and III collagen, elastin, tenascin, and vimentin. The cells remained viable, even when subjected to high viral loads. Similar results were obtained when collagen hydrogels were preloaded with adenovirus prior to insertion into an experimental ACL lesion in vitro. These data confirm the promise of using vector-laden hydrogels for the in situ delivery of genes to cells within damaged ligaments and suggest novel possibilities for the biological repair of the ACL.
Keywords: Anterior cruciate ligament; Collagen hydrogel; Gene transfer; IGF-1; Adenovirus;

The effect of covalent cross-links between the membrane components of microcapsules on the dissemination of encapsulated malignant cells by Julie Dusseault; Geneviève Langlois; Marie-Christine Meunier; Martin Ménard; Claude Perreault; Jean-Pierre Hallé (917-924).
Stem cells and immortalized cells have considerable therapeutic potential but present risks of malignant transformation. Cell microencapsulation allows transplantation without immunosuppression. We have developed a method for microencapsulating living cells within covalently cross-linked membranes that are chemically and mechanically extremely resistant. We provide herein direct evidence that these microcapsules can prevent malignant cell dissemination. When 20,000 or more nonencapsulated EL-4 thymoma cells were implanted intraperitoneally in mice, all recipients died with widespread metastasis within 26.3±1.0 days. All recipients of 250,000 EL-4 cells microencapsulated in covalently cross-linked membranes were living and disease-free, 150 days post-implantation. Encapsulation in standard microcapsules only slightly delayed the recipient death. Pancreatic islets transplanted using either type of microcapsule presented similar survival. We conclude that microencapsulation in covalently cross-linked membranes prevents malignant cell dissemination.
Keywords: Stem cells; Immortalized cells; Malignant transformation; Encapsulation; Islet transplantation;

Label of human bone mesenchymal stem cells with CdSe/ZnS quantum dots (QDs) had been demonstrated to impair cell functions and activities. In the present study, QDs delivered by two different routes, Pep-1-labeled QDs (LQ) and PolyFect transfected QDs (TQ), were utilized to assess the effects of delivery mechanisms on various cellular responses of the QDs-internalized human adipose-derived adult stem cells (hADAS). Examination of labeled cells by flow cytometry and laser scanning confocal microscopy showed that LQ had higher fluorescence intensity due to the cluster formation and their distribution in cytoplasma while TQ were preferentially accumulated at peri-nuclear regions. The fluorescence intensity of the LQ group was still higher than that of the TQ group at 28 days after labeling, though cellular LQ were partitioned after initial cell division. Pep-1 but not PolyFect delivery facilitated QDs to escape from lysosome degradation. Pep-1 delivery of QDs rescued the cells from the negative effects caused by the internalized QDs on cell proliferation and on the expressions of CD29 and CD105 as well as osteogenic and chondrogenic-associated lineage markers. The same effect was also observed in the expression of alkaline phosphatase activity, calcium deposition and secretion of chondrogenic matrices (GAG and collagen type II) in micromass culture. These indicated that Pep-1-delivered QDs may serve appropriately to track the hADAS employed in cell therapy/tissue engineering applications. The results also suggested that the endo-/lysosome degradation of QDs may depend on different surface coatings and critically influence the differentiation of hADAS.
Keywords: Quantum dots; Pep-1; Human adult adipose-derived stem cells (hADAS); Lysosome; CD markers; Differentiation;

Upconversion fluorescence imaging of cells and small animals using lanthanide doped nanocrystals by Dev K. Chatterjee; Abdul J. Rufaihah; Yong Zhang (937-943).
Upconversion fluorescence imaging technique with excitation in the near-infrared (NIR) region has been used for imaging of biological cells and tissues. This has several advantages, including absence of photo-damage to living organisms, very low auto-fluorescence, high detection sensitivity, and high light penetration depth in biological tissues. In this report we demonstrate the use of a new upconversion fluorophore, lanthanide doped nanocrystals, for imaging of cells and some deep tissues in animal. Polyethyleneimine (PEI) coated NaYF4:Yb,Er nanoparticles were synthesized, which produce very strong upconversion fluorescence when excited at 980 nm by a NIR laser. The nanoparticles were shown to be stable in physiologic buffered saline (PBS), non-toxic to bone marrow stem cells, and resistant to photo-bleaching. The nanoparticles delivered into some cell lines or injected intradermally and intramuscularly into some tissues either near the body surface or deep in the body of rats showed visible fluorescence, when exposed to a 980 nm NIR laser. To the best of our knowledge, this represents the first demonstration of use of upconversion fluorophores for cellular and tissue imaging.
Keywords: Nanoparticle; Fluorescence; Surface modification;

Osteoconduction and osteoinduction of low-temperature 3D printed bioceramic implants by Pamela Habibovic; Uwe Gbureck; Charles J. Doillon; David C. Bassett; Clemens A. van Blitterswijk; Jake E. Barralet (944-953).
Rapid prototyping is a valuable implant production tool that enables the investigation of individual geometric parameters, such as shape, porosity, pore size and permeability, on the biological performance of synthetic bone graft substitutes. In the present study, we have employed low-temperature direct 3D printing to produce brushite and monetite implants with different geometries. Blocks predominantly consisting of brushite with channels either open or closed to the exterior were implanted on the decorticated lumbar transverse processes of goats for 12 weeks. In addition, similar blocks with closed channel geometry, consisting of either brushite or monetite were implanted intramuscularly. The design of the channels allowed investigation of the effect of macropore geometry (open and closed pores) and osteoinduction on bone formation orthotopically. Intramuscular implantation resulted in bone formation within the channels of both monetite and brushite, indicating osteoinductivity of these resorbable materials. Inside the blocks mounted on the transverse processes, initial channel shape did not seem to significantly influence the final amount of formed bone and osteoinduction was suggested to contribute to bone formation.
Keywords: Calcium phosphate cement; Three dimensional printing; Osteoconduction; Osteoinduction; In vivo test;