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Biomaterials (v.33, #17)

Editorial board (pp. ifc).
Editorial board (pp. ifc).

Reconstitution of laminin-111 biological activity using multiple peptide coupled to chitosan scaffolds by Kentaro Hozumi; Ayano Sasaki; Yuji Yamada; Dai Otagiri; Kazuki Kobayashi; Chikara Fujimori; Fumihiko Katagiri; Yamato Kikkawa; Motoyoshi Nomizu (pp. 4241-4250).
Laminin-111, a multifunctional matrix protein, has diverse biological functions. Previously, we have identified various biologically active sequences in laminin-111 by a systematic peptide screening. We also demonstrated that peptide-conjugated chitosan matrices enhance the biological functions of the active sequences and are useful as a scaffold. Here, we conjugated sixty biologically active laminin-111 peptides onto chitosan matrices. The twenty-nine peptide-chitosan matrices promoted various biological activities, including cell attachment, spreading, and neurite outgrowth. The biological activities of peptide-chitosan matrices depend on the peptide. These peptide-chitosan matrices are categorized into six groups depending on their biological activities. Next, we conjugated five active peptides, which showed strong cell attachment activity in the each group, onto a single chitosan matrix to mimic the multiple activities of laminin-111. The mixed peptides-chitosan matrix significantly promoted cell attachment and cell spreading over that observed with the individual peptides. We also demonstrated that a mixed peptides-chitosan matrix, using four neurite outgrowth-promoting peptides each from a different group, enhanced the activity. These data suggest that the mixed peptides synergistically induce laminin-like biological activities on a chitosan matrix. The active peptides-chitosan matrices described here have potential for use as biomaterial for tissue engineering and regeneration.

Keywords: Cell adhesion; Chitin/chitosan; ECM (extracellular matrix); Integrin; Peptide; ScaffoldAbbreviations; ECM; Extracellular matrix; BME; Basement membrane extract; EHS; Engelbreth-Holm-Swarm; Fmoc; 9-Fluorenylmethoxycarbonyl; MBS; N; -(; m; -maleimidobenzoyloxy) succinimide; DMF; N,N'; -dimethylformamide; FBS; Fetal bovine serum; BSA; Bovine serum albumin; HDF; Human dermal fibroblast; NGF; Nerve growth factor; DAPI; 4,6-Diamidino-2-phenylindole; FAK; Focal adhesion kinase; ESC; Embryonic stem cell


Reconstitution of laminin-111 biological activity using multiple peptide coupled to chitosan scaffolds by Kentaro Hozumi; Ayano Sasaki; Yuji Yamada; Dai Otagiri; Kazuki Kobayashi; Chikara Fujimori; Fumihiko Katagiri; Yamato Kikkawa; Motoyoshi Nomizu (pp. 4241-4250).
Laminin-111, a multifunctional matrix protein, has diverse biological functions. Previously, we have identified various biologically active sequences in laminin-111 by a systematic peptide screening. We also demonstrated that peptide-conjugated chitosan matrices enhance the biological functions of the active sequences and are useful as a scaffold. Here, we conjugated sixty biologically active laminin-111 peptides onto chitosan matrices. The twenty-nine peptide-chitosan matrices promoted various biological activities, including cell attachment, spreading, and neurite outgrowth. The biological activities of peptide-chitosan matrices depend on the peptide. These peptide-chitosan matrices are categorized into six groups depending on their biological activities. Next, we conjugated five active peptides, which showed strong cell attachment activity in the each group, onto a single chitosan matrix to mimic the multiple activities of laminin-111. The mixed peptides-chitosan matrix significantly promoted cell attachment and cell spreading over that observed with the individual peptides. We also demonstrated that a mixed peptides-chitosan matrix, using four neurite outgrowth-promoting peptides each from a different group, enhanced the activity. These data suggest that the mixed peptides synergistically induce laminin-like biological activities on a chitosan matrix. The active peptides-chitosan matrices described here have potential for use as biomaterial for tissue engineering and regeneration.

Keywords: Cell adhesion; Chitin/chitosan; ECM (extracellular matrix); Integrin; Peptide; ScaffoldAbbreviations; ECM; Extracellular matrix; BME; Basement membrane extract; EHS; Engelbreth-Holm-Swarm; Fmoc; 9-Fluorenylmethoxycarbonyl; MBS; N; -(; m; -maleimidobenzoyloxy) succinimide; DMF; N,N'; -dimethylformamide; FBS; Fetal bovine serum; BSA; Bovine serum albumin; HDF; Human dermal fibroblast; NGF; Nerve growth factor; DAPI; 4,6-Diamidino-2-phenylindole; FAK; Focal adhesion kinase; ESC; Embryonic stem cell


The effect of TNFα secreted from macrophages activated by titanium particles on osteogenic activity regulated by WNT/BMP signaling in osteoprogenitor cells by Sang-Soo Lee; Ashish R. Sharma; Byung-Soo Choi; Jun-Sub Jung; Jun-Dong Chang; Seonghun Park; Eduardo A. Salvati; Edward P. Purdue; Dong-Keun Song; Ju-Suk Nam (pp. 4251-4263).
Wear particles are the major cause of osteolysis associated with failure of implant following total joint replacement. During this pathologic process, activated macrophages mediate inflammatory responses to increase osteoclastogenesis, leading to enhanced bone resorption. In osteolysis caused by wear particles, osteoprogenitors present along with macrophages at the implant interface may play significant roles in bone regeneration and implant osteointegration. Although the direct effects of wear particles on osteoblasts have been addressed recently, the role of activated macrophages in regulation of osteogenic activity of osteoblasts has scarcely been studied. In the present study, we examined the molecular communication between macrophages and osteoprogenitor cells that may explain the effect of wear particles on impaired bone forming activity in inflammatory bone diseases. It has been demonstrated that conditioned medium of macrophages challenged with titanium particles (Ti CM) suppresses early and late differentiation markers of osteoprogenitors, including alkaline phosphatase (ALP) activity, collagen synthesis, matrix mineralization and expression of osteocalcin and Runx2. Moreover, bone forming signals such as WNT and BMP signaling pathways were inhibited by Ti CM. Interestingly, TNFα was identified as a predominant factor in Ti CM to suppress osteogenic activity as well as WNT and BMP signaling activity. Furthermore, Ti CM or TNFα induces the expression of sclerostin (SOST) which is able to inhibit WNT and BMP signaling pathways. It was determined that over-expression of SOST suppressed ALP activity, whereas the inhibition of SOST by siRNA partially restored the effect of Ti CM on ALP activity. This study highlights the role of activated macrophages in regulation of impaired osteogenic activity seen in inflammatory conditions and provides a potential mechanism for autocrine regulation of WNT and BMP signaling mediated by TNFα via induction of SOST in osteprogenitor cells.

Keywords: Particle; TNFα; Osteolysis; WNT; BMP; SOST


The effect of TNFα secreted from macrophages activated by titanium particles on osteogenic activity regulated by WNT/BMP signaling in osteoprogenitor cells by Sang-Soo Lee; Ashish R. Sharma; Byung-Soo Choi; Jun-Sub Jung; Jun-Dong Chang; Seonghun Park; Eduardo A. Salvati; Edward P. Purdue; Dong-Keun Song; Ju-Suk Nam (pp. 4251-4263).
Wear particles are the major cause of osteolysis associated with failure of implant following total joint replacement. During this pathologic process, activated macrophages mediate inflammatory responses to increase osteoclastogenesis, leading to enhanced bone resorption. In osteolysis caused by wear particles, osteoprogenitors present along with macrophages at the implant interface may play significant roles in bone regeneration and implant osteointegration. Although the direct effects of wear particles on osteoblasts have been addressed recently, the role of activated macrophages in regulation of osteogenic activity of osteoblasts has scarcely been studied. In the present study, we examined the molecular communication between macrophages and osteoprogenitor cells that may explain the effect of wear particles on impaired bone forming activity in inflammatory bone diseases. It has been demonstrated that conditioned medium of macrophages challenged with titanium particles (Ti CM) suppresses early and late differentiation markers of osteoprogenitors, including alkaline phosphatase (ALP) activity, collagen synthesis, matrix mineralization and expression of osteocalcin and Runx2. Moreover, bone forming signals such as WNT and BMP signaling pathways were inhibited by Ti CM. Interestingly, TNFα was identified as a predominant factor in Ti CM to suppress osteogenic activity as well as WNT and BMP signaling activity. Furthermore, Ti CM or TNFα induces the expression of sclerostin (SOST) which is able to inhibit WNT and BMP signaling pathways. It was determined that over-expression of SOST suppressed ALP activity, whereas the inhibition of SOST by siRNA partially restored the effect of Ti CM on ALP activity. This study highlights the role of activated macrophages in regulation of impaired osteogenic activity seen in inflammatory conditions and provides a potential mechanism for autocrine regulation of WNT and BMP signaling mediated by TNFα via induction of SOST in osteprogenitor cells.

Keywords: Particle; TNFα; Osteolysis; WNT; BMP; SOST


The fundamental role of subcellular topography in peripheral nerve repair therapies by Eric C. Spivey; Zin Z. Khaing; Jason B. Shear; Christine E. Schmidt (pp. 4264-4276).
Clinical evidence suggests that nano- and microtopography incorporated into scaffolds does not merely improve peripheral nerve regeneration, but is in fact a prerequisite for meaningful restoration of nerve function. Although the biological mechanisms involved are not fully understood, grafts incorporating physical guides that mimic microscopic nerve tissue features (e.g., basal laminae) appear to provide a significant advantage over grafts that rely on purely chemical or macroscopic similarities to nerve tissue. Investigators consistently demonstrate the fundamental importance of nano- and micro-scale physical features for appropriate cell response in a wide range of biological scenarios. Additionally, recent in vivo research demonstrates that nerve regeneration scaffolds with cell-scale physical features are more effective than those that rely only on chemical or macro-scale features. Physical guidance at the cell-scale is especially important for long (>20 mm) nerve defects, for which the only reliable treatment is the autologous nerve graft. The lack of other available options exposes a clear need for the application of nano- and microfabrication techniques that will allow the next generation of engineered nerve guides to more closely mimic native tissue at those scales. This review examines current research to determine what elements of cell-scale topography in experimental scaffolds are most effective at stimulating functional recovery, and then presents an overview of fabrication techniques that could potentially improve future treatment paradigms. Relative advantages and disadvantages of these techniques are discussed, with respect to both clinical adaptation and likely effectiveness. Our intent is to more clearly delineate the remaining obstacles in the development of a next generation nerve guide, particularly for long defects, and offer new perspectives on steering current technologies towards clinically viable solutions.

Keywords: Nerve guide; Microstructure; Scaffold; Nanotopography


The fundamental role of subcellular topography in peripheral nerve repair therapies by Eric C. Spivey; Zin Z. Khaing; Jason B. Shear; Christine E. Schmidt (pp. 4264-4276).
Clinical evidence suggests that nano- and microtopography incorporated into scaffolds does not merely improve peripheral nerve regeneration, but is in fact a prerequisite for meaningful restoration of nerve function. Although the biological mechanisms involved are not fully understood, grafts incorporating physical guides that mimic microscopic nerve tissue features (e.g., basal laminae) appear to provide a significant advantage over grafts that rely on purely chemical or macroscopic similarities to nerve tissue. Investigators consistently demonstrate the fundamental importance of nano- and micro-scale physical features for appropriate cell response in a wide range of biological scenarios. Additionally, recent in vivo research demonstrates that nerve regeneration scaffolds with cell-scale physical features are more effective than those that rely only on chemical or macro-scale features. Physical guidance at the cell-scale is especially important for long (>20 mm) nerve defects, for which the only reliable treatment is the autologous nerve graft. The lack of other available options exposes a clear need for the application of nano- and microfabrication techniques that will allow the next generation of engineered nerve guides to more closely mimic native tissue at those scales. This review examines current research to determine what elements of cell-scale topography in experimental scaffolds are most effective at stimulating functional recovery, and then presents an overview of fabrication techniques that could potentially improve future treatment paradigms. Relative advantages and disadvantages of these techniques are discussed, with respect to both clinical adaptation and likely effectiveness. Our intent is to more clearly delineate the remaining obstacles in the development of a next generation nerve guide, particularly for long defects, and offer new perspectives on steering current technologies towards clinically viable solutions.

Keywords: Nerve guide; Microstructure; Scaffold; Nanotopography


The protection of MSCs from apoptosis in nerve regeneration by TGFβ1 through reducing inflammation and promoting VEGF-dependent angiogenesis by Hailang Luo; Yongjie Zhang; Ziqiang Zhang; Yan Jin (pp. 4277-4287).
Our previous report demonstrated that autologous adipose-derived mesenchymal stem cells (ADSCs) combined with xenogeneic acellular nerve matrix (XANM) can support the regeneration of defective nerves. Although ADSCs had the potential to replace Schwann cells in engineered-tissue nerves, apoptosis easily obstructed the ability to treat serious nerve injury in the host, such as a >50-mm-long nerve defect. In the present study, we found that, in combination with transforming growth factor β1 (TGFβ1), an ADSCs-XANM graft was sufficient to support the regeneration of a 50-mm sciatic nerve defect, which was not achieved using an ADSCs-XANM graft alone. Based on this finding, we further investigated how TGFβ1 coordinated with ADSCs to enhance nerve regeneration. In vitro, cell culture experiments demonstrated that TGFβ1 did not have a direct effect on ADSC proliferation, apoptosis, the cell cycle, or neural differentiation. The expression of VEGF, however, was significantly increased in ADSCs cultured with TGFβ1. In vivo, fluorescence labeling experiments demonstrated that the survival of transplanted ADSCs inoculated with XANM-TGFβ1 was higher than with XANM. Further study showed that TGFβ1 was capable of impairing the host immune response that was trigged by transplanted XANM. Additionally, we discovered that XANM-ADSCs in immunodeficient mice had apoptosis rates similar to XANM-ADSCs-TGFβ1 over a short time course (7 days). Once we blocked VEGF with a neutralizing antibody, the protective effect of TGFβ1 was impaired over a long time course (28 days). These results suggested that TGFβ1 was capable of enhancing the regenerative capacity of an XANM-ADSCs graft, mainly by protecting transplanted ADSCs from apoptosis. This effect was achieved in part through decreasing inflammation and promoting VEGF-dependent angiogenesis.

Keywords: Nerve regeneration; Transforming growth factor β1; Apoptosis; Adipose-derived mesenchymal stem cells; Xenogeneic acellular nerve matrix


The protection of MSCs from apoptosis in nerve regeneration by TGFβ1 through reducing inflammation and promoting VEGF-dependent angiogenesis by Hailang Luo; Yongjie Zhang; Ziqiang Zhang; Yan Jin (pp. 4277-4287).
Our previous report demonstrated that autologous adipose-derived mesenchymal stem cells (ADSCs) combined with xenogeneic acellular nerve matrix (XANM) can support the regeneration of defective nerves. Although ADSCs had the potential to replace Schwann cells in engineered-tissue nerves, apoptosis easily obstructed the ability to treat serious nerve injury in the host, such as a >50-mm-long nerve defect. In the present study, we found that, in combination with transforming growth factor β1 (TGFβ1), an ADSCs-XANM graft was sufficient to support the regeneration of a 50-mm sciatic nerve defect, which was not achieved using an ADSCs-XANM graft alone. Based on this finding, we further investigated how TGFβ1 coordinated with ADSCs to enhance nerve regeneration. In vitro, cell culture experiments demonstrated that TGFβ1 did not have a direct effect on ADSC proliferation, apoptosis, the cell cycle, or neural differentiation. The expression of VEGF, however, was significantly increased in ADSCs cultured with TGFβ1. In vivo, fluorescence labeling experiments demonstrated that the survival of transplanted ADSCs inoculated with XANM-TGFβ1 was higher than with XANM. Further study showed that TGFβ1 was capable of impairing the host immune response that was trigged by transplanted XANM. Additionally, we discovered that XANM-ADSCs in immunodeficient mice had apoptosis rates similar to XANM-ADSCs-TGFβ1 over a short time course (7 days). Once we blocked VEGF with a neutralizing antibody, the protective effect of TGFβ1 was impaired over a long time course (28 days). These results suggested that TGFβ1 was capable of enhancing the regenerative capacity of an XANM-ADSCs graft, mainly by protecting transplanted ADSCs from apoptosis. This effect was achieved in part through decreasing inflammation and promoting VEGF-dependent angiogenesis.

Keywords: Nerve regeneration; Transforming growth factor β1; Apoptosis; Adipose-derived mesenchymal stem cells; Xenogeneic acellular nerve matrix


The role of well-defined patterned substrata on the regeneration of DRG neuron pathfinding and integrin expression dynamics using chondroitin sulfate proteoglycans by Gerald N. Hodgkinson; Patrick A. Tresco; Vladimir Hlady (pp. 4288-4297).
Injured neurons intrinsically adapt to and partially overcome inhibitory proteoglycan expression in the central nervous system by upregulating integrin expression. It remains unclear however, to what extent varying proteoglycan concentrations influence the strength of this response, how rapidly neurons adapt to proteoglycans, and how pathfinding dynamics are altered over time as integrin expression is modulated in response to proteoglycan signals. To investigate these quandaries, we created well-defined substrata in which postnatal DRG neuron pathfinding dynamics and growth cone integrin expression were interrogated as a function of proteoglycan substrata density. DRGs responded by upregulating integrin expression in a proteoglycan dose dependent fashion and exhibited robust outgrowth over all proteoglycan densities at initial time frames. However, after prolonged proteoglycan exposure, neurons exhibited decreasing velocities associated with increasing proteoglycan densities, while neurons growing on low proteoglycan levels exhibited robust outgrowth at all time points. Additionally, DRG outgrowth over proteoglycan density step boundaries, and a brief β1 integrin functional block proved that regeneration was integrin dependent and that DRGs exhibit delayed slowing and loss in persistence after even transient encounters with dense proteoglycan boundaries. These findings demonstrate the complexity of proteoglycan regulation on integrin expression and regenerative pathfinding.

Keywords: Neuronal regeneration; Axonal pathfinding; Growth cone sensing; Micropatterning; Chondroitin sulfate; Integrin


The role of well-defined patterned substrata on the regeneration of DRG neuron pathfinding and integrin expression dynamics using chondroitin sulfate proteoglycans by Gerald N. Hodgkinson; Patrick A. Tresco; Vladimir Hlady (pp. 4288-4297).
Injured neurons intrinsically adapt to and partially overcome inhibitory proteoglycan expression in the central nervous system by upregulating integrin expression. It remains unclear however, to what extent varying proteoglycan concentrations influence the strength of this response, how rapidly neurons adapt to proteoglycans, and how pathfinding dynamics are altered over time as integrin expression is modulated in response to proteoglycan signals. To investigate these quandaries, we created well-defined substrata in which postnatal DRG neuron pathfinding dynamics and growth cone integrin expression were interrogated as a function of proteoglycan substrata density. DRGs responded by upregulating integrin expression in a proteoglycan dose dependent fashion and exhibited robust outgrowth over all proteoglycan densities at initial time frames. However, after prolonged proteoglycan exposure, neurons exhibited decreasing velocities associated with increasing proteoglycan densities, while neurons growing on low proteoglycan levels exhibited robust outgrowth at all time points. Additionally, DRG outgrowth over proteoglycan density step boundaries, and a brief β1 integrin functional block proved that regeneration was integrin dependent and that DRGs exhibit delayed slowing and loss in persistence after even transient encounters with dense proteoglycan boundaries. These findings demonstrate the complexity of proteoglycan regulation on integrin expression and regenerative pathfinding.

Keywords: Neuronal regeneration; Axonal pathfinding; Growth cone sensing; Micropatterning; Chondroitin sulfate; Integrin


Extrahepatic bile duct regeneration in pigs using collagen scaffolds loaded with human collagen-binding bFGF by Qiang Li; Liang Tao; Bing Chen; Haozhen Ren; Xianglin Hou; Siqiao Zhou; Jianxin Zhou; Xitai Sun; Jianwu Dai; Yitao Ding (pp. 4298-4308).
Extrahepatic bile duct defects and their complications are benign lesions but with malignant outcomes. Extrahepatic bile duct regeneration at the injury site could be important for the repair. In our previous work, a human basic fibroblast growth factor (bFGF) fused with a collagen-binding domain (CBD) was produced to activate the collagen membrane to obtain targeted tissue regeneration. This collagen/growth factor functional biomaterial could promote the regeneration of skin, bladder and full-thickness abdominal wall by accelerating vascularization and cellularization of autologous tissues. We speculate that the functional biomaterial could also provide the repairing effect on extrahepatic bile duct injuries. Using a pig extrahepatic bile duct injury model, we found that the collagen/CBD-bFGF composite biomaterial could significantly promote the extrahepatic bile duct regeneration at the injury site without causing structure deformation or hepatic dysfunction during both short- and long-time observations.

Keywords: Collagen; Basic fibroblast growth factor; Extrahepatic bile duct regeneration; Tissue engineering


Extrahepatic bile duct regeneration in pigs using collagen scaffolds loaded with human collagen-binding bFGF by Qiang Li; Liang Tao; Bing Chen; Haozhen Ren; Xianglin Hou; Siqiao Zhou; Jianxin Zhou; Xitai Sun; Jianwu Dai; Yitao Ding (pp. 4298-4308).
Extrahepatic bile duct defects and their complications are benign lesions but with malignant outcomes. Extrahepatic bile duct regeneration at the injury site could be important for the repair. In our previous work, a human basic fibroblast growth factor (bFGF) fused with a collagen-binding domain (CBD) was produced to activate the collagen membrane to obtain targeted tissue regeneration. This collagen/growth factor functional biomaterial could promote the regeneration of skin, bladder and full-thickness abdominal wall by accelerating vascularization and cellularization of autologous tissues. We speculate that the functional biomaterial could also provide the repairing effect on extrahepatic bile duct injuries. Using a pig extrahepatic bile duct injury model, we found that the collagen/CBD-bFGF composite biomaterial could significantly promote the extrahepatic bile duct regeneration at the injury site without causing structure deformation or hepatic dysfunction during both short- and long-time observations.

Keywords: Collagen; Basic fibroblast growth factor; Extrahepatic bile duct regeneration; Tissue engineering


In vivo biocompatibility and biodegradation of 3D-printed porous scaffolds based on a hydroxyl-functionalized poly(ε-caprolactone) by Hajar Seyednejad; Debby Gawlitta; Raoul V. Kuiper; Alain de Bruin; Cornelus F. van Nostrum; Tina Vermonden; Wouter J.A. Dhert; Wim E. Hennink (pp. 4309-4318).
The aim of this study was to evaluate the in vivo biodegradation and biocompatibility of three-dimensional (3D) scaffolds based on a hydroxyl-functionalized polyester (poly(hydroxymethylglycolide-co-ε-caprolactone), PHMGCL), which has enhanced hydrophilicity, increased degradation rate, and improved cell–material interactions as compared to its counterpart poly(ε-caprolactone), PCL. In this study, 3D scaffolds based on this polymer (PHMGCL, HMG:CL 8:92) were prepared by means of fiber deposition (melt-plotting). The biodegradation and tissue biocompatibility of PHMGCL and PCL scaffolds after subcutaneous implantation in Balb/c mice were investigated. At 4 and 12 weeks post implantation, the scaffolds were retrieved and evaluated for extent of degradation by measuring the residual weight of the scaffolds, thermal properties (DSC), and morphology (SEM) whereas the polymer was analyzed for both its composition (1H NMR) and molecular weight (GPC). The scaffolds with infiltrated tissues were harvested, fixed, stained and histologically analyzed. The in vitro enzymatic degradation of these scaffolds was also investigated in lipase solutions. It was shown that PHMGCL 3D-scaffolds lost more than 60% of their weight within 3 months of implantation while PCL scaffolds showed no weight loss in this time frame. The molecular weight (Mw) of PHMGCL decreased from 46.9 kDa before implantation to 23.2 kDa after 3 months of implantation, while the molecular weight of PCL was unchanged in this period.1H NMR analysis showed that the degradation of PHMGCL was characterized by a loss of HMG units. In vitro enzymatic degradation showed that PHMGCL scaffolds were degraded within 50 h, while the degradation time for PCL scaffolds of similar structure was 72 h. A normal foreign body response to both scaffold types characterized by the presence of macrophages, lymphocytes, and fibrosis was observed with a more rapid onset in PHMGCL scaffolds. The extent of tissue–scaffold interactions as well as vascularization was shown to be higher for PHMGCL scaffolds compared to PCL ones. Therefore, the fast degradable PHMGCL which showed good biocompatibility is a promising biomaterial for tissue engineering applications.

Keywords: Functionalized polyester; Polycaprolactone; Biocompatibility; Biodegradation


In vivo biocompatibility and biodegradation of 3D-printed porous scaffolds based on a hydroxyl-functionalized poly(ε-caprolactone) by Hajar Seyednejad; Debby Gawlitta; Raoul V. Kuiper; Alain de Bruin; Cornelus F. van Nostrum; Tina Vermonden; Wouter J.A. Dhert; Wim E. Hennink (pp. 4309-4318).
The aim of this study was to evaluate the in vivo biodegradation and biocompatibility of three-dimensional (3D) scaffolds based on a hydroxyl-functionalized polyester (poly(hydroxymethylglycolide-co-ε-caprolactone), PHMGCL), which has enhanced hydrophilicity, increased degradation rate, and improved cell–material interactions as compared to its counterpart poly(ε-caprolactone), PCL. In this study, 3D scaffolds based on this polymer (PHMGCL, HMG:CL 8:92) were prepared by means of fiber deposition (melt-plotting). The biodegradation and tissue biocompatibility of PHMGCL and PCL scaffolds after subcutaneous implantation in Balb/c mice were investigated. At 4 and 12 weeks post implantation, the scaffolds were retrieved and evaluated for extent of degradation by measuring the residual weight of the scaffolds, thermal properties (DSC), and morphology (SEM) whereas the polymer was analyzed for both its composition (1H NMR) and molecular weight (GPC). The scaffolds with infiltrated tissues were harvested, fixed, stained and histologically analyzed. The in vitro enzymatic degradation of these scaffolds was also investigated in lipase solutions. It was shown that PHMGCL 3D-scaffolds lost more than 60% of their weight within 3 months of implantation while PCL scaffolds showed no weight loss in this time frame. The molecular weight (Mw) of PHMGCL decreased from 46.9 kDa before implantation to 23.2 kDa after 3 months of implantation, while the molecular weight of PCL was unchanged in this period.1H NMR analysis showed that the degradation of PHMGCL was characterized by a loss of HMG units. In vitro enzymatic degradation showed that PHMGCL scaffolds were degraded within 50 h, while the degradation time for PCL scaffolds of similar structure was 72 h. A normal foreign body response to both scaffold types characterized by the presence of macrophages, lymphocytes, and fibrosis was observed with a more rapid onset in PHMGCL scaffolds. The extent of tissue–scaffold interactions as well as vascularization was shown to be higher for PHMGCL scaffolds compared to PCL ones. Therefore, the fast degradable PHMGCL which showed good biocompatibility is a promising biomaterial for tissue engineering applications.

Keywords: Functionalized polyester; Polycaprolactone; Biocompatibility; Biodegradation


The concept of in vivo airway tissue engineering by Philipp Jungebluth; Augustinus Bader; Silvia Baiguera; Susanne Möller; Massimo Jaus; Mei Ling Lim; Kaj Fried; Kristín Rós Kjartansdóttir; Tetsuhiko Go; Heike Nave; Wolfgang Harringer; Vanessa Lundin; Ana I. Teixeira; Paolo Macchiarini (pp. 4319-4326).
We investigated whether decellularized pig tracheas could regenerate in vivo, without being recellularized before transplantation, using the own body as bioreactor. Decellularized pig tracheal scaffolds were intraoperative conditioned with mononuclear cells and growth and differentiation factors. During the postoperative period, the in situ regeneration was boosted by administering bioactive molecules to promote peripheral mobilization and differentiation of stem/progenitor cells and ultimately the regenerative process. Results revealed, after 2 weeks, a nearly normal trachea, with respiratory epithelium and a double-banded cartilage but without any mechanical differences compared to the native tissue. The growth factor administration resulted in a mobilization of progenitor and stem cells into the peripheral circulation and in an up-regulation of anti-apoptotic genes. Isolated stem/progenitor cells could be differentiated in vitro into several cell types, proving their multipotency. We provide evidence that the own body can be used as bioreactor to promote in vivo tissue engineering replacement. Moreover, we demonstrated the beneficial effect of additional pharmaceutical intervention for an improved engraftment of the transplant.

Keywords: Decellularized trachea; In vivo tissue engineering; Intraoperative seeding; Boosting therapy


The concept of in vivo airway tissue engineering by Philipp Jungebluth; Augustinus Bader; Silvia Baiguera; Susanne Möller; Massimo Jaus; Mei Ling Lim; Kaj Fried; Kristín Rós Kjartansdóttir; Tetsuhiko Go; Heike Nave; Wolfgang Harringer; Vanessa Lundin; Ana I. Teixeira; Paolo Macchiarini (pp. 4319-4326).
We investigated whether decellularized pig tracheas could regenerate in vivo, without being recellularized before transplantation, using the own body as bioreactor. Decellularized pig tracheal scaffolds were intraoperative conditioned with mononuclear cells and growth and differentiation factors. During the postoperative period, the in situ regeneration was boosted by administering bioactive molecules to promote peripheral mobilization and differentiation of stem/progenitor cells and ultimately the regenerative process. Results revealed, after 2 weeks, a nearly normal trachea, with respiratory epithelium and a double-banded cartilage but without any mechanical differences compared to the native tissue. The growth factor administration resulted in a mobilization of progenitor and stem cells into the peripheral circulation and in an up-regulation of anti-apoptotic genes. Isolated stem/progenitor cells could be differentiated in vitro into several cell types, proving their multipotency. We provide evidence that the own body can be used as bioreactor to promote in vivo tissue engineering replacement. Moreover, we demonstrated the beneficial effect of additional pharmaceutical intervention for an improved engraftment of the transplant.

Keywords: Decellularized trachea; In vivo tissue engineering; Intraoperative seeding; Boosting therapy


The functional properties of nephronectin: An adhesion molecule for cardiac tissue engineering by Chinmoy Patra; Filomena Ricciardi; Felix B. Engel (pp. 4327-4335).
Despite significant advances in preventive cardiovascular medicine and therapy for acute and chronic heart failure, cardiovascular diseases remain among the leading causes of death worldwide. In recent years cardiac tissue engineering has been established as a possible future treatment option for cardiac disease. However, the quality of engineered myocardial tissues remains poor. In tissue engineering it is important that the scaffold allows cells to attach, spread, maintain their differentiation status or differentiate into functional cells in order to exhibit their physiological function. Here, we have investigated the suitability of the natural cardiac extracellular matrix component nephronectin as an adhesive material for cardiac tissue engineering. Primary neonatal rat cardiomyocytes were seeded on collagen-, fibronectin- or nephronectin-coated glass coverslips and analyzed for cell adhesion, cellular metabolic activity, response to extracellular stimuli, cell-to-cell communication, differentiation and contractility. Our data demonstrate that most neonatal cardiomyocytes attached in an RGD domain-dependent manner within 18 h to nephronectin. The cells exhibited high metabolic activity, responded to growth factor stimuli and maintained their differentiation status. Moreover, nephronectin promoted sarcomere maturation and alignment, cell-to-cell communication and synchronous contractions. In conclusion, our findings demonstrate that nephronectin has excellent properties for cardiomyocyte adhesion and function and thus has the potential to improve current cardiac tissue engineering approaches.

Keywords: Nephronectin; Tissue engineering; RGD; Cell adhesion; Cardiomyocytes; Connexin 43


The functional properties of nephronectin: An adhesion molecule for cardiac tissue engineering by Chinmoy Patra; Filomena Ricciardi; Felix B. Engel (pp. 4327-4335).
Despite significant advances in preventive cardiovascular medicine and therapy for acute and chronic heart failure, cardiovascular diseases remain among the leading causes of death worldwide. In recent years cardiac tissue engineering has been established as a possible future treatment option for cardiac disease. However, the quality of engineered myocardial tissues remains poor. In tissue engineering it is important that the scaffold allows cells to attach, spread, maintain their differentiation status or differentiate into functional cells in order to exhibit their physiological function. Here, we have investigated the suitability of the natural cardiac extracellular matrix component nephronectin as an adhesive material for cardiac tissue engineering. Primary neonatal rat cardiomyocytes were seeded on collagen-, fibronectin- or nephronectin-coated glass coverslips and analyzed for cell adhesion, cellular metabolic activity, response to extracellular stimuli, cell-to-cell communication, differentiation and contractility. Our data demonstrate that most neonatal cardiomyocytes attached in an RGD domain-dependent manner within 18 h to nephronectin. The cells exhibited high metabolic activity, responded to growth factor stimuli and maintained their differentiation status. Moreover, nephronectin promoted sarcomere maturation and alignment, cell-to-cell communication and synchronous contractions. In conclusion, our findings demonstrate that nephronectin has excellent properties for cardiomyocyte adhesion and function and thus has the potential to improve current cardiac tissue engineering approaches.

Keywords: Nephronectin; Tissue engineering; RGD; Cell adhesion; Cardiomyocytes; Connexin 43


Monitoring HSV-TK/ganciclovir cancer suicide gene therapy using CdTe/CdS core/shell quantum dots by Dan Shao; Qinghui Zeng; Zheng Fan; Jing Li; Ming Zhang; Youlin Zhang; Ou Li; Li Chen; Xianggui Kong; Hong Zhang (pp. 4336-4344).
To be able to label a gene and monitor its migration are key important approaches for the clinical application of cancer suicide gene therapy. Photonic nanomaterials are introduced in this work. One of the most promised suicide genes - herpes simplex virus thymidine kinase (HSV-TK) gene - is successfully linked with CdTe/CdS core/shell quantum dots (QDs) via EDC/NHS coupling method. From confocal microscopy it was demonstrated that plasmid TK intracellular trafficking can be effectively and distinctly traced via monitoring the luminescence of the QDs up to 96 h after transfection of QDs-TK conjugates into Hela cells. MTT results show that the QDs-TK conjugates have a high efficient cytotoxicity after adding GCV into Hela cells, whereas the QDs exert no detectable deleterious effects on the cellular processes. The apoptosis induced by QDs-TK conjugates with GCV is distinctly traced partly due to the strong luminescence of the QDs. Our results indicate that photonic nanomaterials, e.g. QDs, provide a tool for monitoring TK gene delivery and anti-cancer activity.

Keywords: Quantum dots; Herpes simplex virus thymidine kinase; Ganciclovir; Gene therapy


Monitoring HSV-TK/ganciclovir cancer suicide gene therapy using CdTe/CdS core/shell quantum dots by Dan Shao; Qinghui Zeng; Zheng Fan; Jing Li; Ming Zhang; Youlin Zhang; Ou Li; Li Chen; Xianggui Kong; Hong Zhang (pp. 4336-4344).
To be able to label a gene and monitor its migration are key important approaches for the clinical application of cancer suicide gene therapy. Photonic nanomaterials are introduced in this work. One of the most promised suicide genes - herpes simplex virus thymidine kinase (HSV-TK) gene - is successfully linked with CdTe/CdS core/shell quantum dots (QDs) via EDC/NHS coupling method. From confocal microscopy it was demonstrated that plasmid TK intracellular trafficking can be effectively and distinctly traced via monitoring the luminescence of the QDs up to 96 h after transfection of QDs-TK conjugates into Hela cells. MTT results show that the QDs-TK conjugates have a high efficient cytotoxicity after adding GCV into Hela cells, whereas the QDs exert no detectable deleterious effects on the cellular processes. The apoptosis induced by QDs-TK conjugates with GCV is distinctly traced partly due to the strong luminescence of the QDs. Our results indicate that photonic nanomaterials, e.g. QDs, provide a tool for monitoring TK gene delivery and anti-cancer activity.

Keywords: Quantum dots; Herpes simplex virus thymidine kinase; Ganciclovir; Gene therapy


Antitumor efficacy following the intracellular and interstitial release of liposomal doxorubicin by Amey Bandekar; Shrirang Karve; Min-Yuan Chang; Qingshan Mu; Jimmy Rotolo; Stavroula Sofou (pp. 4345-4352).
pH-triggered lipid-membranes designed from biophysical principles are evaluated in the form of targeted liposomal doxorubicin with the aim to ultimately better control the growth of vascularized tumors. We compare the antitumor efficacy of anti-HER2/neu pH-triggered lipid vesicles encapsulating doxorubicin to the anti-HER2/neu form of an FDA approved liposomal doxorubicin of DSPC/cholesterol-based vesicles. The HER2/neu receptor is chosen due to its abundance in human breast cancers and its connection to low prognosis. On a subcutaneous murine BT474 xenograft model, superior control of tumor growth is demonstrated by targeted pH-triggered vesicles relative to targeted DSPC/cholesterol-based vesicles (35% vs. 19% decrease in tumor volume after 32 days upon initiation of treatment). Superior tumor control is also confirmed on SKBR3 subcutaneous xenografts of lower HER2/neu expression. The non-targeted form of pH-triggered vesicles encapsulating doxorubicin results also in better tumor control relative to the non-targeted DSPC/cholesterol-based vesicles (34% vs. 41% increase in tumor volume). Studies in BT474 multicellular spheroids suggest that the observed efficacy could be attributed to release of doxorubicin directly into the acidic tumor interstitium from pH-triggered vesicles extravasated into the tumor but not internalized by cancer cells. pH-triggered liposome carriers engineered from gel-phase bilayers that reversibly phase-separate with lowering pH, form transiently defective interfacial boundaries resulting in fast release of encapsulated doxorubicin. Our studies show that pH-triggered liposomes release encapsulated doxorubicin intracellularly and intratumorally, and may improve tumor control at the same or even lower administered doses relative to FDA approved liposomal chemotherapy.

Keywords: pH-triggered liposomes; Targeted liposomal chemotherapy; Doxorubicin; Anti-HER2/neutargeting


Antitumor efficacy following the intracellular and interstitial release of liposomal doxorubicin by Amey Bandekar; Shrirang Karve; Min-Yuan Chang; Qingshan Mu; Jimmy Rotolo; Stavroula Sofou (pp. 4345-4352).
pH-triggered lipid-membranes designed from biophysical principles are evaluated in the form of targeted liposomal doxorubicin with the aim to ultimately better control the growth of vascularized tumors. We compare the antitumor efficacy of anti-HER2/neu pH-triggered lipid vesicles encapsulating doxorubicin to the anti-HER2/neu form of an FDA approved liposomal doxorubicin of DSPC/cholesterol-based vesicles. The HER2/neu receptor is chosen due to its abundance in human breast cancers and its connection to low prognosis. On a subcutaneous murine BT474 xenograft model, superior control of tumor growth is demonstrated by targeted pH-triggered vesicles relative to targeted DSPC/cholesterol-based vesicles (35% vs. 19% decrease in tumor volume after 32 days upon initiation of treatment). Superior tumor control is also confirmed on SKBR3 subcutaneous xenografts of lower HER2/neu expression. The non-targeted form of pH-triggered vesicles encapsulating doxorubicin results also in better tumor control relative to the non-targeted DSPC/cholesterol-based vesicles (34% vs. 41% increase in tumor volume). Studies in BT474 multicellular spheroids suggest that the observed efficacy could be attributed to release of doxorubicin directly into the acidic tumor interstitium from pH-triggered vesicles extravasated into the tumor but not internalized by cancer cells. pH-triggered liposome carriers engineered from gel-phase bilayers that reversibly phase-separate with lowering pH, form transiently defective interfacial boundaries resulting in fast release of encapsulated doxorubicin. Our studies show that pH-triggered liposomes release encapsulated doxorubicin intracellularly and intratumorally, and may improve tumor control at the same or even lower administered doses relative to FDA approved liposomal chemotherapy.

Keywords: pH-triggered liposomes; Targeted liposomal chemotherapy; Doxorubicin; Anti-HER2/neutargeting


Towards personalized medicine with a three-dimensional micro-scale perfusion-based two-chamber tissue model system by Liang Ma; Jeremy Barker; Changchun Zhou; Wei Li; Jing Zhang; Biaoyang Lin; Gregory Foltz; Jenni Küblbeck; Paavo Honkakoski (pp. 4353-4361).
A three-dimensional micro-scale perfusion-based two-chamber (3D-μPTC) tissue model system was developed to test the cytotoxicity of anticancer drugs in conjunction with liver metabolism. Liver cells with different cytochrome P450 (CYP) subtypes and glioblastoma multiforme (GBM) brain cancer cells were cultured in two separate chambers connected in tandem. Both chambers contained a 3D tissue engineering scaffold fabricated with biodegradable poly(lactic acid) (PLA) using a solvent-free approach. We used this model system to test the cytotoxicity of anticancer drugs, including temozolomide (TMZ) and ifosfamide (IFO). With the liver cells, TMZ showed a much lower toxicity to GBM cells under both 2D and 3D cell culture conditions. Comparing 2D, GBM cells cultured in 3D had much high viability under TMZ treatment. IFO was used to test the CYP-related metabolic effects. Cells with different expression levels of CYP3A4 differed dramatically in their ability to activate IFO, which led to strong metabolism-dependent cytotoxicity to GBM cells. These results demonstrate that our 3D-μPTC system could provide a more physiologically realistic in vitro environment than the current 2D monolayers for testing metabolism-dependent toxicity of anticancer drugs. It could therefore be used as an important platform for better prediction of drug dosing and schedule towards personalized medicine.

Keywords: Glioblastoma; 3D cell culture; Micro-scale tissue model system; Two-chamber system; Liver CYP metabolism; Metabolism-dependent toxicity


Towards personalized medicine with a three-dimensional micro-scale perfusion-based two-chamber tissue model system by Liang Ma; Jeremy Barker; Changchun Zhou; Wei Li; Jing Zhang; Biaoyang Lin; Gregory Foltz; Jenni Küblbeck; Paavo Honkakoski (pp. 4353-4361).
A three-dimensional micro-scale perfusion-based two-chamber (3D-μPTC) tissue model system was developed to test the cytotoxicity of anticancer drugs in conjunction with liver metabolism. Liver cells with different cytochrome P450 (CYP) subtypes and glioblastoma multiforme (GBM) brain cancer cells were cultured in two separate chambers connected in tandem. Both chambers contained a 3D tissue engineering scaffold fabricated with biodegradable poly(lactic acid) (PLA) using a solvent-free approach. We used this model system to test the cytotoxicity of anticancer drugs, including temozolomide (TMZ) and ifosfamide (IFO). With the liver cells, TMZ showed a much lower toxicity to GBM cells under both 2D and 3D cell culture conditions. Comparing 2D, GBM cells cultured in 3D had much high viability under TMZ treatment. IFO was used to test the CYP-related metabolic effects. Cells with different expression levels of CYP3A4 differed dramatically in their ability to activate IFO, which led to strong metabolism-dependent cytotoxicity to GBM cells. These results demonstrate that our 3D-μPTC system could provide a more physiologically realistic in vitro environment than the current 2D monolayers for testing metabolism-dependent toxicity of anticancer drugs. It could therefore be used as an important platform for better prediction of drug dosing and schedule towards personalized medicine.

Keywords: Glioblastoma; 3D cell culture; Micro-scale tissue model system; Two-chamber system; Liver CYP metabolism; Metabolism-dependent toxicity


Necrosis of cervical carcinoma by dichloroacetate released from electrospun polylactide mats by Daxing Liu; Shi Liu; Xiabin Jing; Xiaoyuan Li; Wenliang Li; Yubin Huang (pp. 4362-4369).
It is still a great challenge to apply therapeutic concentration of anti-cancer drugs to the tumor site with low system toxicity. An in situ administration strategy was applied to reverse the aerobic glycolysis of tumor in vivo for the first time. Controlled release of therapeutic concentration of dichloroacetate (DCA) from polylactide (PLA) electrospun mats covering the solid tumor locally was designed to suppress the cervical carcinoma in vivo. A dramatic decrease in the volume and weight of tumors was observed for 19 days in tumor-bearing mice, and a totally 96% of the tumor suppression degree was obtained even the initial tumor volume was around 200 mm3. Half of the mice recovered in less than 3 weeks. Necrosis was examined rather than apoptosis on the tumor cells as the main process of cell death induced by the DCA-loaded electrospun mats. A proposed necroptosis mechanism was presented to explain the signal pathways that were induced by the metabolic remodeling of DCA. It provided support for this strategy that target the bio-energy metabolism of the cervical carcinoma locally is a quick and effective pathway to cure the advanced-carcinoma of cervical.

Keywords: Dichloroacetate; Electrospun mats; Metabolism target; ATP depletion; Necrosis


Necrosis of cervical carcinoma by dichloroacetate released from electrospun polylactide mats by Daxing Liu; Shi Liu; Xiabin Jing; Xiaoyuan Li; Wenliang Li; Yubin Huang (pp. 4362-4369).
It is still a great challenge to apply therapeutic concentration of anti-cancer drugs to the tumor site with low system toxicity. An in situ administration strategy was applied to reverse the aerobic glycolysis of tumor in vivo for the first time. Controlled release of therapeutic concentration of dichloroacetate (DCA) from polylactide (PLA) electrospun mats covering the solid tumor locally was designed to suppress the cervical carcinoma in vivo. A dramatic decrease in the volume and weight of tumors was observed for 19 days in tumor-bearing mice, and a totally 96% of the tumor suppression degree was obtained even the initial tumor volume was around 200 mm3. Half of the mice recovered in less than 3 weeks. Necrosis was examined rather than apoptosis on the tumor cells as the main process of cell death induced by the DCA-loaded electrospun mats. A proposed necroptosis mechanism was presented to explain the signal pathways that were induced by the metabolic remodeling of DCA. It provided support for this strategy that target the bio-energy metabolism of the cervical carcinoma locally is a quick and effective pathway to cure the advanced-carcinoma of cervical.

Keywords: Dichloroacetate; Electrospun mats; Metabolism target; ATP depletion; Necrosis


Long-term multimodal imaging of tumor draining sentinel lymph nodes using mesoporous silica-based nanoprobes by Xinglu Huang; Fan Zhang; Seulki Lee; Magdalena Swierczewska; Dale O. Kiesewetter; Lixin Lang; Guofeng Zhang; Lei Zhu; Haokao Gao; Hak Soo Choi; Gang Niu; Xiaoyuan Chen (pp. 4370-4378).
The imaging of sentinel lymph nodes (SLNs), the first defense against primary tumor metastasis, has been considered as an important strategy for noninvasive tracking tumor metastasis in clinics. In this study, we report the development and application of mesoporous silica-based triple-modal nanoprobes that integrate multiple functional moieties to facilitate near-infrared optical, magnetic resonance (MR) and positron emission tomography (PET) imaging. After embedding near-infrared dye ZW800, the nanoprobe was labeled with T1 contrast agent Gd3+ and radionuclide64Cu through chelating reactions. High stability and long intracellular retention time of the nanoprobes was confirmed by in vitro characterization, which facilitate long-term in vivo imaging. Longitudinal multimodal imaging was subsequently achieved to visualize tumor draining SLNs up to 3 weeks in a 4T1 tumor metastatic model. Obvious differences in uptake rate, amount of particles, and contrast between metastatic and contra-lateral sentinel lymph nodes were observed. These findings provide very helpful guidance for the design of robust multifunctional nanomaterials in SLNs' mapping and tumor metastasis diagnosis.

Keywords: Mesoporous silica nanoparticles; Multimodality imaging; Tumor metastasis; Magnetic resonance imaging; Positron emission tomography; Near-infrared fluorescence imaging


Long-term multimodal imaging of tumor draining sentinel lymph nodes using mesoporous silica-based nanoprobes by Xinglu Huang; Fan Zhang; Seulki Lee; Magdalena Swierczewska; Dale O. Kiesewetter; Lixin Lang; Guofeng Zhang; Lei Zhu; Haokao Gao; Hak Soo Choi; Gang Niu; Xiaoyuan Chen (pp. 4370-4378).
The imaging of sentinel lymph nodes (SLNs), the first defense against primary tumor metastasis, has been considered as an important strategy for noninvasive tracking tumor metastasis in clinics. In this study, we report the development and application of mesoporous silica-based triple-modal nanoprobes that integrate multiple functional moieties to facilitate near-infrared optical, magnetic resonance (MR) and positron emission tomography (PET) imaging. After embedding near-infrared dye ZW800, the nanoprobe was labeled with T1 contrast agent Gd3+ and radionuclide64Cu through chelating reactions. High stability and long intracellular retention time of the nanoprobes was confirmed by in vitro characterization, which facilitate long-term in vivo imaging. Longitudinal multimodal imaging was subsequently achieved to visualize tumor draining SLNs up to 3 weeks in a 4T1 tumor metastatic model. Obvious differences in uptake rate, amount of particles, and contrast between metastatic and contra-lateral sentinel lymph nodes were observed. These findings provide very helpful guidance for the design of robust multifunctional nanomaterials in SLNs' mapping and tumor metastasis diagnosis.

Keywords: Mesoporous silica nanoparticles; Multimodality imaging; Tumor metastasis; Magnetic resonance imaging; Positron emission tomography; Near-infrared fluorescence imaging


Surface modified magnetic nanoparticles for immuno-gene therapy of murine mammary adenocarcinoma by Sara Prijic; Lara Prosen; Maja Cemazar; Janez Scancar; Rok Romih; Jaka Lavrencak; Vladimir B. Bregar; Andrej Coer; Mojca Krzan; Andrej Znidarsic; Gregor Sersa (pp. 4379-4391).
Cancer immuno-gene therapy is an introduction of nucleic acids encoding immunostimulatory proteins, such as cytokine interleukin 12 (IL-12), into somatic cells to stimulate an immune response against a tumor. Various methods can be used for the introduction of nucleic acids into cells; magnetofection involves binding of nucleic acids to magnetic nanoparticles with subsequent exposure to an external magnetic field. Here we show that surface modified superparamagnetic iron oxide nanoparticles (SPIONs) with a combination of polyacrylic acid (PAA) and polyethylenimine (PEI) (SPIONs-PAA-PEI) proved to be safe and effective for magnetofection of cells and tumors in mice. Magnetofection of cells with plasmid DNA encoding reporter gene using SPIONs-PAA-PEI was superior in transfection efficiency to commercially available SPIONs. Magnetofection of murine mammary adenocarcinoma with plasmid DNA encoding IL-12 using SPIONs-PAA-PEI resulted in significant antitumor effect and could be further refined for cancer immuno-gene therapy.

Keywords: Nanoparticles; Gene therapy; Cytokine; Polyacrylic acid; Magnetism; Gene transfer


Surface modified magnetic nanoparticles for immuno-gene therapy of murine mammary adenocarcinoma by Sara Prijic; Lara Prosen; Maja Cemazar; Janez Scancar; Rok Romih; Jaka Lavrencak; Vladimir B. Bregar; Andrej Coer; Mojca Krzan; Andrej Znidarsic; Gregor Sersa (pp. 4379-4391).
Cancer immuno-gene therapy is an introduction of nucleic acids encoding immunostimulatory proteins, such as cytokine interleukin 12 (IL-12), into somatic cells to stimulate an immune response against a tumor. Various methods can be used for the introduction of nucleic acids into cells; magnetofection involves binding of nucleic acids to magnetic nanoparticles with subsequent exposure to an external magnetic field. Here we show that surface modified superparamagnetic iron oxide nanoparticles (SPIONs) with a combination of polyacrylic acid (PAA) and polyethylenimine (PEI) (SPIONs-PAA-PEI) proved to be safe and effective for magnetofection of cells and tumors in mice. Magnetofection of cells with plasmid DNA encoding reporter gene using SPIONs-PAA-PEI was superior in transfection efficiency to commercially available SPIONs. Magnetofection of murine mammary adenocarcinoma with plasmid DNA encoding IL-12 using SPIONs-PAA-PEI resulted in significant antitumor effect and could be further refined for cancer immuno-gene therapy.

Keywords: Nanoparticles; Gene therapy; Cytokine; Polyacrylic acid; Magnetism; Gene transfer


Mesoporous carbon@silicon-silica nanotheranostics for synchronous delivery of insoluble drugs and luminescence imaging by Qianjun He; Ming Ma; Chenyang Wei; Jianlin Shi (pp. 4392-4402).
A hierarchical theranostic nanostructure with carbon and Si nanocrystals respectively encapsulated in the mesopores and within the framework of mesoporous silica nanoparticles (CS-MSNs) was constructed by a bottom-up self-assembly strategy combining an in situ one-step carbonization/crystallization approach. CS-MSNs exhibited narrow size distribution, high payload of insoluble drugs and unique NIR-to-Vis luminescence imaging feature. The bio-conjugated CS-MSNs with a PEGylated phospholipid compound and hyaluronic acid showed excellent dispersivity and could specifically target cancer cells overexpressing CD44, deliver insoluble drugs into these cells and consequently kill them effectively, and also fluorescently image them simultaneously in a unique and attractive NIR-to-Vis luminescence imaging fashion, providing a promising opportunity for cancer theranostics.

Keywords: Mesoporous silica; Nanoparticle; Hierarchical nanostructures; Drug delivery; Luminescence imaging; Theranostic nanomedicine


Mesoporous carbon@silicon-silica nanotheranostics for synchronous delivery of insoluble drugs and luminescence imaging by Qianjun He; Ming Ma; Chenyang Wei; Jianlin Shi (pp. 4392-4402).
A hierarchical theranostic nanostructure with carbon and Si nanocrystals respectively encapsulated in the mesopores and within the framework of mesoporous silica nanoparticles (CS-MSNs) was constructed by a bottom-up self-assembly strategy combining an in situ one-step carbonization/crystallization approach. CS-MSNs exhibited narrow size distribution, high payload of insoluble drugs and unique NIR-to-Vis luminescence imaging feature. The bio-conjugated CS-MSNs with a PEGylated phospholipid compound and hyaluronic acid showed excellent dispersivity and could specifically target cancer cells overexpressing CD44, deliver insoluble drugs into these cells and consequently kill them effectively, and also fluorescently image them simultaneously in a unique and attractive NIR-to-Vis luminescence imaging fashion, providing a promising opportunity for cancer theranostics.

Keywords: Mesoporous silica; Nanoparticle; Hierarchical nanostructures; Drug delivery; Luminescence imaging; Theranostic nanomedicine


A mPEG-PLGA- b-PLL copolymer carrier for adriamycin and siRNA delivery by Peifeng Liu; Hui Yu; Ying Sun; Mingjie Zhu; Yourong Duan (pp. 4403-4412).
A amphiphilic block copolymer composed of conventional monomethoxy (polyethylene glycol)-poly (d,l-lactide- co-glycolide)-poly (l-lysine) (mPEG-PLGA- b-PLL) was synthesized. The chemical structure of this copolymer and its precursors was confirmed by Fourier Transform Infrared Spectroscopy (FTIR),1H Nuclear Magnetic Resonance (1H NMR) and Gel Permeation Chromatography (GPC). The copolymer was used to prepare nanoparticles (NPs) that were then loaded with either the anti-cancer drug adriamycin or small interfering RNA-negative (siRNA) using a double emulsion method. MTT assays used to study the in vitro cytotoxicity of mPEG-PLGA- b-PLL NPs showed that these particles were not toxic in huh-7 hepatic carcinoma cells. Confocal laser scanning microscopy (CLSM) and flow cytometer analysis results demonstrated efficient mPEG-PLGA- b-PLL NPs-mediated delivery of both adriamycin and siRNA into the cells. In vivo the targeting delivery of adriamycin or siRNA mediated by mPEG-PLGA- b-PLL NPs in the huh-7 hepatic carcinoma-bearing mice was evaluated using a fluorescence imaging system. The targeting delivery results and froze section analysis confirmed that drug or siRNA is deliver to tumor more efficiently by mPEG-PLGA- b-PLL NPs than free drug or Lipofectamine™2000. The high efficiency delivery of mPEG-PLGA- b-PLL NPs mainly due to the enhancement of cellular uptake. These results imply that mPEG-PLGA- b-PLL NPs have a great potential to be used as an effective carriers for adriamycin or siRNA.

Keywords: Copolymer; Nanoparticles; Carrier for delivery; Drug delivery; Gene delivery


A mPEG-PLGA- b-PLL copolymer carrier for adriamycin and siRNA delivery by Peifeng Liu; Hui Yu; Ying Sun; Mingjie Zhu; Yourong Duan (pp. 4403-4412).
A amphiphilic block copolymer composed of conventional monomethoxy (polyethylene glycol)-poly (d,l-lactide- co-glycolide)-poly (l-lysine) (mPEG-PLGA- b-PLL) was synthesized. The chemical structure of this copolymer and its precursors was confirmed by Fourier Transform Infrared Spectroscopy (FTIR),1H Nuclear Magnetic Resonance (1H NMR) and Gel Permeation Chromatography (GPC). The copolymer was used to prepare nanoparticles (NPs) that were then loaded with either the anti-cancer drug adriamycin or small interfering RNA-negative (siRNA) using a double emulsion method. MTT assays used to study the in vitro cytotoxicity of mPEG-PLGA- b-PLL NPs showed that these particles were not toxic in huh-7 hepatic carcinoma cells. Confocal laser scanning microscopy (CLSM) and flow cytometer analysis results demonstrated efficient mPEG-PLGA- b-PLL NPs-mediated delivery of both adriamycin and siRNA into the cells. In vivo the targeting delivery of adriamycin or siRNA mediated by mPEG-PLGA- b-PLL NPs in the huh-7 hepatic carcinoma-bearing mice was evaluated using a fluorescence imaging system. The targeting delivery results and froze section analysis confirmed that drug or siRNA is deliver to tumor more efficiently by mPEG-PLGA- b-PLL NPs than free drug or Lipofectamine™2000. The high efficiency delivery of mPEG-PLGA- b-PLL NPs mainly due to the enhancement of cellular uptake. These results imply that mPEG-PLGA- b-PLL NPs have a great potential to be used as an effective carriers for adriamycin or siRNA.

Keywords: Copolymer; Nanoparticles; Carrier for delivery; Drug delivery; Gene delivery


Co-delivery of SOX9 genes and anti-Cbfa-1 siRNA coated onto PLGA nanoparticles for chondrogenesis of human MSCs by Su Yeon Jeon; Ji Sun Park; Han Na Yang; Dae Gyun Woo; Keun-Hong Park (pp. 4413-4423).
Some genes expressed in stem cells interrupt and/or enhance differentiation. Therefore, the aim of this study was to inhibit the expression of unnecessary genes and enhance the expression of specific genes involved in stem cell differentiation by using small interfering RNA (siRNA) and plasmid DNA (pDNA) incorporated into cationic polymers as co-delivery factors. To achieve co-delivery of siRNA and pDNA to human mesenchymal stem cells (hMSCs), two different genes were complexed with poly(ethyleneimine) (PEI) and then coated onto poly(lactide-co-glycolic acid) (PLGA) nanoparticles (NP). To evaluate co-delivery of siRNA and pDNA into hMSCs, cells were transfected with green fluorescence protein (GFP) pDNA (GFP pDNA) and GFP siRNA (GFP siRNA). The percentage of GFP-expressing hMSCs decreased from 25.35 to 3.7% after transfection with GFP-DNA/PLGA NP (NPs) or GFP siRNA/PLGA NPs, whereas GFP-DNA/PLGA NPs and scramble siRNA (MOCK)/PLGA NPs had no effect on GFP expression. hMSCs cotransfected with coSOX9-pDNA/NPs and Cbfa-1-siRNA/NPs were tested both in vitro and in vivo using gel retardation, dynamic light scattering (DLS), and scanning electron microscope (SEM). The expression of genes and proteins associated with chondrogenesis was evaluated by FACS, RT-PCR, real time-qPCR, Western blotting, immunohistochemistry, and immunofluorescence imaging.

Keywords: hMSCs; PLGA NP; PEI; SOX-9; siRNA


Co-delivery of SOX9 genes and anti-Cbfa-1 siRNA coated onto PLGA nanoparticles for chondrogenesis of human MSCs by Su Yeon Jeon; Ji Sun Park; Han Na Yang; Dae Gyun Woo; Keun-Hong Park (pp. 4413-4423).
Some genes expressed in stem cells interrupt and/or enhance differentiation. Therefore, the aim of this study was to inhibit the expression of unnecessary genes and enhance the expression of specific genes involved in stem cell differentiation by using small interfering RNA (siRNA) and plasmid DNA (pDNA) incorporated into cationic polymers as co-delivery factors. To achieve co-delivery of siRNA and pDNA to human mesenchymal stem cells (hMSCs), two different genes were complexed with poly(ethyleneimine) (PEI) and then coated onto poly(lactide-co-glycolic acid) (PLGA) nanoparticles (NP). To evaluate co-delivery of siRNA and pDNA into hMSCs, cells were transfected with green fluorescence protein (GFP) pDNA (GFP pDNA) and GFP siRNA (GFP siRNA). The percentage of GFP-expressing hMSCs decreased from 25.35 to 3.7% after transfection with GFP-DNA/PLGA NP (NPs) or GFP siRNA/PLGA NPs, whereas GFP-DNA/PLGA NPs and scramble siRNA (MOCK)/PLGA NPs had no effect on GFP expression. hMSCs cotransfected with coSOX9-pDNA/NPs and Cbfa-1-siRNA/NPs were tested both in vitro and in vivo using gel retardation, dynamic light scattering (DLS), and scanning electron microscope (SEM). The expression of genes and proteins associated with chondrogenesis was evaluated by FACS, RT-PCR, real time-qPCR, Western blotting, immunohistochemistry, and immunofluorescence imaging.

Keywords: hMSCs; PLGA NP; PEI; SOX-9; siRNA


Tumor vasculature targeting following co-delivery of heparin-taurocholate conjugate and suberoylanilide hydroxamic acid using cationic nanolipoplex by Ji-young Kim; Gayong Shim; Hyun-woo Choi; Jooho Park; Seung Woo Chung; Sunil Kim; Kwangmeyung Kim; Ick Chan Kwon; Chan-Wha Kim; Sang Yoon Kim; Victor C. Yang; Yu-Kyoung Oh; Youngro Byun (pp. 4424-4430).
The chemical conjugate of low molecular weight heparin with taurocholate (LHT7) was previously designed to offer anticancer activity while minimizing the anticoagulant activity. In the present study, we found that the systemic administration of LHT7 in nanolipoplex could substantially enhance tumor vasculature targeting and anticancer effects. Moreover, we found that co-delivery of LHT7 with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, in nanolipoplex could provide synergistic antitumor effect. LHT7/SAHA nanolipoplex was formulated by encapsulating SAHA inside cationic liposomes, followed by complexation of negatively charged LHT7 onto the cationic surfaces of SAHA-loaded liposomes (SAHA-L). LHT7/SAHA nanolipoplex was positively charged with a mean diameter of 117.6 nm, and stable in serum. The nanolipoplex form of LHT7 could alter its pharmacokinetics and biodistribution. Compared to the free form of LHT7, LHT7 in the nanolipoplex showed 1.9-fold higher mean residence time, and higher tumor vasculature accumulation after its intravenous administration. LHT7/SAHA nanolipoplex showed highest antitumor efficacy in SCC-bearing mice, compared to LHT7, SAHA-L and sequential co-administration of LHT7 and SAHA-L. Consistent with the enhanced antitumor effect, the reduction of abnormal vessels in the tumor site was also the highest in the LHT7/SAHA nanolipoplex-treated group. These results suggested the potential of LHT7/SAHA nanolipoplex for enhanced tumor vasculature targeting, and the importance of nanolipoplex-mediated co-delivery with a histone deacetylase inhibitor for maximal anticancer effect.

Keywords: Heparin-taurocholate conjugate; Angiogenesis inhibitor; Histone deacetylase inhibitor; Cationic nanolipoplex; Tumor vasculature targeting


Tumor vasculature targeting following co-delivery of heparin-taurocholate conjugate and suberoylanilide hydroxamic acid using cationic nanolipoplex by Ji-young Kim; Gayong Shim; Hyun-woo Choi; Jooho Park; Seung Woo Chung; Sunil Kim; Kwangmeyung Kim; Ick Chan Kwon; Chan-Wha Kim; Sang Yoon Kim; Victor C. Yang; Yu-Kyoung Oh; Youngro Byun (pp. 4424-4430).
The chemical conjugate of low molecular weight heparin with taurocholate (LHT7) was previously designed to offer anticancer activity while minimizing the anticoagulant activity. In the present study, we found that the systemic administration of LHT7 in nanolipoplex could substantially enhance tumor vasculature targeting and anticancer effects. Moreover, we found that co-delivery of LHT7 with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, in nanolipoplex could provide synergistic antitumor effect. LHT7/SAHA nanolipoplex was formulated by encapsulating SAHA inside cationic liposomes, followed by complexation of negatively charged LHT7 onto the cationic surfaces of SAHA-loaded liposomes (SAHA-L). LHT7/SAHA nanolipoplex was positively charged with a mean diameter of 117.6 nm, and stable in serum. The nanolipoplex form of LHT7 could alter its pharmacokinetics and biodistribution. Compared to the free form of LHT7, LHT7 in the nanolipoplex showed 1.9-fold higher mean residence time, and higher tumor vasculature accumulation after its intravenous administration. LHT7/SAHA nanolipoplex showed highest antitumor efficacy in SCC-bearing mice, compared to LHT7, SAHA-L and sequential co-administration of LHT7 and SAHA-L. Consistent with the enhanced antitumor effect, the reduction of abnormal vessels in the tumor site was also the highest in the LHT7/SAHA nanolipoplex-treated group. These results suggested the potential of LHT7/SAHA nanolipoplex for enhanced tumor vasculature targeting, and the importance of nanolipoplex-mediated co-delivery with a histone deacetylase inhibitor for maximal anticancer effect.

Keywords: Heparin-taurocholate conjugate; Angiogenesis inhibitor; Histone deacetylase inhibitor; Cationic nanolipoplex; Tumor vasculature targeting


Long-term fate of silica nanoparticles interacting with human dermal fibroblasts by Sandrine Quignard; Gervaise Mosser; Michel Boissière; Thibaud Coradin (pp. 4431-4442).
The long-term fate of fluorescent non-porous FITC-SiO2 nanoparticles of various sizes (10–200 nm) and charge is studied in the presence of human dermal fibroblasts. Particle aggregates are formed in the culture medium and uptaken, at least partially, by macropinocytosis. The smallest particles have a strong impact on cell viability and genotoxic effects can be observed for negatively-charged colloids 10 nm in size. Largest particles do not impact on cellular activity and can be monitored in cellulo via fluorescence and transmission electron microscopy studies over two weeks. These observations reveal a significant decrease in the size of silica particles located in endocytic vesicles. The dissolution process is confirmed by monitoring the cell culture medium that contains both colloidal and soluble silica species. Such dissolution can be explained on the sole basis of silica solubility and has great implication for the use of non-porous silica particles as intra-cellular drug release systems.

Keywords: Silica; Nanoparticles; Intra-cellular dissolution; Fibroblasts


Long-term fate of silica nanoparticles interacting with human dermal fibroblasts by Sandrine Quignard; Gervaise Mosser; Michel Boissière; Thibaud Coradin (pp. 4431-4442).
The long-term fate of fluorescent non-porous FITC-SiO2 nanoparticles of various sizes (10–200 nm) and charge is studied in the presence of human dermal fibroblasts. Particle aggregates are formed in the culture medium and uptaken, at least partially, by macropinocytosis. The smallest particles have a strong impact on cell viability and genotoxic effects can be observed for negatively-charged colloids 10 nm in size. Largest particles do not impact on cellular activity and can be monitored in cellulo via fluorescence and transmission electron microscopy studies over two weeks. These observations reveal a significant decrease in the size of silica particles located in endocytic vesicles. The dissolution process is confirmed by monitoring the cell culture medium that contains both colloidal and soluble silica species. Such dissolution can be explained on the sole basis of silica solubility and has great implication for the use of non-porous silica particles as intra-cellular drug release systems.

Keywords: Silica; Nanoparticles; Intra-cellular dissolution; Fibroblasts

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