Peer-Reviewed Research Publications
Spatial distribution of mineralized bone matrix produced by marrow mesenchymal stem cells in self-assembling peptide hydrogel scaffold. J Biomed Mater Res A. 2008 Jan;84(1):128-36.
Hamada K, Hirose M, Yamashita T, Ohgushi H.
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 3-11-46 Nakoji, Amagasaki, Hyogo 661-0974, Japan.
We evaluated the osteogenic differentiation of mesenchymal stem cells (MSCs) using a new class of synthetic self-assembling peptide hydrogels, RADA 16, as a scaffold for three-dimensional culture. MSCs derived from rat bone marrow were culture-expanded and seeded into the hydrogel and further cultured in osteogenic medium containing beta-glycerophosphate, ascorbic acid, and dexamethasone for 2-4 weeks. High alkaline phosphatase activity and osteocalcin (OC) contents were detected at both the protein and gene expression levels during the culture periods. Both calcium and the OC contents increased over time, indicating the growth of a mineralized extracellular matrix within the hydrogel. Moreover, the process of the growth of the mineralized matrix determined by three-dimensional microarchitecture images was obtained by confocal laser scanning microscopy. The findings show that MSCs can differentiate into mature osteoblasts to form mineralized matrices within the hydrogel scaffold. Importantly, the differentiation can occur three dimensionally within the hydrogel, indicating that RADA 16 can be considered attractive synthetic biomaterial for use in bone tissue engineering.
Temperature and pH effects on biophysical and morphological properties of self-assembling peptide RADA16-I. J Pept Sci. 2008 Jan 14;14(2):152-162 [Epub ahead of print]
Ye Z, Zhang H, Luo H, Wang S, Zhou Q, DU X, Tang C, Chen L, Liu J, Shi YK, Zhang EY, Ellis-Behnke R, Zhao X.
Institute for NanoBiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Science Park No 1, Ke Yuan 4th St., Gao Peng Road, Hi-tech Industrial Development Zone, Chengdu, 610041, Sichuan, China.
It has been found that the self-assembling peptide RADA 16-I forms a beta-sheet structure and self-assembles into nanofibers and scaffolds in favor of cell growth, hemostasis and tissue-injury repair. But its biophysical and morphological properties, especially for its beta-sheet and self-assembling properties in heat- and pH-denatured conditions, remain largely unclear. In order to better understand and design nanobiomaterials, we studied the self-assembly behaviors of RADA16-I using CD and atomic force microscopy (AFM) measurements in various pH and heat-denatured conditions. Here, we report that the peptide, when exposed to pH 1.0 and 4.0, was still able to assume a typical beta-sheet structure and self-assemble into long nanofiber, although its beta-sheet content was dramatically decreased by 10% in a pH 1.0 solution. However, the peptide, when exposed to pH 13.0, drastically lost its beta-sheet structure and assembled into different small-sized globular aggregates. Similarly, the peptide, when heat-denatured from 25 to 70 degrees C, was still able to assume a typical beta-sheet structure with 46% content, but self-assembled into small-sized globular aggregates at much higher temperature. Titration experiments showed that the peptide RADA16-I exists in three types of ionic species: acidic (fully protonated peptide), zwitterionic (electrically neutral peptide carrying partial positive and negative charges) and basic (fully deprotonated peptide) species, called 'super ions'. The unordered structure and beta-turn of these 'super ions' via hydrogen or ionic bonds, and heat Brownian motion under the above denatured conditions would directly affect the stability of the beta-sheet and nanofibers. These results help us in the design of future nanobiomaterials, such as biosensors, based on beta-sheets and environmental changes. These results also help understand the pathogenesis of the beta-sheet-mediated neuronal diseases such as Alzheimer's disease and the mechanism of hemostasis. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.
Incorporation of a matrix metalloproteinase-sensitive substrate into self-assembling peptides - A model for biofunctional scaffolds. Biomaterials. 2008 Jan 11 [Epub ahead of print]
Chau Y, Luo Y, Cheung AC, Nagai Y, Zhang S, Kobler JB, Zeitels SM, Langer R.
Department of Chemical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.
Controlling and guiding cell behavior requires scaffolding materials capable of programming the three-dimensional (3-D) extracellular environment. In this study, we devised a new self-assembling peptide template for synthesizing nanofibrous hydrogels containing cell-responsive ligands. In particular, the insertion of a matrix metalloproteinase-2 (MMP-2) labile hexapeptide into the self-assembling building blocks of arginine-alanine-aspartate-alanine (RADA) was investigated. A series of peptides, varied by the position of the MMP-2 hexapeptide substrate and the length of RADA blocks, were prepared by parallel synthesis. Their self-assembling capabilities were characterized and compared by circular dichroism spectroscopy and dynamical mechanical analysis. Among all the different insertion patterns, the sequence comprising a centrically positioned MMP-2 substrate was flanked with three RADA units on each side self-assembled into a hydrogel matrix, with mechanical properties and nanofiber morphology comparable to the native material built with (RADA)(4) alone. Exposure of the new gel to MMP-2 resulted in peptide cleavage, as confirmed by mass spectroscopy, and a decrease in surface hardness, as detected by nanoindentor, indicating that the enzyme mediated degradation was localized to the gel surface. The new design can be used for introducing biological functions into self-assembling peptides to create scaffolding materials with potential applications in areas such as tissue engineering and regenerative medicine.
Self-assembling Peptide nanofiber scaffolds accelerate wound healing. PLoS ONE. 2008 Jan 9;3(1):e1410.
Schneider A, Garlick JA, Egles C.
Division of Cancer Biology and Tissue Engineering, Department of Oral and Maxillofacial Pathology, Tufts University, School of Dental Medicine, Boston, Massachusetts, United States of America.
Cutaneous wound repair regenerates skin integrity, but a chronic failure to heal results in compromised tissue function and increased morbidity. To address this, we have used an integrated approach, using nanobiotechnology to augment the rate of wound reepithelialization by combining self-assembling peptide (SAP) nanofiber scaffold and Epidermal Growth Factor (EGF). This SAP bioscaffold was tested in a bioengineered Human Skin Equivalent (HSE) tissue model that enabled wound reepithelialization to be monitored in a tissue that recapitulates molecular and cellular mechanisms of repair known to occur in human skin. We found that SAP underwent molecular self-assembly to form unique 3D structures that stably covered the surface of the wound, suggesting that this scaffold may serve as a viable wound dressing. We measured the rates of release of EGF from the SAP scaffold and determined that EGF was only released when the scaffold was in direct contact with the HSE. By measuring the length of the epithelial tongue during wound reepithelialization, we found that SAP scaffolds containing EGF accelerated the rate of wound coverage by 5 fold when compared to controls without scaffolds and by 3.5 fold when compared to the scaffold without EGF. In conclusion, our experiments demonstrated that biomaterials composed of a biofunctionalized peptidic scaffold have many properties that are well-suited for the treatment of cutaneous wounds including wound coverage, functionalization with bioactive molecules, localized growth factor release and activation of wound repair.
Primary sequence of ionic self-assembling peptide gels affects endothelial cell adhesion and capillary morphogenesis. J Biomed Mater Res A. 2008 Jan 9 [Epub ahead of print]
Sieminski AL, Semino CE, Gong H, Kamm RD.
Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139.
Appropriate choice of biomaterial supports is critical for the study of capillary morphogenesis in vitro as well as to support vascularization of engineered tissues invivo. Self-assembling peptides are a class of synthetic, ionic, oligopeptides that spontaneously assemble into gels with an ECM-like microarchitecture when exposed to salt. In this paper, the ability of four different self-assembling peptide gels to promote endothelial cell adhesion and capillary morphogenesis is explored. Human umbilical vein endothelial cells (HUVECs) were cultured within ionic self-assembling peptide family members, RAD16-I ((RADA)(4)), RAD16-II ((RARADADA)(2)), KFE-8 ((FKFE)(2)), or KLD-12 ((KLDL)(3)). HUVECs suspended in RAD16-I or RAD16-II gels elongated and formed interconnected capillary-like networks resembling in vivo capillaries, while they remained round and formed clusters within KFE-8 or KLD-12 gels. As HUVECs attach to RAD16-I and RAD16-II significantly better than the other peptides, these differences appear to be explained by differences in cell adhesion. Although adhesion likely occurs via bound adhesion proteins, there appears to be no difference in protein binding to the peptides investigated. Results indicate that, although these oligopeptides have similar mechanisms of self- assembly, their primary sequence can greatly affect cell adhesion. Additionally, a subset of these biomimetic ECM-like materials support capillary morphogenesis and thus may be useful for supporting vascularization. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2008.
Three-Dimensional Primary Hepatocyte Culture in Synthetic Self-Assembling Peptide Hydrogel. Tissue Eng. 2008 Jan 3 [Epub ahead of print]
Wang S, Nagrath D, Chen PC, Berthiaume F, Yarmush ML.
Center for Engineering in Medicine, Massachusetts General Hospital, Shriners Burn Hospital, and Harvard Medical School, Boston, Massachusetts., Present address: Biomedical Engineering Department, The City College of New York, CUNY, New York, New York.
Drug metabolism studies and liver tissue engineering necessitate stable hepatocyte cultures that express liver functions for a minimum of 4 days to 3 weeks. Current techniques, using different biomaterials and geometries, that maintain hepatocellular function in vitro exhibit a low cell density and functional capacity per unit volume. Herein we investigated a well-defined synthetic peptide that can self-assemble into three-dimensional interweaving nanofiber scaffolds to form a hydrogel, PuraMatrix, as a substrate for hepatocyte culture. Freshly isolated primary rat hepatocytes attached, migrated, and formed spheroids within 3 days after seeding on PuraMatrix. Hepatocytes expressed the apical membrane marker dipeptidyl peptidase IV at cell-cell contacts. Compared to the collagen sandwich, albumin and urea secretion on PuraMatrix were higher for the first week, and cytochrome P450IA1 activity was higher throughout the culture period. Mitochondrial membrane potential 1 day after seeding was higher on PuraMatrix than in the collagen sandwich, suggesting better preservation of the metabolic machinery. PuraMatrix and Matrigel showed similar albumin and urea production. PuraMatrix is an attractive system for generating hepatocyte spheroids that quickly restore liver functions after seeding. This system is also amenable to scale-up, which makes it suitable for in vitro toxicity, hepatocyte transplantation, and bioartificial liver development studies.
Compatibility of human fetal neural stem cells with hydrogel biomaterials in vitro. Brain Res. 2008 Jan 2;1187:42-51. Epub 2007 Oct 26.
Thonhoff JR, Lou DI, Jordan PM, Zhao X, Wu P.
Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555-0620, USA.
Stroke and spinal cord or brain injury often result in cavity formation. Stem cell transplantation in combination with tissue engineering has the potential to fill such a cavity and replace lost neurons. Several hydrogels containing unique features particularly suitable for the delicate nervous system were tested by determining whether these materials were compatible with fetal human neural stem cells (hNSCs) in terms of toxicity and ability to support stem cell differentiation in vitro. The hydrogels examined were pluronic F127 (PF127), Matrigel and PuraMatrix. We found that PF127, in a gelated (30%) form, was toxic to hNSCs, and Matrigel, in a gelated (1-50%) form, prevented hNSCs' normal capacity for neuronal differentiation. In contrast, PuraMatrix was the most optimal hydrogel for hNSCs, since it showed low toxicity when gelated (0.25%) and retained several crucial properties of hNSCs, including migration and neuronal differentiation. Further optimization and characterization of PuraMatrix is warranted to explore its full potential in assisting neural regeneration in vivo.
Cartilage tissue engineering using human auricular chondrocytes embedded in different hydrogel materials. J Biomed Mater Res A. 2006 Jul;78(1):1-11.
Yamaoka H, Asato H, Ogasawara T, Nishizawa S, Takahashi T, Nakatsuka T, Koshima I, Nakamura K, Kawaguchi H, Chung UI, Takato T, Hoshi K.
Department of Fujisoft ABC Cartilage and Bone Regeneration, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-0033, Japan.
To seek a suitable scaffold for cartilage tissue engineering, we compared various hydrogel materials originating from animals, plants, or synthetic peptides. Human auricular chondrocytes were embedded in atelopeptide collagen, alginate, or PuraMatrix, all of which are or will soon be clinically available. The chondrocytes in the atelopeptide collagen proliferated well, while the others showed no proliferation. A high-cell density culture within each hydrogel enhanced the expression of collagen type II mRNA, when compared with that without hydrogel. By stimulation with insulin and BMP-2, collagen type II and glycosaminoglycan were significantly accumulated within all hydrogels. Chondrocytes in the atelopeptide collagen showed high expression of beta1 integrin, seemingly promoting cell-matrix signaling. The N-cadherin expression was inhibited in the alginate, implying that decrease in cell-to-cell contacts may maintain chondrocyte activity. The matrix synthesis in PuraMatrix was less than that in others, while its Young's modulus was the lowest, suggesting a weakness in gelling ability and storage of cells and matrices. Considering biological effects and clinical availability, atelopeptide collagen may be accessible for clinical use. However, because synthetic peptides can control the risk of disease transmission and immunoreactivities, some improvement in gelling ability would provide a more useful hydrogel for ideal cartilage regeneration.
Self-assembling peptide nanofiber as a novel culture system for isolated porcine hepatocytes. Cell Transplant. 2006;15(10):921-7.
Navarro-Alvarez N, Soto-Gutierrez A, Rivas-Carrillo JD, Chen Y, Yamamoto T, Yuasa T, Misawa H, Takei J, Tanaka N, Kobayashi N.
Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan.
Freshly isolated porcine hepatocytes are a very attractive cell source in the cell-based therapies to treat liver failure because of unlimited availability. However, due to the loss of hepatocyte functions in vitro, there is a need to develop a functional culture system to keep the cells metabolically active. Here we compared the effect of a self-assembling peptide nanofiber (SAPNF) as an extracellular matrix (ECM) with collagen type I on hepatocyte metabolic and secretion activities following hepatocyte isolation. Isolated porcine hepatocytes were cultured in SAPNF and collagen type I. Morphological assessment at different time points was performed by using SEM and phase contrast microscope. Metabolic and secretion activities were comparatively performed in the groups, by means of ammonia, lidocaine, and diazepam as well as albumin. Hepatocytes cultured on SAPNF revealed a three-dimensional spheroidal formation, thus maintaining cell differentiation status during 2 weeks of culture. On the other hand, hepatocytes in collagen revealed a spread shape, and by day 14 no hepatocyte-like cells were observed, but cells with long shape were present, thus revealing a degree of dedifferentiation in collagen culture. Hepatocytes in SAPNF were capable of drug-metabolizing activities and albumin secretion in higher ratio than those cultured on collagen. The present work clearly demonstrates the usefulness of SAPNF for maintaining differentiated functions of porcine hepatocytes in culture.
PuraMatrix facilitates bone regeneration in bone defects of calvaria in mice. Cell Transplant. 2006;15(10):903-10.
Misawa H, Kobayashi N, Soto-Gutierrez A, Chen Y, Yoshida A, Rivas-Carrillo JD, Navarro-Alvarez N, Tanaka K, Miki A, Takei J, Ueda T, Tanaka M, Endo H, Tanaka N, Ozaki T.
Department of Orthopeadic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan.
Artificial bones have often used for bone regeneration due to their strength, but they cannot provide an adequate environment for cell penetration and settlement. We therefore attempted to explore various materials that may allow the cells to penetrate and engraft in bone defects. PuraMatrix is a self-assembling peptide scaffold that produces a nanoscale environment allowing both cellular penetration and engraftment. The objective of this study was to investigate the effect of PuraMatrix on bone regeneration in a mouse bone defect model of the calvaria. Matrigel was used as a control. The expression of bone-related genes (alkaline phosphatase, Runx2, and Osterix) in the PuraMatrix-injected bone defects was stronger than that in the Matrigel-injected defects. Soft X-ray radiographs revealed that bony bridges were clearly observed in the defects treated with PuraMatrix, but not in the Matrigel-treated defects. Notably, PuraMatrix treatment induced mature bone tissue while showing cortical bone medullary cavities. The area of newly formed bones at the site of the bone defects was 1.38-fold larger for PuraMatrix than Matrigel. The strength of the regenerated bone was 1.72-fold higher for PuraMatrix (146.0 g) than for Matrigel (84.7 g). The present study demonstrated that PuraMatrix injection favorably induced functional bone regeneration.
The Use of 3-D Culture in Peptide Hydrogel for Analysis of Discoidin Domain Receptor 1-Collagen Interaction. Cell Adhesion & Migration 2006; Col. 1 (2) 92-98.
Daizo Yoshida and Akira Teramoto.
Department of Neurosurgery, Nippon Medical School; Nippon Medical School, Tokyo, Japan.
The aim of this study is to examine a novel drop culture model using a biologically inspired self-assembling peptide: hydrogel (RAD16-I, also called PuraMatrix), which produces a nanoscale environment similar to native extracellular matrix (ECM) for a cell line weakly adherent to a plastic surface during cell culture. Our work investigates quantitatively analyzing discoidin domain receptor (DDR) 1-mediated protein interactions between collagen type I and matrix metalloproteinase (MMP)-2 or -9, as well as cell invasion, using, as a scaffold, PuraMatrix, a novel peptide hydrogel. Results demonstrate that the dynamic cell culture technique produced a highly stable reharvesting of cells throughout the constructs with HP-75, human pituitary adenoma cell line when compared to the traditional seeding methods. Secretion of MMP via collagen type I was observed quantitatively in the supernatant (EC50; MMP-2, 50.4 ng/ml: MMP-9, 57.6 ng/ml). In PuraMatrix gel impregnated with 50 ng/ml of collagen type I, transfection of the vector encoding full-length DDR1 or siRNA targeting DDR1 up- or down-regulated respectively secretion of MMP-2 and -9, and cell invasion. Our results show that incorporation of this peptide with each ECM component provides a more permissive environment to elucidate ECM to cell signal interaction.
Controlled delivery of PDGF-BB for myocardial protection using injectable self-assembling peptide nanofibers. J Clin Invest. 2006 Jan;116(1):237-48. Epub 2005 Dec 15.
Hsieh PC, Davis ME, Gannon J, MacGillivray C, Lee RT.
Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Endothelial cells can protect cardiomyocytes from injury, but the mechanism of this protection is incompletely described. Here we demonstrate that protection of cardiomyocytes by endothelial cells occurs through PDGF-BB signaling. PDGF-BB induced cardiomyocyte Akt phosphorylation in a time- and dose-dependent manner and prevented apoptosis via PI3K/Akt signaling. Using injectable self-assembling peptide nanofibers, which bound PDGF-BB in vitro, sustained delivery of PDGF-BB to the myocardium at the injected sites for 14 days was achieved. A blinded and randomized study in 96 rats showed that injecting nanofibers with PDGF-BB, but not nanofibers or PDGF-BB alone, decreased cardiomyocyte death and preserved systolic function after myocardial infarction. A separate blinded and randomized study in 52 rats showed that PDGF-BB delivered with nanofibers decreased infarct size after ischemia/reperfusion. PDGF-BB with nanofibers induced PDGFR-beta and Akt phosphorylation in cardiomyocytes in vivo. These data demonstrate that endothelial cells protect cardiomyocytes via PDGF-BB signaling and that this in vitro finding can be translated into an effective in vivo method of protecting myocardium after infarction. Furthermore, this study shows that injectable nanofibers allow precise and sustained delivery of proteins to the myocardium with potential therapeutic benefits.
Nano neuro knitting: peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision. Proc Natl Acad Sci U S A. 2006 Mar 28;103(13):5054-9. Epub 2006 Mar 20.
Ellis-Behnke RG, Liang YX, You SW, Tay DK, Zhang S, So KF, Schneider GE.
Department of Brain and Cognitive Science, Massachusetts Institute of Technology, 77 Massachusett Avenue, Cambridge, MA 02139-4307, USA.
Nanotechnology is often associated with materials fabrication, microelectronics, and microfluidics. Until now, the use of nanotechnology and molecular self assembly in biomedicine to repair injured brain structures has not been explored. To achieve axonal regeneration after injury in the CNS, several formidable barriers must be overcome, such as scar tissue formation after tissue injury, gaps in nervous tissue formed during phagocytosis of dying cells after injury, and the failure of many adult neurons to initiate axonal extension. Using the mammalian visual system as a model, we report that a designed self-assembling peptide nanofiber scaffold creates a permissive environment for axons not only to regenerate through the site of an acute injury but also to knit the brain tissue together. In experiments using a severed optic tract in the hamster, we show that regenerated axons reconnect to target tissues with sufficient density to promote functional return of vision, as evidenced by visually elicited orienting behavior. The peptide nanofiber scaffold not only represents a previously undiscovered nanobiomedical technology for tissue repair and restoration but also raises the possibility of effective treatment of CNS and other tissue or organ trauma.
Controlled delivery of PDGF-BB for myocardial protection using injectable self-assembling peptide nanofibers. J Clin Invest. 2006 Jan;116(1):237-48. Epub 2005 Dec 15.
Hsieh PC, Davis ME, Gannon J, MacGillivray C, Lee RT.
Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Endothelial cells can protect cardiomyocytes from injury, but the mechanism of this protection is incompletely described. Here we demonstrate that protection of cardiomyocytes by endothelial cells occurs through PDGF-BB signaling. PDGF-BB induced cardiomyocyte Akt phosphorylation in a time- and dose-dependent manner and prevented apoptosis via PI3K/Akt signaling. Using injectable self-assembling peptide nanofibers, which bound PDGF-BB in vitro, sustained delivery of PDGF-BB to the myocardium at the injected sites for 14 days was achieved. A blinded and randomized study in 96 rats showed that injecting nanofibers with PDGF-BB, but not nanofibers or PDGF-BB alone, decreased cardiomyocyte death and preserved systolic function after myocardial infarction. A separate blinded and randomized study in 52 rats showed that PDGF-BB delivered with nanofibers decreased infarct size after ischemia/reperfusion. PDGF-BB with nanofibers induced PDGFR-beta and Akt phosphorylation in cardiomyocytes in vivo. These data demonstrate that endothelial cells protect cardiomyocytes via PDGF-BB signaling and that this in vitro finding can be translated into an effective in vivo method of protecting myocardium after infarction. Furthermore, this study shows that injectable nanofibers allow precise and sustained delivery of proteins to the myocardium with potential therapeutic benefits.
Overexpression of the Epidermal Growth Factor Receptor
Confers Migratory Properties to Nonmigratory Postnatal
Neural Progenitors The Journal of Neuroscience, November 30, 2005 • 25(48):11092–11106
Adan Aguirre (1), Tilat A. Rizvi (2), Nancy Ratner (2), and Vittorio Gallo (1)
(1) Center for Neuroscience Research, Children’s Research Institute, Children’s National Medical Center, Washington, DC 20010, and
(2) Division of
Experimental Hematology, Department of Pediatrics, Cincinnati Children’s Hospital Research Foundation, University of Cincinnati College of Medicine,
Cincinnati, Ohio 45229
Approaches to successful cell transplantation therapies for the injured brain involve selecting the appropriate neural progenitor type and optimizing the efficiency of the cell engraftment. Here we show that epidermal growth factor receptor (EGFR) expression enhances postnatal neural progenitor migration in vitro and in vivo. Migratory NG2-expressing (NG2) progenitor cells of the postnatal subventricular zone (SVZ) express higher EGFR levels than nonmigratory, cortical NG2cells. The higher endogenous EGFR expression in SVZ NG2 cells is causally related with their migratory potential in vitro as well as in vivo after cell engraftment. EGFR overexpression in cortical NG2 cells by transient transfection converted these cells to a migratory phenotype in vitro and in vivo. Finally, cortical NG2 cells purified from a transgenic mouse in which the EGFR is overexpressed under the CNP promoter exhibited enhanced migratory capability. These findings reveal a new role for EGFR in the postnatal brain and open new avenues to optimize cell engraftment for brain repair.
Key words: CNP-EGFP mouse; cell transplantation; cell migration; rostral migratory stream; white matter; hippocampus; olfactory bulb
The enhancement of osteoblast growth and differentiation in vitro on a peptide hydrogel-polyHIPE polymer hybrid material Biomaterials 2005 Sep; 26(25):5198-208
Bokhari MA, Akay G, Zhang S, Birch MA.
School of Chemical Engineering and Advanced Materials, University of Newcastle, Newcastle upon Tyne, NE1 7RU, UK.
The objective of this study was to investigate the effect of combining two biomaterials on osteoblast proliferation, differentiation and mineralised matrix formation in vitro. The first biomaterial has a well-defined architecture and is known as PolyHIPE polymer (PHP). The second biomaterial is a biologically inspired self-assembling peptide hydrogel (RAD16-I, also called PuraMatrix) that produces a nanoscale environment similar to native extracellular matrix (ECM). Our work investigates the effect of combining RAD16-I with two types of PHP (HA (Hydroxyapatite)-PHP and H (Hydrophobic)-PHP) and evaluates effects on osteoblast growth and differentiation. Results demonstrated successful incorporation of RAD16-I into both types of PHP. Osteoblasts were observed to form multicellular layers on the combined biomaterial surface and also within the scaffold. Dynamic cell seeding and culturing techniques were compared to static seeding methods and produced a more even distribution of cells throughout the constructs. Cells were found to penetrate the scaffold to a maximum depth of 3 mm after 35 days in culture. There was a significant increase in cell number in H-PHP constructs coated with RAD16-I compared to H-PHP alone. Our results show that RAD16-I enhances osteoblast differentiation and indicates that the incorporation of this peptide provides a more permissive environment for osteoblast growth. We have developed a microcellular polymer containing a nanoscale environment to enhance cell: biomaterial interactions and promote osteoblast growth in vitro.
The
effect of functionalized self-assembling peptide scaffolds on human aortic
endothelial cell function Biomaterials 26
(2005) 3341–3351
Elsa Genové (a), Colette Shen (b), Shuguang Zhang (a,c), Carlos E. Semino (a,c)
(a) Center
for Biomedical Engineering, Massachusetts Institute of Technology,
77 Massachusetts Avenue, Cambridge, MA 02139, USA
(b) Harvard
University, Cambridge, MA, 02138, USA
(c) Biotechnology Process Engineering
Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Abstract
A class of designed self-assembling peptide
nano.ber scaffolds with more than 99% water content has been shown to
be a goodbiological material for cell
culture. Here, we report the functionalization of one of these peptide
scaffolds, by direct solid phase synthesis extension at the amino terminal
with three shortsequencemotifs. These motifs
are present in two major protein components of the basement membrane,
laminin 1 (YIGSR, RYVVLPR) and collagen IV (TAGSCLRKFSTM). These motifs
have been previously shown to promote speci.c biological activities including
endothelial cell adhesion, spreading, and tubular formation. Therefore,
the generic functionalized peptide developed was AcN–X–GG-RAD16–CONH2 with each motif represented
by ‘‘X’’. We show in this work that these tailor-made
peptide scaffolds enhance the formation of con.uent cell monolayers of
human aortic endothelial cells (HAEC) in culture. Moreover, additional
assays designed to evaluate endothelial cell function showed that HAEC
monolayers obtained on these scaffolds not only maintained LDL uptake
activity but also enhanced nitric oxide release and elevated laminin
1 and collagen IV deposition. These results suggest that this new scaffold
provide a better physiological substrate for endothelial cell culture
and suggest its further application for biomedical research, cancer biology
and regenerative biology.
Keywords: Self-assembly; Biomimetic material; Cell proliferation; Extracellular matrix; Endotheliar monolayer
Self-assembling
short oligopeptides and the promotion of angiogenesis Biomaterials
26 (2005) 4837–4846
Daria A. Narmoneva (a, b), Olumuyiwa Oni (b), Alisha L. Sieminski (b), Shugang Zhang (b), Jonathan P. Gertler (c), Roger D. Kamm (b), Richard T. Lee (a,b)
(a) Cardiovascular Division, Brigham and Women’s Hospital & Harvard
Medical School, Boston, MA 02139, USA
(b) Division of Biological Engineering, Massachusetts Institute of Technology,
Cambridge, MA 02139, USA
(c) Vascular Surgery Research Laboratory, Division of Vascular Surgery, Massachusetts
General Hospital and Harvard Medical School, Boston, MA 02114, USA
Abstract
Because an adequate blood supply to and within tissues is an essential factor
for successful tissue regeneration, promoting a
functional microvasculature is a crucial factor for biomaterials. In this study,
we demonstrate that short self-assembling peptides
form scaffolds that provide an angiogenic environment promoting long-term cell
survival and capillary-like network formation in
three-dimensional cultures of human microvascular endothelial cells. Our data
show that, in contrast to collagen type I, the peptide
scaffold inhibits endothelial cell apoptosis in the absence of added angiogenic
factors, accompanied by enhanced gene expression of
the angiogenic factor VEGF. In addition, our results suggest that the process
of capillary-like network formation and the size and
spatial organization of cell networks may be controlled through manipulation
of the scaffold properties, with a more rigid scaffold
promoting extended structures with a larger inter-structure distance, as compared
with more dense structures of smaller size
observed in a more compliant scaffold. These .ndings indicate that self-assembling
peptide scaffolds have potential for engineering
vascularized tissues with control over angiogenic processes. Since these peptides
can be modi.ed in many ways, they may be
uniquely valuable in regeneration of vascularized tissues.
Keywords: Angiogenesis; Endothelial cell; Peptide; Scaffold; Myocardial regeneration
Mauck RL,
Helm JM, Tuan RS
Cartilage
Biology and Orthopaedics Branch, National
Institute of Arthritis and Musculoskeletal and Skin Disorders, National
Institutes of Health, Bethesda,
MD 20892
The goals of the present study were to first determine if chondrogenic differentiation of human MSCs was possible in a self-assembling peptide hydrogel (Puramatrix, (REDA)4, 3DM, Inc.), secondly to compare the level of chondrogenic differentiation that occurs in this hydrogel to that which occurs under standard pellet culture, and thirdly to determine if chondrogenesis within this matrix will lead to the production of functional mechanical properties with long term culture.
Preliminary studies using three separate human donor MSCs embedded in Puramatrix demonstrated a pronounced chondrogenic differentiation after 21 days in the presence of TGF-â3 (Figure 1). Beads became increasingly opaque and palpably stiffer over the duration of the experiment with pronounced induction of chondrogenic mRNA transcripts (Figure 1). Histology showed an even distribution of cells throughout the bead and abundant proteoglycan and collagen deposition (Figure 2). In subsequent studies with four additional human donor MSCs, comparison of mRNA transcript levels using real time PCR demonstrated a marked upregulation of aggrecan and type II collagen transcript levels with the addition of TGF-â3 in both traditional cell pellet culture and in Puramatrix beads (Figure 3). Interestingly, aggrecan transcript levels in Puramatrix beads were similar to that of cell pellets for different donors (0.5-4 fold ratio between (+) conditions) while for type II collagen there was a marked increase in transcript levels in Puramatrix beads compared to cell pellets for every donor (5-38 fold). Puramatrix disks seeded with human MSCs (from donor 3) cultured in chondrogenic medium with TGF-â3 showed a marked increase in mechanical properties between days 21 and 42 with accompanying increases in GAG content (Figure 4, p<0.01, day 21 vs. day 42 for all properties).
Michael E. Davis, PhD; J.P. Michael Motion, BS; Daria A. Narmoneva, PhD; Tomosaburo Takahashi, MD, PhD; Daihiko Hakuno, MD, PhD; Roger D. Kamm, PhD; Shuguang Zhang, PhD; Richard T. Lee, MD
Background—Promoting survival of transplanted cells or endogenous precursors is an important goal. We hypothesized that a novel approach to promote vascularization would be to create injectable microenvironments within the myocardium that recruit endothelial cells and promote their survival and organization.
Methods and Results—In this study we demonstrate that self-assembling peptides can be injected and that the resulting nanofiber microenvironments are readily detectable within the myocardium. Furthermore, the self-assembling peptide nanofiber microenvironments recruit progenitor cells that express endothelial markers, as determined by staining with isolectin and for the endothelial-specific protein platelet– endothelial cell adhesion molecule-1. Vascular smooth muscle cells are recruited to the microenvironment and appear to form functional vascular structures. After the endothelial cell population, cells that express -sarcomeric actin and the transcription factor Nkx2.5 infiltrate the peptide microenvironment. When exogenous donor green fluorescent protein–positive neonatal cardiomyocytes were injected with the self-assembling peptides, transplanted cardiomyocytes in the peptide microenvironment survived and also augmented endogenous cell recruitment.
Conclusions—These experiments demonstrate that self-assembling peptides can create nanofiber microenvironments in the myocardium and that these microenvironments promote vascular cell recruitment. Because these peptide nanofibers may be modified in a variety of ways, this approach may enable injectable tissue regeneration strategies. (Circulation. 2005; 111:442-450.)
Key Words: tissue engineering, microenvironment, regeneration
Endothelial
cells promote cardiac myocyte survival and spatial reorganization: implications
for cardiac regeneration. Circulation. 2004 Aug 24;110(8):962-8.
Narmoneva DA, Vukmirovic R, Davis ME, Kamm RD, Lee RT. Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, USA.
Endothelial-cardiac myocyte (CM) interactions play a key role in regulating cardiac function, but the role of these interactions in CM survival is unknown. This study tested the hypothesis that endothelial cells (ECs) promote CM survival and enhance spatial organization in a 3-dimensional configuration. METHODS AND RESULTS: Microvascular ECs and neonatal CMs were seeded on PEPTIDE HYDROGELS in 1 of 3 experimental configurations: CMs alone, CMs mixed with ECs (coculture), or CMs seeded on preformed EC networks (prevascularized). Capillary-like networks formed by ECs promoted marked CM reorganization along the EC structures, in contrast to limited organization of CMs cultured alone. The presence of ECs markedly inhibited CM apoptosis and necrosis at all time points. In addition, CMs on preformed EC networks resulted in significantly less CM apoptosis and necrosis compared with simultaneous EC-CM seeding (P<0.01, ANOVA). Furthermore, ECs promoted synchronized contraction of CMs as well as connexin 43 expression. CONCLUSIONS: These results provide direct evidence for a novel role of endothelium in survival and organization of nearby CMs. Successful strategies for cardiac regeneration may therefore depend on establishing functional CM-endothelium interactions.
Entrapment
of migrating hippocampal neural cells in three-dimensional peptide nanofiber
scaffold. Tissue Engineering. 2004 Mar-Apr;10(3-4):643-55.
Semino CE, Kasahara J, Hayashi Y, Zhang S. Center for Biomedical
Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts,
USA.
Isolation and expansion of self-renewing neural cells ex
vivo are required for neural tissue repair in regenerative medicine. Neurogenesis
occurs in restricted areas of postnatal mammalian brain including dentate
gyrus and subventricular zone. We developed a simple method to entrap migrating
neural cells (potential neuroprogenitors) from postnatal hippocampal organotypic
cultures in three-dimensional (3-D) peptide nanofiber scaffolds. A few hours
after placing the hippocampal slices in culture, cell proliferation activity
at the "interface zone" between the tissue slice and the membrane
culture surface was observed. Pulse-chase experiments using 5-bromodeoxyuridine
(BrdU), which measures mitotic activity, showed that a number of cells incorporated
BrdU at the interface zone. The number of BrdU(+) cells increased exponentially
during the first 3 days of exposure to the label. The BrdU(+) cells also
stained positive for glial fibrillary acidic protein (2.2 +/- 0.5%), a marker
for astroglia; and for betaIII tubulin (7.3 +/- 2.8%) and nestin (2.7 +/-
0.9%), markers for neural progenitors. When hippocampal slices were cultured
on a peptide nanofiber scaffold layer (~500 microm thick), a more extended
interface zone between each tissue slice and the scaffold was formed. Moreover,
the migrating BrdU(+) cell population entrapped in the 3-D peptide scaffold
was readily isolated by mechanically disrupting the scaffold and then used
for conventional 2-D culture systems for further studies. This simple method
may be useful not only in developing technology for neural progenitor cell
isolation and enrichment in vitro, but also for expanding cells for cell-based
therapies of regenerative medicine.
Effects
of dynamic compressive loading on chondrocyte biosynthesis in self-assembling
peptide scaffolds. Journal of Biomechanics (May 2004)
Kisiday JD, Jin M, DiMicco MA, Kurz B, Grodzinsky AJ.; Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA, USA.
Dynamic mechanical loading has been reported to affect chondrocyte biosynthesis in both cartilage explant and chondrocyte-seeded constructs. In this study, the effects of dynamic compression on chondrocyte-seeded peptide hydrogels were analyzed for extracellular matrix synthesis and retention over long-term culture. Initial studies were conducted with chondrocyte-seeded agarose hydrogels to explore the effects of various non-continuous loading protocols on chondrocyte biosynthesis. An optimized alternate day loading protocol was identified that increased proteoglycan (PG) synthesis over control cultures maintained in free-swelling conditions. When applied to chondrocyte-seeded peptide hydrogels, alternate day loading stimulated PG synthesis up to two-fold higher than that in free-swelling cultures. While dynamic compression also increased PG loss to the medium throughout the 39-day time course, total PG accumulation in the scaffold was significantly higher than in controls after 16 and 39 days of loading, resulting in an increase in the equilibrium and dynamic compressive stiffness of the constructs. Viable cell densities of dynamically compressed cultures differed from free-swelling controls by less than 20%, demonstrating that changes in PG synthesis were due to an increase in the average biosynthesis per viable cell. Protein synthesis was not greatly affected by loading, demonstrating that dynamic compression differentially regulated the synthesis of PGs. Taken together, these results demonstrate the potential of dynamic compression for stimulating PG synthesis and accumulation for applications to in vitro culture of tissue engineered constructs prior to implantation.
Functional
differentiation of hepatocyte-like spheroid structures from putative liver
progenitor cells in three dimensional peptide scaffolds. Differentiation
(Dec 2003)
Semino CE, Kasahara J, Hayashi Y, Zhang S.; Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Isolation and expansion of self-renewing neural cells ex vivo are required for neural tissue repair in regenerative medicine. Neurogenesis occurs in restricted areas of postnatal mammalian brain including dentate gyrus and subventricular zone. We developed a simple method to entrap migrating neural cells (potential neuroprogenitors) from postnatal hippocampal organotypic cultures in three-dimensional (3-D) peptide nanofiber scaffolds. A few hours after placing the hippocampal slices in culture, cell proliferation activity at the "interface zone" between the tissue slice and the membrane culture surface was observed. Pulse-chase experiments using 5-bromodeoxyuridine (BrdU), which measures mitotic activity, showed that a number of cells incorporated BrdU at the interface zone. The number of BrdU(+) cells increased exponentially during the first 3 days of exposure to the label. The BrdU(+) cells also stained positive for glial fibrillary acidic protein (2.2 +/- 0.5%), a marker for astroglia; and for betaIII tubulin (7.3 +/- 2.8%) and nestin (2.7 +/- 0.9%), markers for neural progenitors. When hippocampal slices were cultured on a peptide nanofiber scaffold layer (~500 microm thick), a more extended interface zone between each tissue slice and the scaffold was formed. Moreover, the migrating BrdU(+) cell population entrapped in the 3-D peptide scaffold was readily isolated by mechanically disrupting the scaffold and then used for conventional 2-D culture systems for further studies. This simple method may be useful not only in developing technology for neural progenitor cell isolation and enrichment in vitro, but also for expanding cells for cell-based therapies of regenerative medicine.
Self-assembling peptide hydrogel
fosters chondrocyte extracellular matrix production and cell division: Implications
for cartilage tissue repair. Proceedings of the National Academy of Science
(July 2002)
Kisiday J, Jin M, Kurz B, Hung H, Semino C, Zhang S, Grodzinsky AJ.; Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA, USA.
Emerging medical technologies for effective and lasting repair of articular cartilage include delivery of cells or cell-seeded scaffolds to a defect site to initiate de novo tissue regeneration. Biocompatible scaffolds assist in providing a template for cell distribution and extracellular matrix (ECM) accumulation in a three-dimensional geometry. A major challenge in choosing an appropriate scaffold for cartilage repair is the identification of a material that can simultaneously stimulate high rates of cell division and high rates of cell synthesis of phenotypically specific ECM macromolecules until repair evolves into steady-state tissue maintenance. We have devised a self-assembling peptide hydrogel scaffold for cartilage repair and developed a method to encapsulate chondrocytes within the peptide hydrogel. During 4 weeks of culture in vitro, chondrocytes seeded within the peptide hydrogel retained their morphology and developed a cartilage-like ECM rich in proteoglycans and type II collagen, indicative of a stable chondrocyte phenotype. Time-dependent accumulation of this ECM was paralleled by increases in material stiffness, indicative of deposition of mechanically functional neo-tissue. Taken together, these results demonstrate the potential of a self-assembling peptide hydrogel as a scaffold for the synthesis and accumulation of a true cartilage-like ECM within a three-dimensional cell culture for cartilage tissue repair.
Control of self-assembling oligopeptide matrix formation through systematic variation of amino acid sequence. Biomaterials (Jan 2002)
Caplan MR, Schwartzfarb EM, Zhang S, Kamm RD, Lauffenburger DA. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
In order to elucidate design principles for biocompatible materials that can be created by in situ transformation from self-assembling oligopeptides, we investigate a class of oligopeptides that can self-assemble in salt solutions to form three-dimensional matrices. This class of peptides possesses a repeated sequence of amino acid residues with the type: hydrophobic/negatively-charged/hydrophobic/positively-charged. We systematically vary three chief aspects of this sequence type: (1) the hydrophobic side chains: (2) the charged side-chains: and (3) the number of repeats. Employing a rheometric assay to judge matrix formation, we determine the critical concentration of NaCl salt solution required to drive transformation from viscous state to gel state. We find that increasing side-chain hydrophobicity decreases the critical salt concentration in accord with our previous validation of DLVO theory for explaining this self-assembly phenomenon Caplan et al. (Biomacromolecules 1 (2000) 627). Further, we find that increasing the number of repeats yields a biphasic dependence-first decreasing, then increasing, the critical salt concentration. We believe that this result is likely due to an unequal competition between a greater hydrophobic (favorable) effect and a greater entropic (unfavorable) effect as the peptide length is increased. Finally, we find that we can use this understanding to rationally alter the charged side-chains to create a self-assembling oligopeptide sequence that at pH 7 remains viscous in the absence of salt but gels in the presence of physiological salt concentrations, a highly useful property for technological applications.
Extensive neurite outgrowth and active
synapse formation on self-assembling peptide scaffold. Proceedings of
the National Academy of Science (June 2000)
Holmes TC, de Lacalle S, Su X, Liu G, Rich A, Zhang S.; Center for Biomedical Engineering, Department of Biology, Center for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA, USA.
A new type of self-assembling peptide (sapeptide) scaffolds that serve as substrates for neurite outgrowth and synapse formation is described. These peptide-based scaffolds are amenable to molecular design by using chemical or biotechnological syntheses. They can be tailored to a variety of applications. The sapeptide scaffolds are formed through the spontaneous assembly of ionic self-complementary beta-sheet oligopeptides under physiological conditions, producing a hydrogel material. The scaffolds can support neuronal cell attachment and differentiation as well as extensive neurite outgrowth. Furthermore, they are permissive substrates for functional synapse formation between the attached neurons. That primary rat neurons form active synapses on such scaffold surfaces in situ suggests these scaffolds could be useful for tissue engineering applications. The buoyant sapeptide scaffolds with attached cells in culture can be transported readily from one environment to another. Furthermore, these peptides did not elicit a measurable immune response or tissue inflammation when introduced into animals. These biological materials created through molecular design and self assembly may be developed as a biologically compatible scaffold for tissue repair and tissue engineering.
Biological surface engineering: a simple system for cell pattern formation. Biomaterials. (Jul 1999)
Zhang S, Yan L, Altman M, Lassle M, Nugent H, Frankel F, Lauffenburger DA, Whitesides GM, Rich A.; Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Biological surface engineering using synthetic biological materials has a great potential for advances in our understanding of complex biological phenomena. We developed a simple system to engineer biologically relevant surfaces using a combination of self-assembling oligopeptide monolayers and microcontact printing (muCP). We designed and synthesized two oligopeptides containing a cell adhesion motif (RADS)n (n = 2 and 3) at the N-terminus, followed by an oligo(alanine) linker and a cysteine residue at the C-terminus. The thiol group of cysteine allows the oligopeptides to attach covalently onto a gold-coated surface to form monolayers. We then microfabricated a variety of surface patterns using the cell adhesion peptides in combination with hexa-ethylene glycol thiolate which resist non-specific adsorption of proteins and cells. The resulting patterns consist of areas either supporting or inhibiting cell adhesion, thus they are capable of aligning cells in a well-defined manner, leading to specific cell array and pattern formations.
Mechancial properties of
a self-assembling ogliopeptide matrix. Journal of Biomaterials Science,
Polymer Edition (1998)
Leon EJ, Verma N, Zhang S, Lauffenburger DA, Kamm RD. Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge MA, USA.
We have begun studies of a novel type of biomaterial derived from a recently-discovered class of ionic self-complementary oligopeptides. These short peptides (typically 8, 16, 24, or 32 amino acid residues with internally-repeating sequences) self-assemble in aqueous salt solution into three-dimensional matrices capable of favorable interactions with cells, and offer promise for useful bioengineering design based on rational changes in sequence. In this paper we present preliminary results on mechanical properties, combining experimental and theoretical approaches, of one particular example of these peptide materials, EFK8. The static elastic modulus was measured using an apparatus designed to allow sample fabrication and mechanical testing in the same system with the sample in aqueous solution. The material microstructure was examined by SEM and the measurements interpreted with the aid of a model for cellular solids. Values for the elastic modulus increased from 1.59 +/- 0.06 to 14.7 +/- 1.0 kPa for peptide concentrations increasing from 2.7 to 10 mg ml-1. SEM photographs showed the microstructure to consist of a relatively homogeneous lattice with fiber thickness of 10-30 nm independent of peptide concentration, but with fiber density increasing with peptide concentration. This behavior is consistent with scaling predictions from the cellular solids model and yields an estimate for the individual fiber elastic modulus in the range of 1-20 MPa. We therefore have provided some initial physical principles for guiding improvement of the mechanical properties of these new materials.
Self-complementary oligopeptide matrices
support mammalian cell attachment. Biomaterials (Dec 1995)
Zhang S, Holmes TC, DiPersio CM, Hynes RO, Su X, Rich A; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
A new class of ionic self-complementary oligopeptides is described. These oligopeptides consist of regular repeats of alternating ionic hydrophilic and hydrophobic amino acids and associate to form stable beta-sheet structures in water. The addition of buffers containing millimolar amounts of monovalent salts or the transfer of a peptide solution into physiological solutions results in the spontaneous assembly of the oligopeptides into a stable, macroscopic membranous matrix. The matrix is composed of ordered filaments which form porous enclosures. A variety of mammalian cell types are able to attach to both membranous matrices. These matrices provide a novel experimental system for analysing mechanisms of in vitro cell attachment and may have applications in in vivo studies of tissue regeneration, tissue transplantation and would healing.
Conference Presentations & Abstracts
8TH International Congress of the Cell Transplant Society (May, 2006)
PuraMatrix facilitates osteogenic differentiation of mouse ES cells in vitro.
Haruo Misawa*, Naoya Kobayashi, Yan Chin, Matsato Tanaka, Aki Yoshida, Rie Tokunou, Hirosuke Endou, Toshifumi Ozaki
Okayama University Graduate School of Medicine, Department of Orthopaedic Surgery, Japan
Objective: Puramatrix is the self-assembling hydro- peptide gel formed with peptide nanofibers. The RAD arrangement of Puramatrix work as an extra-cellular matrix. It makes three-dimensional cellular cultivation possible by that three-dimensional structure. Puramatrix has reported it promotes the differentiation of hepatocyts, neurocyts, and so on. There is Matrigel as a similar product. Matrigel has an abundance of extra-cellular matrixes such as type four collagen and Laminin, and transcription factors such as EGF and PDGF, and it promotes differentiations of many cells. We compared the effects of Puramatrix and Matrigel in the differentiation from the ES cells to the osteoblasts.
Material and Method: We used mouse ES cells. The differentiation to the osteoblasts went by the medium containing dexamethasone, glycerophosphate, and ascorbic acid. Cells were cultivated on the PTE plate and the gels of Puramatrix and Matrigel. RNA was extracted with time, and the expression of bone-related genes were evaluated by PCR. A cellular form was observed by scanning electron microscope.
Result: In the comparing by PCR, both of Puramatrix and Matrigel have higher expressions of the bone-related genes than that of the PTE plate. The gene expressions of Puramatrix were equally close to that of Matrigel. The good embryoid body formations were observed on both of Puramatrix and matrigel by scanning electron microscope. The stitch of nanofibers was seen with Puramatrix, and cells formed the three-dimensional structure inside of the stitch.
Conclusion: Puramatrix has the effect on promoting the differentiation from the ES cells to the osteoblasts nearly to Matrigel in vitro. Puramatrix is not biologic origin, and there is no danger such as zoonosis in it. It is a very favorable point for clinical use. Puramatrix is a very useful product for regeneration medicine.
AMERICAN INSTITUTE OF CHEMICAL ENGINEERS 2005 ANNUAL MEETING (November, 2005)
Long Term 3D Primary Hepatocyte Culture in Nano-Scaffold Hydrogel for Bioartificial Liver
Sihong Wang, Deepak Nagrath, Francois Berthiaume, and Martin L. Yarmush.
Massachusetts General Hospital/Shriners Burn Hospital/ Harvard Medical School, 51 Blossom Street, Boston, MA 02114
Extracorporeal bioartificial liver devices (BAL) have been in the development to help recovery from acute liver failure and provide a bridge to liver transplantation. One challenge of BAL is to maintain primary isolated hepatocytes healthy and fully functional. The most common culture method for hepatocytes is collagen double gel sandwich culture (it stabilizes many liver-specific functions). However, there is a limitation in scale up in this method because of the restriction of low surface cell density. Matrigel is another commonly used method, however, it is extracted from animals and hence may have issue of immune rejection. In this paper, we have investigated long term primary hepatocyte culture using a peptide based synthetic self- assembling nanostructure hydrogel. It has an average pore size of 50~200nm and promotes cell attachment. The enlarged cell growth area in 3D makes it possible to increase the cell density of hepatocytes in bioartificial liver thus increasing the hepatic function. Our experimental results show that hepatocytes attached to 3 dimensional nanofibers after seeding and migrated to form stable hepatocyte spheroids within 5 to 6 days. Albumin and urea functions of clustered hepatocytes were similar to the collagen sandwich configuration. Moreover, we observed an improved cytochrome P450 function of hepatocytes in this hydrogel as compared to collagen sandwich configuration. Furthermore, because of its bio-compatibility, and 3D culture conditions the peptide based scaffold culture method may be of immense use in the area of hepatocyte transplantation.
American Society for Cell Biology (ASCB) Annual Meeting (December, 2005)
Engineered 3D Models to Study Force-Dependent Mammary Morphogenesis and Malignancy
J. B. Leach,1 J. L. Leight,2 K. R. Johnson,2 N. Zahir,2 M. J. Paszek, 2 A. Sieminski,3 L. Spirio,4 J. Y. Wong,5 V. M. Weaver6
1Chemical & Biochemical Engineering, University of Maryland, Baltimore County, Baltimore, MD, 2Bioengineering, University of Pennsylvania, Philadelphia, PA, 3Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA, 43DM, Inc., Boston, MA, 5Biomedical Engineering, Boston University, Boston, MA, 6Department of Pathology and Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA
Epithelial morphogenesis proceeds within a three dimensional (3D) tissue in which soluble, cellular and physical cues operate to direct tissue form and function. Although we understand much about the role of soluble factors and cell interactions in tissue homeostasis and tumorigenesis, we know little about the role of spatial organization and mechanical force in these processes. Key to understanding tissue behavior is the availability of tractable model systems in which biochemical, spatial and mechanical variables can be precisely and independently manipulated. Here we report the development, analysis and morphogenetic assessment of various extracellular matrix (ECM) 2D and 3D systems for the study of mammary epithelial cell (MEC) behavior in culture including: collagen I/reconstituted basement membrane gels (COL/rBM-gel), rBM-crosslinked polyacrylamide-gels (rBM- PA-gels), self-assembling peptide/rBM-gels (peptide/rBM-gel), and peptide-conjugated PEG-gels (PEG-Conj-gels). Previously we have demonstrated that MEC morphogenesis can be recapitulated using rBM- gels, COL/rBM-gels, and rBM-PA-gels with elastic moduli similar to that of the normal mammary gland. However, rBM-gels and COL/rBM-gels cannot model ECMs with an elastic modulus similar to mammary tumors in vivo (5,000 Pa), and rBM-PA-gels are not compatible for 3D embedment and in vivo studies. These limitations demonstrate the need for biocompatible synthetic ECM model systems whose mechanical properties are easily manipulated; hence we are exploring peptide/rBM- gels and PEG-Conj-gels. Thus far we have found that 3D peptide/rBM- gels support mammosphere formation, however they cannot be remodeled by embedded MECs. Therefore, laminin peptide PEG-Conj-gels with collagenase-digestible-peptides comprise the optimal model ECM system for further development. Accordingly, experiments in which the chemistry, spatial organization and EM of the PEG-Conj-gels and their variants are underway and a comprehensive analysis of the effect of matrix stiffness on normal and malignant MEC behavior in 2D versus 3D are in progress. (Supported by: W81XWH-05-1-330 to VMW and JYW).
American Society for Cell Biology (ASCB) Annual Meeting (December, 2005)
Microenvironmental Regulators of Metastasis: Role of Matrix- and Mechanotransduction in Control of MT1-MMP Expression.
M. Barbolina,1 E. Ariztia,2 M. S. Stack1
1Cell and Molecular Biology, Northwestern University, Chicago, IL, 2Obstetrics and Gynecology, New York University Medical Center, New York, NY
Epithelial ovarian carcinoma is the leading cause of death from gynecologic malignancy. Due to difficulties diagnosing ovarian cancer in early stages, most patients present when metastasis had already occurred. Thus, it is important to understand mechanisms of invasion and develop strategies to prevent tumor cells from spreading. The metastatic phenotype is characterized by disruption of cell-cell contacts and loss of extracellular matrix (ECM) constraints due to upregulation of ECM-degrading matrix metalloproteinases (MMP) and alterations in matrix- and mechano-transduction. We previously showed that metastatic ovarian cancer cells interact with interstitial collagens via a2ß1- and a3ß1-integrins, resulting in increased expression of membrane type 1-MMP (MT1-MMP). The goal of our research was to establish the role of epigenetic factors, particularly matrix status, in the transcriptional regulation MT1-MMP. We used real-time RT-PCR, western blotting and zymography to monitor changes in MT1-MMP and MMP2 levels in ovarian cancer cell lines cultured a) on 2D vs 3D collagen I; b) in a synthetic 3D Hydrogel (BD PuraMatrix); c) under conditions of static strain. Our results indicate an upregulation of MT1-MMP protein and activity following 3D collagen culture or static strain. Corresponding to the increased protein levels, an 8-fold increase in MT1-MMP mRNA was detected following 4 h of collagen culture using RT-RT-PCR. Upregulation of the transcription factors Egr-1 and -2 (20- and 3-fold, respectively) was observed at 2 h of collagen culture, suggesting that these factors participate in transcriptional control of MT1-MMP. These data implicate signal transduction through collagen binding integrins in control of MT1-MMP expression. As we have previously demonstrated that MT1-MMP co- localizes with a2ß1 and a3ß1 integrins at the cell-matrix interface, these results provide additional data in support of the hypothesis that matrix status influences matrix degrading potential.
American Society for Cell Biology (ASCB) Annual Meeting (December, 2005)
Robust Differentiation of PC12 Cells and ES-D3 Murine Embryonic Stem Cells on Substrates Coated with Photo Reagents.
T. J. Naqvi, G. W. Opperman, M. A. Lodhi
Diagnostics and Drug Discovery, SurModics, Inc., Eden Prairie, MN
In the past several years there has been a paradigm shift in developing synthetic biomaterials to grow cells or harvest cell products, primarily for applications in tissue culture, tissue engineering and regenerative medicine. For in vitro applications, physical and chemical properties as well as the structure of the substrate have been modified to closely mimic the natural microenvironment found in vivo. Cell attachment, proliferation, differentiation and cell-cell interaction are greatly affected by these physical and chemical properties of the substrate. We designed several experiments to test the effect of surface properties on the differentiation of PC12 and ES-D3 cells. PuraMatrix™ and Matrigel™ were used as substrates in addition to the substrates coated with photo-reagents containing 3-aminopropylmethacrylate (APMA), polyethylenimine (PEI), fibronectin, laminin, collagen and RGD peptides.
Society for Neuroscience Annual Conference (2005)
NEURONAL-GLIAL INTERACTIONS IN A TISSUE-ENGINEERED THREE-DIMENSIONAL ARCHITECTURE USING SYNTHETIC NANOFIBER SCAFFOLDS OF PURAMATRIX PEPTIDE HYDROGEL
A.C.Shivachar 1. Dept. Pharmaceutical Sci., 2. Col. of Pharmacy, Texas Southern Univ., Houston, TX, USA
Advances in tissue-engineering technology has shown that nanometer- sized peptide scaffolds provide a suitable support for in vitro cell growth and differentiation in a 3-dimensional(3-D)architecture. The peptide scaffolds provide a defined cell culture condition for cell migration, nutrient diffusion, and cell harvesting. The purpose of this study was to develop and characterize a novel three-dimensional (3-D), neuronal-glial
Self-assembling peptides and proteins for nanotechnological applications. Curr Opin Struct Biol. 2004 Aug;14(4):480-6.
Rajagopal K, Schneider JP.
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA. Photolithography enables the precise construction of nanodevices in two-dimensional formats. However, self-assembly of designed molecules serves as an alternative for the construction of three-dimensional nanoscale systems and is particularly appealing in that material properties can potentially be engineered at the molecular level. Peptides and proteins hold promise as building blocks for self- assembled systems because of their exquisite three-dimensional structures and evolutionarily fine-tuned functions.
Hydrogels:
Let Wet or Die. Nature Materials (Jan 2004)
Zhang S.; Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
New self-assembling peptide hydrogels provide a stable aqueous environment in which to study proteins, their structure and their interactions. These hydrogels allow the proper three dimensional conformation and folding, with applications in DNA and protein microarrays, proteomics, cell surface signaling and many applications involving the study of proteins in physiological-like microenvironments..
Fabrication
of novel biomaterials through molecular self-assembly Focus on Nanobiotechnology.
Nature Biotechnology (Oct 2003)
Zhang S.; Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Two complementary strategies can be used in the fabrication of molecular biomaterials. In the 'top-down' approach, biomaterials are generated by stripping down a complex entity into its component parts (for example, paring a virus particle down to its capsid to form a viral cage). This contrasts with the 'bottom-up' approach, in which materials are assembled molecule by molecule (and in some cases even atom by atom) to produce novel supramolecular architectures. The latter approach is likely to become an integral part of nanomaterials manufacture and requires a deep understanding of individual molecular building blocks and their structures, assembly properties and dynamic behaviors. Two key elements in molecular fabrication are chemical complementarity and structural compatibility, both of which confer the weak and noncovalent interactions that bind building blocks together during self-assembly. Using natural processes as a guide, substantial advances have been achieved at the interface of nanomaterials and biology, including the fabrication of nanofiber materials for three-dimensional cell culture and tissue engineering, the assembly of peptide or protein nanotubes and helical ribbons, the creation of living microlenses, the synthesis of metal nanowires on DNA templates, the fabrication of peptide, protein and lipid scaffolds, the assembly of electronic materials by bacterial phage selection, and the use of radiofrequency to regulate molecular behaviors.
Building
from the bottom up MaterialsToday (May 2003)
Zhang S.; Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Emerging biological materials through
molecular self-assembly. Biotechnol Adv. (2002 Dec)
Zhang S.; Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Understanding of new materials at the molecular level has become increasingly critical for a new generation of nanomaterials for nanotechnology, namely, the design, synthesis and fabrication of nanodevices at the molecular scale. New technology through molecular self-assembly as a fabrication tool will become tremendously important in the coming decades. Basic engineering principles for microfabrication can be learned by understanding the molecular self-assembly phenomena. Self-assembly phenomenon is ubiquitous in nature. The key elements in molecular self-assembly are chemical complementarity and structural compatibility through noncovalent interactions. We have defined the path to understand these principles. Numerous self-assembling systems have been developed ranging from models to the study of protein folding and protein conformational diseases, to molecular electronics, surface engineering, and nanotechnology. Several distinctive types of self-assembling peptide systems have been developed. Type I, "molecular Lego" forms a hydrogel scaffold for tissue engineering; Type II, "molecular switch" as a molecular actuator; Type III, "molecular hook" and "molecular velcro" for surface engineering; Type IV, peptide nanotubes and nanovesicles, or "molecular capsule" for protein and gene deliveries and Type V, "molecular cavity" for biomineralization. These self-assembling peptide systems are simple, versatile and easy to produce. These self-assembly systems represent a significant advance in the molecular engineering for diverse technological innovations.
Design of nanostructured biological
materials through self-assembly of peptides and proteins. Current
Opinion in Chemical Biology (December 2002)
Shuguang Zhang, 1, Davide M. Marini, Wonmuk Hwang and Steve Santoso; Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
Several self-assembling peptide and protein systems that form nanotubes, helical ribbons and fibrous scaffolds have recently emerged as biological materials. Peptides and proteins have also been selected to bind metals, semiconductors and ions, inspiring the design of new materials for a wide range of applications in nano-biotechnology. Recently discovered self-assembling peptide and protein systems represent simple, versatile, and modular construction units for producing nanoscale scaffolds and materials for a broad range of technological innovation and applications. Author Keywords: self-assembly; peptides; biomaterials; amphiphilic; surfactant; nanofibers; 3-D Matrix; regenerative biology
Novel peptide-based biomaterial
scaffold for tissue engineering. Trends in Biotechnology(Jan 2002)
T Holmes. Department of Biology, New York University, New York, NY 10003, USA.
Biomaterial scaffolds are components of cell-laden artificial tissues and transplantable biosensors. Some of the most promising new synthetic biomaterial scaffolds are composed of self-assembling peptides that can be modified to contain biologically active motifs. Peptide-based biomaterials can be fabricated to form two- and three-dimensional structures. Recent studies show that biomaterial promotion of multi-dimensional cell-cell interactions and cell density are crucial for proper cellular differentiation and for subsequent tissue formation. Other refinements in tissue engineering include the use of stem cells, cell pre-selection and growth factor pre-treatment of cells that are used for seeding scaffolds. These cell-culture technologies, combined with improved processes for defining the dimensions of peptide-based scaffolds, might lead to further improvements in tissue engineering. Novel peptide-based biomaterial scaffolds seeded with cells show promise for tissue repair and for other medical applications.
Advances in Biomedical Engineering. Journal of the American Medical Association (Feb 2001)
Linda Griffith, Alan Grodzinsky. Massachusetts Institute of Technology, Cambridge, MA, USA.
The most visible contributions of biomedical engineering to clinical practice involve instrumentation for diagnosis, therapy, and rehabilitation. Cell and tissue engineering, some enabled by novel biomaterial technology, also have emerged as clinical realities. Products for skin replacement are in clinical use and progress has been made in developing technologies for repair of cartilage, bone, liver, kidney, skeletal muscle, blood vessels, the nervous system, and urological disorders. In the next 25 years, advances in electronics, optics, materials, and miniaturization will accelerate development of more sophisticated devices for diagnosis and therapy, such as imaging and virtual surgery. The emerging new field of bioengineering-engineering based in the science of molecular cell biology-will greatly expand the scope of biomedical engineering to tackle challenges in molecular and genomic medicine.
Biomimetic Materials:
Properties and Processing Encyclopedia of Materials: Science and
Technology
P. Calvert
Molecular
Self-Assembly in Nature & Emerging Applications Encyclopedia
of Materials: Science and Technology
S. Zhang
Tissue Engineering and Reparative Medicine:
Overview of the 2001 NIH Bioengineering Consortium (BECON) symposium on "Reparative
Medicine: Growing Tissues and Organs"
Sipe J, National Institutes of Health, Bethesda, MD, USA
Guidelines
& Protocol for Culturing Cells in PuraMatrix™
BD
PuraMatrix™ Peptide Hydrogel Brochure
Puramatrix™: Self-assembling Peptide Nanofiber Scaffolds. Spirio L, Chu Z, Zhang S.
A Chapter in Scaffolding in Tissue Engineering, edited by Peter X. Ma and Jenniffer Elisseeff, published by Marcel Dekker, Inc.
Over the past eight years, a new class of biologically-inspired polypeptide biomaterials have been discovered and tested in the context of cell culture, stem cell biology and tissue engineering. These self-assembling peptide hydrogel scaffolds, which we call PuraMatrix, have been used successfully as a synthetic in vitro and in vivo ECM, proving themselves as a critical component to successful 3-D cell growth.
Molecular Self-assembly, Encyclopedia of Materials: Science and Technology. Shuguang Zhang
Self-Assembling Peptide Systems in Biology, Medicine and Engineering
Published by Kluwer and edited by Amalia Aggeli University of Leeds, UK
Neville Boden University of Leeds, UK
Shuguang Zhang Massachusetts Institute of Technology, Cambridge, USA