Jan P. Stegemann

Gelatin microspheres crosslinked with genipin for local delivery of growth factors

A main challenge in tissue engineering and regenerative medicine is achieving local and efficient growth factor release to guide cell function. Gelatin is a denatured form of collagen that cells can bind to and degrade through enzymatic action. In this study, gelatin microspheres were used to release bone morphogenetic protein 2 (BMP2). Spherical microparticles with diameters in the range of 2–6 µm were created by an emulsification process and were stabilized by crosslinking with the small molecule genipin. The degree of crosslinking was varied by controlling the incubation time in genipin solution. Loading rate studies, using soy bean trypsin inhibitor as a model protein, showed rapid protein uptake over the first 24 h, followed by a levelling off and then a further increase after approximately 3 days, as the microspheres swelled. Growth factor release studies using microspheres crosslinked to 20%, 50% and 80% of saturation and then loaded with BMP2 showed that higher degrees of crosslinking resulted in higher loading efficiency and slower protein release. After 24 h, the concentration profiles produced by all microsphere formulations were steady and approximately equal. Microspheres incubated with adult human mesenchymal stem cells accumulated preferentially on the cell surface, and degraded over time in culture. BMP2‐loaded microspheres caused a three‐ to eight‐fold increase in expression of the bone sialoprotein gene after 14 days in culture, with more crosslinked beads producing a greater effect. These results demonstrate that genipin‐crosslinked gelatin microspheres can be used to deliver growth factors locally to cells in order to direct their function. Copyright

Thermogelling chitosan and collagen composite hydrogels initiated with β-glycerophosphate for bone tissue engineering

Chitosan and collagen type I are naturally derived materials used as cell carriers because of their ability to mimic the extracellular environment and direct cell function. In this study beta-glycerophosphate (beta-GP), an osteogenic medium supplement and a weak base, was used to simultaneously initiate gelation of pure chitosan, pure collagen, and chitosan-collagen composite materials at physiological pH and temperature. Adult human bone marrow-derived stem cells (hBMSC) encapsulated in such hydrogels at chitosan/collagen ratios of 100/0, 65/35, 25/75, and 0/100 wt% exhibited high viability at day 1 after encapsulation, but DNA content dropped by about half over 12 days in pure chitosan materials while it increased twofold in materials containing collagen. Collagen-containing materials compacted more strongly and were significantly stiffer than pure chitosan gels. In monolayer culture, exposure of hBMSC to beta-GP resulted in decreased cell metabolic activity that varied with concentration and exposure time, but washing effectively removed excess beta-GP from hydrogels. The presence of chitosan in materials resulted in higher expression of osterix and bone sialoprotein genes in medium with and without osteogenic supplements. Chitosan also increased alkaline phosphatase activity and calcium deposition in osteogenic medium. Chitosan-collagen composite materials have potential as matrices for cell encapsulation and delivery, or as in situ gel-forming materials for tissue repair.

Strategies for directing the structure and function of three-dimensional collagen biomaterials across length scales.

Collagen type I is a widely used natural biomaterial that has found utility in a variety of biological and medical applications. Its well-characterized structure and role as an extracellular matrix protein make it a highly relevant material for controlling cell function and mimicking tissue properties. Collagen type I is abundant in a number of tissues, and can be isolated as a purified protein. This review focuses on hydrogel biomaterials made by reconstituting collagen type I from a solubilized form, with an emphasis on in vitro studies in which collagen structure can be controlled. The hierarchical structure of collagen from the nanoscale to the macroscale is described, with an emphasis on how structure is related to function across scales. Methods of reconstituting collagen into hydrogel materials are presented, including molding of macroscopic constructs, creation of microscale modules and electrospinning of nanoscale fibers. The modification of collagen biomaterials to achieve the desired structures and functions is also addressed, with particular emphasis on mechanical control of collagen structure, creation of collagen composite materials and crosslinking of collagenous matrices. Biomaterials scientists have made remarkable progress in rationally designing collagen-based biomaterials and in applying them both to the study of biology and for therapeutic benefit. This broad review illustrates recent examples of techniques used to control collagen structure and thereby to direct its biological and mechanical functions.

Carbon nanotubes in neural interfacing applications

Carbon nanotubes (CNT) are remarkable materials with a simple and inert molecular structure that gives rise to a range of potentially valuable physical and electronic properties, including high aspect ratio, high mechanical strength and excellent electrical conductivity. This review summarizes recent research on the application of CNT-based materials to study and control cells of the nervous system. It includes the use of CNT as cell culture substrates, to create patterned surfaces and to study cell-matrix interactions. It also summarizes recent investigations of CNT toxicity, particularly as related to neural cells. The application of CNT-based materials to directing the differentiation of progenitor and stem cells toward neural lineages is also discussed. The emphasis is on how CNT surface chemistry and nanotopography can be altered, and how such changes can affect neural cell function. This knowledge can be applied to creating improved neural interfaces and devices, as well as providing new approaches to neural tissue engineering and regeneration.

Glyoxal Crosslinking of Cell-Seeded Chitosan/Collagen Hydrogels for Bone Regeneration

Chitosan and collagen are natural biomaterials that have been used extensively in tissue engineering, both separately and as composite materials. Most methods to fabricate chitosan/collagen composites use freeze drying and chemical crosslinking to create stable porous scaffolds, which subsequently can be seeded with cells. In this study, we directly embedded human bone marrow stem cells (hBMSC) in chitosan/collagen materials by initiating gelation using β-glycerophosphate at physiological temperature and pH. We further examined the use of glyoxal, a dialdehyde with relatively low toxicity, to crosslink these materials and characterized the resulting changes in matrix and cell properties. The cytocompatibility of glyoxal and the crosslinked gels were investigated in terms of hBMSC metabolic activity, viability, proliferation and osteogenic differentiation. These studies revealed that glyoxal was cytocompatible at concentrations below about 1mM for periods of exposure up to 15 h, though the degree of cell spreading and proliferation were dependent on matrix composition. Glyoxal-crosslinked matrices were stiffer and compacted less than uncrosslinked controls. It was further demonstrated that hBMSC can attach and proliferate in three-dimensional matrices composed of 50/50 chitosan/collagen, and that these materials supported osteogenic differentiation in response to stimulation. Such glyoxal-crosslinked chitosan/collagen composite materials may find utility as cell delivery vehicles for enhancing the repair of bone defects.

Reduced serum content and increased matrix stiffness promote the cardiac myofibroblast transition in 3D collagen matrices.

INTRODUCTION The fibroblast-myofibroblast transition is an important event in the development of cardiac fibrosis and scar formation initiated after myocardial ischemia. The goals of the present study were to better understand the contribution of environmental factors to this transition and determine whether myofibroblasts provide equally important feedback to the surrounding environment. METHODS The influence of matrix stiffness and serum concentration on the myofibroblast transition was assessed by measuring message levels of a panel of cardiac fibroblast phenotype markers using quantitative reverse transcriptase polymerase chain reaction. Cell-mediated gel compaction measured the influence of environmental factors on cardiac fibroblast contractility. Immunohistochemistry characterized alpha-smooth muscle actin expression and cell morphology, while static and dynamic compression testing evaluated the effect of the cell response on the mechanical properties of the cell-seeded collagen hydrogels. RESULTS Both reduced serum content and increased matrix stiffness contributed to the myofibroblast transition, as indicated by contractile compaction of the gels, increased message levels of col3α1 and alpha-smooth muscle actin, and a less stellate morphology. However, the effects of serum and matrix stiffness were not additive. Mechanical testing indicated that reduced serum content increased the initial elastic modulus of cell-seeded gels and that gels lost their viscous character with time. CONCLUSIONS The results suggest that reduced serum and increased matrix stiffness promote the myofibroblast phenotype in the myocardium. This transition both enhances and is promoted by matrix stiffness, indicating the presence of positive feedback that may contribute to the pathogenesis of cardiac fibrosis.

Delivery of mesenchymal stem cells in chitosan/collagen microbeads for orthopedic tissue repair.

Microencapsulation and delivery of stem cells in biomaterials is a promising approach to repairing damaged tissue in a minimally invasive manner. An appropriate biomaterial niche can protect the embedded cells from the challenging environment in the host tissue, while also directing stem cell differentiation toward the desired lineage. In this study, adult human mesenchymal stem cells (MSC) were embedded in hydrogel microbeads consisting of chitosan and type I collagen using an emulsification process. Glyoxal and β-glycerophosphate were used as chemical and physical crosslinkers to initiate copolymerization of the matrix materials. The average size and size distribution of the microbeads could be varied by controlling the emulsification conditions. Spheroidal microbeads ranging in diameter from 82 ± 19 to 290 ± 78 µm were produced. Viability staining showed that MSC survived the encapsulation process (>90% viability) and spread inside the matrix over a period of 9 days in culture. Induced osteogenic differentiation using medium supplements showed that MSC increased gene expression of osterix and osteocalcin over time in culture, and also deposited calcium mineral. Bone sialoprotein and type I collagen gene expression were not affected. Delivery of microbeads through standard needles at practically relevant flow rates did not adversely affect cell viability, and microbeads could also be easily molded into prescribed geometries for delivery. Such protein-based microbeads may have utility in orthopedic tissue regeneration by allowing minimally invasive delivery of progenitor cells in microenvironments that are both protective and instructive.

Interstitial fluid flow and cyclic strain differentially regulate cardiac fibroblast activation via AT1R and TGF-β1

Cardiac fibroblasts are exposed to both cyclic strain and interstitial fluid flow in the myocardium. The balance of these stimuli is affected by fibrotic scarring, during which the fibroblasts transition to a myofibroblast phenotype. The present study investigates the mechanisms by which cardiac fibroblasts seeded in three-dimensional (3D) collagen gels differentiate between strain and fluid flow. Neonatal cardiac fibroblast-seeded 3D collagen gels were exposed to interstitial flow and/or cyclic strain and message levels of collagens type I and III, transforming growth factor β1 (TGF-β1), and α-smooth muscle actin (α-SMA) were assessed. Flow was found to significantly increase and strain to decrease expression of myofibroblast markers. Corresponding immunofluorescence indicated that flow and strain differentially regulated α-SMA protein expression. The effect of flow was inhibited by exposure to losartan, an angiotensin II type 1 receptor (AT1R) blocker, and by introduction of shRNA constructs limiting AT1R expression. Blocking of TGF-β also inhibited the myofibroblast transition, suggesting that flow-mediated cell signaling involved both AT1R and TGF-β1. Reduced smad2 phosphorylation in response to cyclic strain suggested that TGF-β is part of the mechanism by which cardiac fibroblasts differentiate between strain-induced and flow-induced mechanical stress. Our experiments show that fluid flow and mechanical deformation have distinct effects on cardiac fibroblast phenotype. Our data suggest a mechanism in which fluid flow directly acts on AT1R and causes increased TGF-β1 expression, whereas cyclic strain reduces activation of smad proteins. These results have relevance to the pathogenesis and treatment of heart failure.

Wrapping and dispersion of multiwalled carbon nanotubes improves electrical conductivity of protein–nanotube composite biomaterials

Composites of extracellular matrix proteins reinforced with carbon nanotubes have the potential to be used as conductive biopolymers in a variety of biomaterial applications. In this study, the effect of functionalization and polymer wrapping on the dispersion of multiwalled carbon nanotubes (MWCNT) in aqueous media was examined. Carboxylated MWCNT were wrapped in either Pluronic(®) F127 or gelatin. Raman spectroscopy and X-ray photoelectron spectroscopy showed that covalent functionalization of the pristine nanotubes disrupted the carbon lattice and added carboxyl groups. Polymer and gelatin wrapping resulted in increased surface adsorbed oxygen and nitrogen, respectively. Wrapping also markedly increased the stability of MWCNT suspensions in water as measured by settling time and zeta potential, with Pluronic(®)-wrapped nanotubes showing the greatest effect. Treated MWCNT were used to make 3D collagen-fibrin-MWCNT composite materials. Carboxylated MWCNT resulted in a decrease in construct impedance by an order of magnitude, and wrapping with Pluronic(®) resulted in a further order of magnitude decrease. Functionalization and wrapping also were associated with maintenance of fibroblast function within protein-MWCNT materials. These data show that increased dispersion of nanotubes in protein-MWCNT composites leads to higher conductivity and improved cytocompatibility. Understanding how nanotubes interact with biological systems is important in enabling the development of new biomedical technologies.

Effects of hydroxyapatite on endothelial network formation in collagen/fibrin composite hydrogels in vitro and in vivo

Co-culture of endothelial cells (EC) and mesenchymal stem cells (MSC) results in robust vascular network formation in constrained 3-D collagen/fibrin (COL/FIB) composite hydrogels. However, the ability to form endothelial networks is lost when such gels are allowed to compact via cell-mediated remodeling. In this study, we created co-cultures of human EC and human MSC in both constrained and unconstrained COL/FIB matrices and systematically added nanoparticulate hydroxyapatite (HA, 0-20 mg ml(-1)), a bone-like mineral that has been shown to have pro-vasculogenic effects. Constructs cultured for 7 days were assayed for gel compaction, vascular network formation, and mechanical properties. In vitro, robust endothelial network formation was observed in constrained COL/FIB constructs without HA, but this response was significantly inhibited by addition of 5, 10, or 20 mg ml(-1) HA. In unconstrained matrices, network formation was abolished in pure COL/FIB constructs but was rescued by 1.25 or 2.5 mg ml(-1) HA, while higher levels again inhibited vasculogenesis. HA inhibited gel compaction in a dose-dependent manner, which was not correlated to endothelial network formation. HA affected initial stiffness of the gels, but gel remodeling abrogated this effect. Subcutaneous implantation of COL/FIB with 0, 2.5 or 2 0mg ml(-1) HA in the mouse resulted in increased perfusion at the implant site, with no significant differences between materials. Histology at day 7 showed both host and human CD31-stained vasculature infiltrating the implants. These findings are relevant to the design of materials and scaffolds for orthopedic tissue engineering, where both vasculogenesis and formation of a mineral phase are required for regeneration.