Kay C Dee

Mechanical Characterization of Collagen Fibers and Scaffolds for Tissue Engineering

Engineered tissues must utilize scaffolding biomaterials that support desired cellular functions and possess or can develop appropriate mechanical characteristics. This study assessed properties of collagen as a scaffolding biomaterial for ligament replacements. Mechanical properties of extruded bovine achilles tendon collagen fibers were significantly affected by fiber diameter, with smaller fibers displaying higher tangent moduli and peak stresses. Mechanical properties of 125 micrometer-diameter extruded fibers (tangent modulus of 359.6+/-28.4MPa; peak stress of 36.0+/-5.4MPa) were similar to properties reported for human ligaments. Scaffolds of extruded fibers did not exhibit viscoelastic creep properties similar to natural ligaments. Collagen fibers from rat tail tendon (a well-studied comparison material) displayed characteristic strain-softening behavior, and scaffolds of rat tail fibers demonstrated a non-intuitive relationship between tangent modulus and specimen length. Composite scaffolds (extruded collagen fibers cast within a gel of Type I rat tail tendon collagen) were maintained with and without fibroblasts under standard culture conditions for 25 days; cell-incorporated scaffolds displayed significantly higher tangent moduli and peak stresses than those without cells. Because tissue-engineered products must possess appropriate mechanical as well as biological/chemical properties, data from this study should help enable the development of improved tissue analogues.

Conditions which promote mineralization at the bone-implant interface: a model in vitro study

This in vitro study was an investigation of osteoblast functions on glass substrates modified with the bioactive peptide Arg-Gly-Asp-Ser (RGDS) in the absence and presence of recombinant human Osteogenic Protein-1 (OP-1); control substrates were plain glass, glass modified with amine groups, and glass modified with the non-adhesive peptide Arg-Asp-Gly-Ser. In serum-free cell culture medium, osteoblasts adhered in greater numbers (P < 0.1) to glass modified with RGDS, compared to adhesion on all other substrate types tested in the present study. In the presence of serum proteins, osteoblasts adhered similarly to all substrate types examined, in the absence or presence of 100 ng ml-1 OP-1. The presence of 100 ng ml-1 OP-1 inhibited (P < 0.1) 72 h proliferation of sparsely seeded (2500 cells cm-2) cultures on all substrates examined in the present study. OP-1 (100 ng ml-1) promoted 21 day mineralization on all substrates examined; in addition, mineralization was further enhanced in osteoblast cultures grown on glass modified with the adhesive peptide RGDS. The present study establishes conditions which can be utilized in the design of dental/orthopaedic biomaterials which elicit timely, specific responses from surrounding bone tissue.

Endothelial cell migration on surfaces modified with immobilized adhesive peptides

Endothelial cell (EC) migration has been studied on aminophase surfaces with covalently bound RGDS and YIGSRG cell adhesion peptides. The fluorescent marker dansyl chloride was used to quantify the spatial distribution of the peptides on the modified surfaces. Peptides appeared to be distributed in uniformly dispersed large clusters separated by areas of lower peptide concentrations. We employed digital time-lapse video microscopy and image analysis to monitor EC migration on the modified surfaces and to reconstruct the cell trajectories. The persistent random walk model was then applied to analyze the cell displacement data and compute the mean root square speed, the persistence time, and the random motility coefficient of EC. We also calculated the time-averaged speed of cell locomotion. No differences in the speed of cell locomotion on the various substrates were noted. Immobilization of the cell adhesion peptides (RGDS and YIGSRG), however, significantly increased the persistence of cell movement and, thus, the random motility coefficient. These results suggest that immobilization of cell adhesion peptides on the surface of implantable biomaterials may lead to enhanced endothelization rates.

Osteoblast population migration characteristics on substrates modified with immobilized adhesive peptides.

The process of cell migration is inextricably linked with the process of cell adhesion and, therefore, with cell/substrate adhesiveness. The present study adapted an under-agarose cell migration assay to quantitatively examine population migration characteristics of osteoblasts, on substrates modified with adhesive peptides, in the absence and presence of growth factors. Short-term, that is, 48 h osteoblast migration distances on substrates modified with adhesive Arg-Gly-Asp-Ser peptides were significantly (P < 0.05) less than migration distances on substrates modified with non-adhesive Arg-Asp-Gly-Ser peptides, demonstrating that osteoblast population haptokinesis was significantly decreased on substrates modified with adhesive peptides. Random motility coefficients calculated in the present study for osteoblast populations were an order of magnitude lower than a published random motility coefficient for leukocytes, proving quantitatively that, compared to leukocytes, osteoblasts migrate via haptokinesis more slowly. The 48 and 72 h osteoblast population migration differentials in the presence of an initial mass of 60 ng of basic Fibroblast Growth Factor, on substrates modified with Arg-Gly-Asp-Ser or with Arg-Asp-Gly-Ser, were larger than all other chemotactic differentials on these substrates. Quantitative investigations (such as the present study) of cell population migration characteristics on model biomaterial surfaces will become increasingly necessary as the discipline of cell/tissue engineering matures.

Development of Ligament-Like Structural Organization and Properties in Cell-Seeded Collagen Scaffolds in vitro

Acute anterior cruciate ligament (ACL) injuries lead to poor joint function, instability, and eventually osteoarthritis if left untreated. Current surgical treatment options are not ideal; however, tissue engineering may provide mechanically sound, biocompatible reconstructions. Collagen fiber scaffolds were combined with fibroblast-seeded collagen gels and maintained in culture for up to 20 days. The tensile and viscoelastic behavior of the constructs closely mimicked that of natural ligament. Constructs’ mechanical and viscoelastic properties did not degrade over time in culture, and peak stress was significantly higher for constructs with embedded fibroblasts. Immunocytochemical and histological analyses demonstrated cell proliferation and ligament-like organization. We have created an engineered tissue that closely approaches key mechanical and viscoelastic properties of the ACL, does not degrade after 20 days in culture, and is histologically similar to the native tissue. This study should aid in developing effective treatments for ACL injury.

Mini-review: Proactive biomaterials and bone tissue engineering

Recent advances in cell isolation and culture procedures, combined with growing understanding and use of molecular biology and biochemistry techniques, have resulted in the establishment of a new field of biological/biomedical research: cellular and tissue engineering. In the biomaterials field, cell and tissue bioengineers are investigating the development of proactive biomaterials (for example, bioceramics, chemically modified implant metals, and biodegradable tissue scaffolds) which utilize cellular‐ or molecular‐level methods of manipulating cell/tissue behavior in order to encourage clinically desirable biological events at the tissue‐implant interface. In vitro investigations utilizing osteoblasts, osteoclasts, and appropriate precursor cells, combined with long‐term (i.e., years) tissue engineering studies in vivo are needed to enhance current understanding of the many mechanisms involved in bone formation and regulation. Such understanding will allow the development of proactive biomaterials for use in bone, which can elicit specific, timely, and clinically desirable responses from surrounding cells and tissues.

Short Collagen Fibers Provide Control of Contraction and Permeability in Fibroblast-Seeded Collagen Gels

Tissue engineering may allow for the reconstruction of breast, facial, skin, and other soft tissue defects in the human body. Cell-seeded collagen gels are a logical choice for creating soft tissues because they are biodegradable, mimic the natural tissue, and provide a three-dimensional environment for the cells. The main drawback associated with this approach, however, is the subsequent contraction of the gel by the constituent cells, which severely reduces permeability, initiates apoptosis, and precludes control of the resulting shape and size of the construct. In this study, type I collagen gels were seeded with fibroblasts and cast either with or without the addition of short collagen fibers. Gel contraction was monitored and permeability was assessed after 7 and 14 days in culture. The addition of short collagen fibers both significantly limited contraction and increased permeability of fibroblast-seeded collagen gels. The addition of short collagen fibers had no detrimental effect on cell proliferation, and there were a high number of viable fibroblasts in gels with fibers and gels without fibers. Gels containing short collagen fibers demonstrated permeabilities that were 100 to 1000 times greater than controls and also closely maintained their casting dimensions (never less than 96% of original). By limiting contraction and maintaining permeability, the incorporation of short collagen fibers should enable the creation of larger constructs by allowing for greater nutrient diffusion, and permit the creation of more complicated shapes during gel casting.

Research report: learning styles of biomedical engineering students.

AbstractExamining students’ learning styles can yield information useful to the design of learning activities, courses, and curricula. A variety of measures have been used to characterize learning styles, but the literature contains little information specific to biomedical engineering (BMEN) students. We, therefore, utilized Felder’s Index of Learning Styles to investigate the learning style preferences of BMEN students at Tulane University. Tulane BMEN students preferred to receive information visually (preferred by 88% of the student sample) rather than verbally, focus on sensory information (55%) instead of intuitive information, process information actively (66%) instead of reflectively, and understand information globally (59%) rather than sequentially. These preferences varied between cohorts (freshman, sophomore, etc.) and a significantly higher percentage of female students preferred active and sensing learning styles. Compared to other engineering student populations, our sample of Tulane BMEN students contained the highest percentage of students preferring the global learning style. Whether this is a general trend for all BMEN students or a trait specific to Tulane engineers requires further investigation. Regardless, this study confirms the existence of a range of learning styles within biomedical engineering students, and provides motivation for instructors to consider how well their teaching style engages multiple learning styles.

An assessment of the strength of NG108-15 cell adhesion to chemically modified surfaces.

The strength of adhesion of NG108-15 cells to glass substrates modified with adsorbed proteins (laminin and poly-ornithine) or modified with covalently bound peptides (tri-ornithine and Tyr-Ile-Gly-Ser-Arg) was quantitatively assessed, by determining the shear stresses necessary to denude the cells from substrates using a spinning disk device. The shear stresses required to detach NG108-15 cells from glass modified with either adsorbed poly-ornithine or with both poly-ornithine and laminin were significantly (P < 0.05) higher than the shear stresses required to detach the cells from plain glass substrates. Covalent surface modifications resulted in higher strengths of NG108-15 adhesion than were exhibited on surfaces modified with adsorbed proteins. NG108-15 cell adhesion strength was maximal on surfaces covalently modified with only amine groups (without any peptides or proteins). These results indicate that general (i.e., not necessarily receptor-specific) surface modification strategies, which increase the net surface charge of a substrate, will elicit strong adhesion of NG108-15 cells.

A Device for Long Term, In Vitro Loading of Three-Dimensional Natural and Engineered Tissues

AbstractIn vitro studies of mechanical loads applied to three-dimensional tissue constructs are important to the design and production of functional, engineered bone tissue. This study reports the development and characterization of a mechanical device capable of subjecting a three-dimensional section of natural or engineered tissue to precise, reproducible four-point bending deformations over a range of programmable magnitudes and frequencies. To test the biological and mechanical capabilities of the system, a low-cycle (360 cycles/day), medium-range strain (2500 microstrain), long-term (16 day) loading regime was applied to rat bone marrow stromal cells cultured in porous DL-polylactic acid scaffolds. Cells proliferated in culture throughout the experiment, and with time showed an increase in alkaline phosphatase expression per cell. Calcium and phosphorus mineral deposition by the unloaded group was significantly greater (p < 0.05) than that deposited by the loaded group. The molar ratio of calcium to phosphorus in the unloaded group (0.94:1) was significantly greater (p < 0.05) than that of the loaded group (0.41:1). The loading device presented here is a tool which can be used to help elucidate contributions of mechanical loading/fatigue on biodegradable materials, as well as study the effects of mechanical loading on natural or engineered tissues in vitro.