Research

Aaron Goldstein

Tissue engineering is a field of research that applies the principles of engineering and life sciences toward the development of biological substitutes to restore the function of damaged tissues and organs. As such, it is intrinsically interdisciplinary, and integrates aspects of material sciences, biological sciences, engineering and medical sciences. Although the specific approaches depend on the target tissue, the general paradigm is that an engineered tissue can be constructed by combining a biomaterial scaffold, cells, and pharmaceutic agents together into a construct, which can subsequently be conditioned in vitro prior to implantation.

Central to the efficacy of this paradigm is the recognition that the properties of the cell microenvironment, within this construct, provides a set of stimuli that can guide cell proliferation, differentiation, and tissue maturation (Figure 2).

Over the past decade, our research efforts in the Skeletal Tissue Engineering and Mechanobiology Laboratory have involved developing novel biomaterials and in vitro culturing conditions for the creation of biologically active materials that can guide regeneration of bone and tendon/ligament tissues. Concurrently, we have probed the mechanisms of mechanotransduction in osteoblasts and created an optical reporter system for visually assessing osteoblast differentiation. As such our focus has been on the following facets of tissue engineering.

Cells
Scaffold Fabrication
Bioreactors and Mechanotransduction
Imaging

Cells

Mesenchymal stem cells are a class of pluripotent adult stem cells that can be isolated from bone marrow, fat, skin, blood, and muscle biopsies, expanded in culture, and directed to differentiate into the various cell phenotypes found in bone, cartilage, muscle, tendons and ligaments and fat (Figure 3). In addition, there is evidence that these same cells can differentiate in to nerve, liver, and insulin-secreting beta cells. Currently, we are using these cells to test our biomaterial and bioreactor strategies for forming engineered bone and ligament tissue. However, in clinical practice, these cells can be obtained from the patient, seeded into the biomaterials and subsequently implanted back into the patient.

Figure 3. The various tissue types that have been shown to develop from mesenchymal stem cells.

Scaffold Fabrication

Scaffolds are three dimensional structures with extensive surface area for cell adhesion and tissue formation. We have been forming porous foam and fiber structures from a variety of natural (e.g., alginate, chitosan, cellulose) and synthetic materials (e.g., polycaprolactone, polyurethane) for application in bone and ligament regeneration. Figure 4a shows a polycaprolactone fibers with embedded hydroxyapatite nanoparticles (scale bar = 10 μm). Figure 4b shows a tyramine-based polyurethane, where the cube-shaped pores are 300-500 μm in size. Figure 4c shows sintered poly(lactic-co-glycolic acid) microspheres decorated with amorphous calcium phosphates. Figure 4d shows a bacteria cellulose scaffold that grown by fermentation of Acteobacter xylinum around paraffin wax microspheres.

Figure 4. Various biomaterial scaffolds. a) Electrospun polycaprolactone fibers containing hydroxyapatite nanoparticles. b) Polyurethane elastomer foam. c) Fused poly(lactic-co-glycolic acid) microspheres decorated with amorphous calcium phosphate particles. d) Porous bacteria cellulose foam.

Our electrospinning approach, in particular, has allowed us to create fused-fiber structures that can orient cells. By collecting fibers on a spinning mandrel the degree of fiber orientation can be controlled by the rate of mandrel rotation, O (Figure 5a-c). We have shown that cells form multiple attachments to the fibers (Figure 5d, green) that serve as anchors for the actin cytoskeleton (blue). Consequently, we have found that cells exhibit a rounder morphology on randomly oriented fibers (Figure 5e) and align parallel to oriented fibers (Figure 5f).

Figure 5. Electrospinning of micron-diameter fibers. a) Diagram of apparatus. b) Osteoblast on fibers. Vinculin protein (blue) involved in adhesion of the actin cytoskeleton (blue) to fibers (red). c) Randomly oriented fibers result in d) round/randomly oriented adherent cells. e) Aligned fibers guide f) orientation and elongation of adherent cells.

Further, co-electrospinning different materials from offset spinnerets can be used to produce spatial gradient of chemistry, mechanical properties, and architecture (Figure 6). We hypothesize that this approach can be used to construct scaffolds for the repair of complex tissues with spatial gradients of biological and mechanical properties.

Figure 6. Co-electrospinning two different polymers: a) depicted graphically, b) a set of SEM images showing PCL with hydroxyapatite (top) polyurethane (bottom) and a mixture (middle), and c) fluorescent images collected using two different dyes.

Bioreactors and Mechanotransduction

Bioreactors are an effective tool for long-term in vitro culture of cells within three-dimensional porous scaffolds. Perfusion, in particular, generates convective currents that delivers oxygen and nutrient to cells. In addition, we have shown that it can stimulate osteoblastic maturation and the production of a biologically active extracellular matrix (ECM). Further, we have recently shown that dynamic (pulsatile) perfusion can enhance osteoblastic cells to express protein markers of maturation (Figure 7). The significance of this is that we can stimulate the osteoblasts to secrete the very growth factors that are involved in bone formation. Although we have not yet tested it, this should - in theory - make the cell/biomaterial construct more biologically active when it is implanted into a bone defect.

Figure 7: Dynamic perfusion bioreactor: a) image of device, b) schematic of scaffold, c) dynamic flow profile, and effects of dynamic perfusion on d) alkaline phosphatase activity, and e) mRNA expression of osteopontin.

The significance of this is that we can stimulate the osteoblasts to secrete the very growth factors that are involved in bone formation. Although we have not yet tested it, this should - in theory - make the cell/biomaterial construct more biologically active when it is implanted into a bone defect.

Imaging

Bioreactors provide a method for maintaining cell viability and stimulating cells within three dimensional biomaterial scaffolds over periods of days to weeks. During this period, the cells are able to proliferate and mature. However, as the bioreactor is effectively a black box and most assays of cell proliferation and maturation are destructive, new non-destructive tools to assess cell viability and maturation in situ are needed. As a proof-of-principle, we created a reporter cell line in which the MC3T3-E1 osteoprogenitor cell line was stably transfected with a luciferase reporter gene under a BMP-2 promoter. These cells were seeded into a translucent chitosan scaffolds, maintained for several days in a perfusion bioreactor and were imaged for bioluminescence activity by the addition of beetle luciferin (Figure 8). The diffuse glow (light blue) of the scaffold is consistent with cell viability and expression of BMP-2 throughout the scaffold.

Figure 8: Bioluminescent imaging: a) perfusion bioreactor system, b) light images of the four faces of a scaffold within the bioreactor, and c) and corresponding false-color images of luciferase activity.