Projects
If you would like to learn more about the active projects within the Laboratory for Tissue Regeneration, please review the projects below
3D Cell Culturing
- Broad Objectives
i) to develop technologies that facilitate the production of 3D cell composites with high precision and reliability, for adaption in large scale preparations, ii) to develop synthetic 3D surrogates that mimic cell functions similar to that in the body, and iii) to use these conditions in regenerating tissue of interest. Our hypothesis is that various matrix elements in the body contribute different signals to the cells and they have to be present in an appropriate composition for recreating needed function. Successful completion of this project will have significant impact on i) the development of synthetic surrogates to test disease states (mechanism of wound healing), and ii) for toxicology studies and to test the effect of pathogens i.e., as real time sensors for detecting biological agents.
- Background
The body's functions are determined by the cells. Understanding the dynamic changes in cellular activity is of significant importance in a many applications including occurrence of diseases and developmental biology. Petri dish cultures were developed in the early 1900s to understand cellular activity in an environment of reduced complexity when evaluated in the whole body. Although these insights have been helpful in understanding many concepts, there are many problems with two-dimensional (2D) tissue culture technique. Tissue culture plastic surface, cells are restricted to spread on a rigid surface. Hence, effects of biophysical properties of the matrix that provide a spatio-temporal effect in the body are not part of the effect. However, biophysical properties significantly influence cell adhesion and functions in 3D environment. Cells do respond differently in attachment, morphology, migration, and proliferation on 3D porous structures. There are also significant differences in many proteins responsible for cell-matrix interactions. Such differences in cell adhesion between 2D and 3D structures trigger different signaling mechanisms. Since cells exist in 3D spaces in the human body, developing systems to for the cell colonization in 3D is necessary. Porous structures generated from natural and synthetic polymers or after removing the cellular components from xenogeneic tissues have been used to support and guide the in-growth of cells. Examples include small intestinal submucosa (SIS), and acellular dermis. On the other hand, manufacturing porous templates using pure components allows formation of matrices with required features in addition to large scale production. Scaffolds are generated from synthetic and natural polymers using various techniques such as controlled rate freezing and lyophilization, porogen-leaching technique, free-form printing and electrospraying. Each technique has advantages and disadvantages and many not be suitable for all polymers. Also, altered surface texture and surface charge affects cell spreading. Current bottleneck in the field is the dearth of biomaterial scaffolds that elicit controlled cellular responses and possess essential mechanical properties.
- Our Approach
Our group focuses on innovative methods of dispersing polymeric systems without chemical reactions. We work on generating scaffolds and injectable hydrogels from blends of natural and synthetic polymers, based on the desired properties. We innovate and adapt many novel techniques to obtain desirable scaffolds. We have formed emulsions and blends of synthetic and natural polymers using unique solvents. We form co-axial and tri-axial fibers to obtain reinforced composites. We utilize controlled release of stimulants locally to create a heterogeneous microenvironment form differentiating stem cells. We have formulated novel temperature sensitive injectable hydrogels. We use bioprinting to form blood vessels and then co-culture other types of cells. We perform a wide variety of mechanical tests including tensile, compression, cyclical, stress relaxation testing under physiological conditions and evaluate various mechanical properties.
Bioreactors for Tissue Regeneration
- Broad Objectives
develop the governing conditions for designing bioreactors during the process of regeneration. Successful development of principles will significantly advance the regeneration of high quality tissues “off the shelf” by variety of contributions including a methodology to monitor the regenerative process non-destructively.
- Background
In a traditional cell culture, cells are populated in a batch culture on flat plates with a defined amount of nutrients which distribute within the porous structure by diffusion, dictated by Fick’s first law. As cells consume nutrients, relying on diffusion alone with depleting concentration gradient leads to starvation and non- uniform distribution. A way to improve the nutrient distribution is by constant mixing and/or constant replenishment via fluid flow. In addition to improving the nutrient distribution, fluid flow also applies shear force on the cells, which is important for certain tissues. Many parts of the body are exposed to stresses either due to the weight they carry (such as bone), the function they perform (such as bladder and cartilage) or due to the flow of fluid (lung, blood vessels). In these cases, there is evidence that application of certain magnitude of stress helps alters how cells behave. One such effect is shown below on endothelial cells, which line the inner lumen of blood vessels.
Different types of bioreactors have been designed to regenerate tissues with the while applying mechanical stimuli. However, the design principles in developing these bioreactors are not well developed. For example, selecting a specific shape of bioreactor, the inlets and outlet locations, and the media flow rates have remained random. There is limited understanding of the nutrient distribution within these bioreactors during the entire process of tissue regeneration. The kinetic models are often over-simplified because mass and structural complexities have been ignored. Tissue regeneration is a dynamic process where the porous characteristics change due to cell growth, assembly of newly secreted matrix components, and degradation of the porous architecture. These changes affect the transport characteristics which ultimately determine the quality of the regenerated tissue. Non-uniform flow patterns within the reactor could lead to i) poor distribution of nutrients and ii) non-uniform shear stress distribution. These factors affect cellular colonization, proliferation, and function. Thus to develop improved quality tissues, one has to understand the influence of these factors.
- Our Approach
We design bioreactors for regenerating tissues using the tools and governing equations, proven to work in other engineering disciplines. We use a set of integrated tools: i) computational fluid dynamic (CFD) software such as COMSOL and CFX to understand the effect of flow configurations on fluid distribution through the porous structure, ii) different scaffold preparations preparations, and iii) cell culture experiments in bioreactors with specifications identical to simulation. Some factors evaluated thus far include i) reactor shapes (rectangular, circular, spherical), ii) flow rate, iii) inlet-outlet location, iv) inlet-outlet size which regulate velocities, v) nutrient consumption (oxygen, glucose, estrogen) kinetics, vi) cell types (smooth muscle cells, chondrocytes, hepatocytes, fibroblasts, cord blood stem cells), vii) scaffold mechanical properties, and viii) scaffold permeability properties using Kozeny-Carman definition. We also combine residence time distribution analysis with parametric models to calculate the outlet oxygen concentration. We assess the effect of changing porous architecture due to cell growth and deposited matrix elements on fluid distribution, shear stresses and pressure drop. We measure diffusivities in porous structures, and account for changes in dimensions of the scaffolds using mechanical properties. We believe these efforts will help develop strategies to run the bioreactor during the entire regenerative cycle. Our current efforts are focused on integrating mechanical effects on nutrient distribution.
Composite Tissue Regeneration
- Broad Objectives
i) to understand the influence of structural features, chemical signals, and mechanical signals on the proliferation and differentiation of stem cells from various sources and ii) to utilize these conditions in forming composite tissue grafts. Successful completion of this project will have significant impact on i) the regeneration and transplantation of high quality tissues on-demand, ii) the development of synthetic surrogates to test disease states (mechanism of wound healing), toxicology studies and effect of pathogens i.e., as real time sensors for detecting biological agents, and iii) importantly changes the way cell culture experiments will be performed in the future worldwide.
- Background
With the advent of bone marrow transplantation to cure various hematologic disorders, cell based therapies have see significant attention. In particular stem cells from various have been harvested and explored for use. In addition, the plastic nature of stem cells is well demonstrated in a variety of models; bone marrow cells can differentiate into liver cells or muscle cells based on the location of their recruitment. Based on these success, many cell-based therapies have been investigated in clinical trials. For example, bone, cardiac tissue and cartilage. However, stem cell based therapies have not seen significant success due to a major problem of poor regeneration attributed to attrition of injected cells. Non-invasive approaches using injectable hydrogels have shown marginal improvements as cells could be dispersed instead of injecting pellets. However, significant loss of cells remains a problem and hydrogels are also very weak relative to native tissues. In addition, current products obtainable by tissue engineering are limited to tissues/organs without blood vessels due to the technological limitations in developing organized vasculature. Using stem cells to differentiate to diverse cell types, including endothelial cells i.e., the cells forming the vascular networks is an option. However, the primary challenge is to transcribe the in vivo microenvironment into in vitro conditions so that vascularized tissues can be regenerated in 3D configuration. Evaluating 3D configurations are critical a) to understand the spatio-temporal effects, b) to evaluate the reorganization of various compartments and organ formation, and c) to develop devices that can be used in clinical applications. Thus, exploring the possibility of differentiating cells in 3D will significantly help as an alternative cell source in tissue regeneration. A number of 3D models are explored using matrigels obtained from a mouse sarcoma. However, matrigels do not accurately reflect the diversity of proteins of most tissues; for example, Laminin-1 is not present at high quantities in most adult tissues. Our innovation is that we mimic by introducing tissue-specific matrix elements without using any cross-linkers.
- Our Approach
With the advent of genetically inducing pluripotency in mature cells (called induced pluripotent stem cells), we explore differentiating adult cells by controlling the scaffold architecture and signaling. We utilize adult stem cells from different tissues such as bone marrow, cord blood and adipose tissue. We evaluate the regeneration patterns and compare how stem cells would perform at two levels a) microscale and b) nanoscale using novel technologies. Human foreskin fibroblasts are explored to form cartilage. We alter hydrogel formulation to suite a required tissue, chemically crosslink one component selectively within the hydrogel to improve mechanical properties, and incorporate nutrients including oxygen releasing molecules. We use anisotropic injectable hydrogels that can be used with minimally invasive surgical procedure. In addition, we evaluate bioreactor configurations that could apply different stress to different types of cells within the scaffold. We evaluate cell differentiation using various cell specific markers. We perform many evaluations including cell growth, attachment, and assembly and maturation of matrix. We use the injectable hydrogels to form vasculatures and other types of tissues within the same template.
Drug Delivery
- Broad Objective
is to develop strategies to delivery drugs by different routes and approaches. Few projects are described below
- Delivery Vehicles
Many therapeutic molecules have short half-lives and one way to extend their life span is encapsulation. Encapsulating regulatory factors and delivering them at a controlled rate provide opportunities: i) to create heterogeneous microenvironments within the matrix, unlike exogenous supplementation which creates a homogeneous microenvironment; ii) to continuously deliver regulatory factor, minimizing the time dependent variation in concentration, attributed to the frequency of the media change and half-lives of different growth factors; iii) to address the concerns related to incomplete secretion of growth factors into the cell culture medium in addition to the efficacy of genetically modified cellular components. Optimization of growth factor delivery and evaluation of cellular interactions in the matrices will help their usage in tissue healing.
Nanoparticle-based delivery. Nanoparticles are employed in various therapeutic approaches for innovative drug delivery strategies. Various techniques exist to form nanoparticles from biodegrable polymers to liposomes with selective targeting. We use double emulsion technique to form PLGA and PCL nanoparticles. We form chitosan nanoparticles using tripolyphosphate (TPP) anions. Similar technology also exists to form gelatin nanoparticles. In addition, we form liposomes and derivatize with holo-transferrin and polyethylene glycol. We use these particles in combination with various porous templates in tissue regeneration.
Microfiber-based delivery. Alternative to nanoparticle-based encapsulation is to form drug-containing micro or nanofibers. We work on using novel electrospinning technology to form various drug-containing fibers. In order to control the release rate, we create co-axial and triaxial fibers from combinations of polymers that possess both required mechanical properties and biological properties. One could use these mats of fibers along with hydrogels and create a heterogeneous environment. Further, we can release various molecules selectively and sequentially for the differentiation of cells.
- Transdermal Drug Delivery
Development of transdermal drugs requires the identification of suitable delivery mechanisms. Much effort has been directed towards the search for specific chemical or chemical combinations that could enhance drug penetration. However, effective chemical penetration enhancers (CPE) themselves permeate skin thereby eliciting some undesired reactions. Reliable and quantitative models for predicting precutaneous penetration and irritation as a function of chemical structure are required. The development of these models enables us to virtually screen for viable penetration enhancers thereby reducing the need for expensive experiments. The major goal of this project is to integrate non-linear, theory-based quantitative-structure-property-relationship (QSPR) modeling and robust genetic algorithms (GAs) to facilitate the design of improved CPEs. This is a collaborative project with Dr. Khaled Gasem at OSU. Currently, a project assessing the transdermal delivery of insulin is pending in NIH. If successful, this will have a significant impact on delivery systems.
- Oral Delivery
In this cutting edge technology, pH-sensitive hydrogels are loaded with insulin and can be administered through the oral cavity. Unique feature of these hydrogels are that they form interpolymer complexes in an acidic environment and protect the insulin from the harsh stomach environment, a challenge to overcome for oral drug delivery. Insulin is safely released in the intestine due to the swelling of the carrier matrix at high pH and the delivery rate is influenced by particle size. Oral administration of insulin showed responses similar to subcutaneous delivery, suggesting the successful delivery of insulin orally.