Development of Functional Synthetic Alveoli through Novel Polymer Perfusion Scaffold

Melvin Du, Luke Wang, Averee Chang, Jing Yu

Introduction

End-stage lung disease is a major healthcare challenge. As of June 2007, there are over 2,800 patients on the waiting list for a lung donation. Lung transplantation remains the definitive treatment, yet rejection and donor organ shortage limit its broader clinical impact. Engineering bioartificial lungs could lead to alternative treatments. Although many challenges remain, important early milestones towards translation have been met like advanced culture

conditions that facilitate the formation of 3-D function tissues and bioartificlal grafts that provide gas exchange. Current challenges include the creation of ideal scaffolding materials, differentiation and expansion of lung-specific cell populations and full maturation of engineered constructs to provide graph longevity after implantation in vivo.

Objectives and Rationale

As of right now, the extent of lung research is to use lungs harvested from one organism, flushing out the cells and seeding allogenic or autogenic cells onto the “natural scaffold” of the remaining ECM. So far, this technique, while proven to be effective in mice, is not applicable to a large clinical setting. With this scaffold design, we envision being able to create alveolar and eventually entire lungs from synthetic constructs by passing the immunogenic effects of the “natural scaffold.” Currently, no known research seeks to develop lung tissue in this manner. This is a scaffold design that has a very specific application into lung development. We first obtained Poloxamer, a thermosensitive, biodegradable polymer and using the perfusion conduction pipettes to create alveolar like structure using cyclic perfusion pump. This will create our “inverted” scaffold, which will then be seeded with ATII and ATI cell on top of the feeder layer.

Materials

Thermosensitive, Biodegradable Polymer

        Due to the nature of the experimental design, the use of a polymer with ideal characteristics is critical. The polymer must be non-cytotoxic, bio-activee, thermosensitive, thermoreversible, stable at temperatures, viscous in liquid phase, stable and biocompatible in solid phase. Ideally, the polymer in question should be in a solid state at 37°C, a viscous gel state around room temperature (~23°C) and a liquid state below room temperature.  

Poloxamers are a series of ABA block copolymers of polyethylene oxide and polypropylene oxide. They are amphiphilic in character, being comprised of a central polypropylene oxide (PO) block, which is hydrophobic, sandwiched between two hydrophilic poyethylene oxide (EO) blocks (Figure 1).

Fig 1: molecular structure of poloxamers

The ABA type of copolymer exhibited by poloxamers shows characteristic properties of thermoreversible gelation. In other words, they are liquid when refrigerated, but turn into gel form when at room temperature. The gel formed can be reversible on again cooling[4]. When the pluronic is placed into cold water, the hydration layer that surrounds the poloxramer molecule and hydrophobic portions are separated due to hydrogen bonding. With increasing temperature, desolvation of the hydrophilic chains occurs are the result of breakage of hydrogen bonds. This results into hydrophobic interactions amongst the polyoxypropylene domains and a gel forms. Due to this unique property, poloxamers, also known by the trade name Pluronic® macromolecules, have been studied in other biomedical applications[5] and will be ideal for our requirements since they have a temperature dependent gelling process essential to capturing the shape of the scaffold established in the perfusion step.

There are are 60 Pluronic® macromolecules available due to the fact that that lengths of the polymer blocks can be customized and thus a wide variety of poloxamers exists with slightly different properties. In the Pluronic tradename nomenclature, the first letter of each designated poloxamer define its physical form at room temperature (L=liquid, P=paste, F=flake (solid)) followed by two or three digits. The first digit in the numerical designation, multiplied by 300, indicates the approximate molecular weight of the hydrophobe, and the last digit, multiplied by 10, gives the percentage polyoxyethylene content.  Considering these requirements, Pluronic P85 or Pluronic P94 would be ideal for our purposes.  Both have a melting point of 29°C and 27°C, respectively, and therefore would be most suitable for our experimental design[6].

Perfusion conduction Pipettes

        These tubes serve as a conduit for perfusion from the motor to the poloxamer and form the bubbles that are vital to the scaffold structure. Ideally, they will mirror the diameter of the terminal bronchiole around 0.5 mm and have slight perforations at the terminus. The fibers need not be bioactive or cellularly conducive in anyway as they do not directly interact with the cell culture. They are lined on the inside with a temperature dependent, degradable, cellularly active surfactant. The pipettes are textured on the outside so as to imprint a microtexture onto the surfactant, aiding the adhesion and therefore viability of the cell culture.

Cells

        Feeder Layer/Epithelium Cells

        Type I Pneumocytes

        Type II Pneumocytes

        Alveolar Macrophages

Cyclic Perfusion Pump

        To produce the scaffold structure, a cyclic pump is employed to perfuse the conducting pipettes. The pump is also capable of generating cyclic pressure not exceeding 500 mmHg to mimic the cyclic pressure associated with tidal breathing.

Methods

Verification and Optimization

        Optimization and verification assessments occur before any scaffold or perfusion steps  can commence. For the Cyclic Perfusion Pump, optimal pump perfusion pressure, cyclic pump pressure and pumps per minute must be obtained. In the poloxamer, optimal temperature range must be found to maintain the proper viscosity. The poloxamer must be a viscosity sufficiently high so that the air bubbles produced through the perfusion conduction pipets are stable and maintained. Basic environmental and cell culture optimizations can be obtained through literature.

Alveolar Structure through Perfusion

        Once optimization has been achieved, the building of the poloaxmer scaffold structure can begin. A bath of liquid poloxamer will be maintained at temperature <23°C (to ensure that the poloxamer will be in a low viscosity liquid phase) in bioreactor. In the bath, bundles of the perfusion conduction pipettes are lowered (spearheaded by the perforated terminus) into the thermosensitive at optimal depth in the polymer and the cyclic perfusion pump will pressurize the perfusion conduction pipettes producing the bubbles that mimic the size and shape of alveoli.

Fig 2: Schematic modeling of the air/liquid interface where z is the position of the bubble top. The curvature radius is assumed to be constant for z<z0 [8]

The flow and pressure rate that will be used to obtain the desired alveolar shape and size will be based on the studies of air bubbling done with bifurcation diagrams[8]. Essentially, the growth rate of the bubble volume is proportional to the pressure distance to the equilibrium pressure and the pressure growth rate is proportional to the difference between air flow and volume grown (multiplied by a reference pressure po).

While the perfusion pipettes create air bubbles, the temperature of the poloxamer will be slowly raised, over a period of time, to 37°C in order to ‘capture’ the air bubble morphology within the scaffold. Each individual air bubble should have a diameter around 200 um. This achieves an “inverted” alveolar scaffold that is ready for cell seeding.

Cell Seeding and Alveolar Development

Previous study from Wang et al 2007, showed repopulation of lung alveolar from Alveolar Type II cells derived from HESC. With ES cells isolated from the ICM of embryos, the ATII cells are derived with known methods via embryonic body formation or coculture of EBs with pulmonary mesenchyme. [1][2] It is important to note that this method is not efficient, generating only small percentage of ESC derived ATII cells. [3 ] Not only so, a mixed population will not be suitable for transplantation into the inverted scaffold. After obtaining the necessary amount of ATII cell, they are seeded on top of the feeder layer on the inverted scaffold. As for the ATI cells, they are obtained from method used by Fuchs et el 2002. [4] The ATI cell are then seeded on top of the ATII layer. Then we will seed the alveolar macrophages as well as following transcriptional factors: Nitric Oxide, prostaglandins, interleukin-4 and -10, and TGF-beta. These are secreted by macrophages to prevent uncontrolled inflammation and prevent collateral damage to ATI and ATII cell. [5][6][7][8] In case of our method fails, there is a study by Ridge et el, 2003 showin g the expression of alpha-2-Na,K-ATPase, which helps to lung liquid clearance for ATI cells. We will add ATPase with the other transcriptional factors, however, they were not tested on humans and only on the mouse. We will also attempt to change the concentration of ATI and ATII cells seeded. In in vivo setting, ATI and ATII represent approximately 95% and 5% of the alveolar surface area, respectively. we will change the variation of the concentration.[9]

Scaffold Degradation and Alveoli Extraction

        The synthetic functional alveoli can be extracted once the ATI/II cells have fully developed and are functional. Degradation of poloxamers can be achieved through gentle organic solvents and a sustained decrease of temperature to below 23°C, which will induce to poloxamer to enter liquid form. The fully functional Alveoli can be stored in media and bioreactor held at 37ºC in high partial pressure of oxygen environments till needed.

Product Characterization and Validation

Characterization

Ideally, our lung scaffold would be synonymous with actual alveoli and lung tissue. In terms of cellular composition, this should hold true since the scaffold is seeded with the exact lung cells we wish to mimic, either allogeneic or autologous. The growth factors that we apply to the scaffold will also maintain the cellular integrity of our product.  Furthermore, thanks to our pressure-driven bubbling technique, the scaffold’s morphological detail resembles an alveolar network, but the bubbles we form are ultimately fairly random and crude. It is difficult to faithfully reconstruct the intricacies of an actual alveolar network. However, making them as true to form as possible is viable, but requires a very fine degree of control in scaffold development and bubble formation.

In characterizing our product, some parameters are unknown until further testing and validation. For example, the structural integrity of the ECM and scaffold as a whole is potentially fragile – and as of yet, we do not know the degree to which this is true. And while we are fairly certain that our scaffold looks like alveolar tissue and is made from the right cells, another feature we must verify is whether or not this scaffold can perform and support real alveolar functions, such as dealing with perpetual blood flow and gas exchange.

Validation

We can validate the characterization of our alveolar scaffold through histological analysis and comparison to organic alveoli. To test oxygen perfusion of our product, we can submerge our scaffold into blood or some blood-like fluid and pump oxygen into the alveoli, mimicking gas exchange in the body. Alternatively, we can pump oxygen poor/rich blood through the scaffold continuously to simulate the flow of carbon dioxide rich blood through the lungs. These tests may or may not be performed in a bioreactor – either is feasible. Measuring the concentration of oxygen inside the bath/blood before and after oxygen diffusion can give us an idea of whether or not our lung scaffold effectively engages in oxygen perfusion.

As a post-experimental analysis, we may also section a scaffold sample to perform additional experiments and assays to verify various properties of product. We can measure and observe the size of the bubbles to give us insight into any morphological change or structural weaknesses in the scaffold. We may perform various stains/concentration measurements as well. We would need to section carefully, however, to ensure minimal loss of structural integrity. We may use fixation/freezing and preservation of the scaffold in formaldehyde solution prior to sectioning/processing, then recover the original state by dehydration with solvents and a heat bath. We may also use very precise equipment, such as a microtome knife, for precise cutting.

Discussion

        This proposal highlights a novel technique which involves the creation of an inverted scaffold with the use of reverse thermal gelation in order to develop a model that will mimic and structure and function of alveoli. This method, called bubbling, is made possible with the use of thermosensitive poloxamers and pipettes before the seeding the scaffold with Alveolar Type II cells. After incubation, the inverted scaffold dissolves, leaving behind alveolar-like structures which can then be used as pathogenic or clinical models.

        Considering that this design introduces a novel technique and hence there are yet no publications that can demonstrate the weakness of such technique, we have yet to know what improvement are to be made to our existing design. However, a number of modifications can be made from the use of polymer to seeding techniques. While poloxamer have been well used in the industry since their formulation in 1973, we are open to the idea of using alternative polymers that exhibit reverse thermal gelation such as poly (ethylene gloycol)-block-poly(alanine-co-phenyl alanine) (PEG-PAF) [12]. While promising for biomedical applications, this polymer was recently developed and more studies need to be done in order to deem it compatible with our needs. The pipette material may also be modified to a material like chitosan[13] in order to optimize the biocompatibility and the mechanical stability of the alveolar model.

        All the materials that will be used for this design experiment exists and have been validated by previous studies[6][7][11]. However, the validation of materials hardly correlates to feasibility of the overall design. Preliminary studies must be made that will determine the optimal diameter of perfusion pipettes, the optimal pressure range and viscosity range (between 23°C and 37°C) that will form the air bubbles the same size and shape as alveoli, and seeding techniques that will develop favorable ECM formation. While this design holds promise as a model to better understand the specific mechanics of alveoli and, more specifically, to better understand the exchange properties between a normal alveoli and pathological alveoli, a limiting factor to take into consideration is the time it may take to develop a single alveolar structure. An alternative to this design is to focus on better imaging techniques in order to study alveolar gas exchange properties in vivo, but even with the advancement in computer tomography (CT), magnetic resonance imaging (MRI)[14], and fourier domain optical coherence tomography (FDOCT)[15] there is still a lack of quality in in vivo alveolar resolution and thus, makes it difficult to study the mechanics of functioning alveoli.

References

Basic Physiology

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Poloxamers

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Lung on a Chip

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Cell Seeding

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Discussion

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