Lung Tissue Engineering The Current State of Play

The biological function of the lung is to facilitate exchange of oxygen and carbon dioxide between the cardiovascular circulation and the environment. The lung is a physiologically and anatomically complex vital organ that is hallmarked by a compartmentalized tissue architecture. It is convenient to divide the lung into two anatomically and functionally distinct units, namely the proximal and distal airways. The proximal airways (and proximal pulmonary arteries) are smooth muscle-lined conduits that control the amount of air (and blood) reaching the distal gas exchange region; they are also known as the alveoli. The functional components of the alveoli are the distal epithelium and microvasculature.

The use of tissue engineering to generate respiratory tissue models for both clinical and basic science applications has focused primarily on reconstruction of the trachea and proximal airway structures (bronchial tissue). These models consisted of collagen gels pre-seeded with tissue-derived fibroblasts on top of which an epithelial layer is seeded following a fibroblast conditioning period. The selection of collagen type I as a matrix is straightforward because the cell-cell interactions between fibroblasts and epithelial cells are critical for the formation of an organotypic basement membrane and the differentiated epithelium. Depending on the cell source used, engineered bronchial tissue models are capable of re capitulating both normal physiological as well as asthmatic features [128-130]. Models of proximal lung tissue constructs might be used for therapeutic purposes; an example would be as bronchial tissue patches to repair damaged bronchial tissue, and/or for HTS of respiratory toxicants enhancing lung permeability, or for developing drugs to treat respiratory diseases.

In the distal lung, the alveolar epithelium is composed of two distinct cell types, alveolar type I (AE1) and alveolar type II (AE2) cells. Although both cell types are found in approximately equal numbers, AE1 cells cover 95% of the alveolar surface, which makes their presence especially pertinent to the study of transport across the air-blood barrier. The essential physiological function of AE1 cells is establishment of the air-blood interface along with capillary endothelial cells (ECs). The majority of the AE2 cells lie basal to the alveolar surface, and perform numerous essential functions in the alveoli, including the production and secretion of surfactant lipids and proteins into the alveolus (i.e., the lubricant required for alveolar protection, gas exchange and antimicrobial defense). AE2 cells are critical in repair and remodeling following injury, serving as "progenitor cells" that divide, migrate and differentiate in response to insult or injury. In attempting to design an organotypic alveolar tissue model, the situation is further complicated by the contributions of the microvascular ECs and interstitial fibroblasts to the microenvironment.

In traditional 2D culture, AE2 cells lose many of their specialized features such as the ability to produce surfactant [131], which suggests that AE2 cells rapidly dedifferentiate when not provided with an appropriate cellular microenvironment. The optimization of ECM composition [132], growth factor [133] and hormone [134] composition of the medium has improved the maintenance of differentiated AE2 functions in vitro.

During the past year, the first reports have emerged of lung alveolar tissue models generated by tissue engineering approaches. Chen et al. [135] reported the formation of lung-alveolar-like structures following extended in-vitro culture of rat fetal pulmonary cells on collagen-chrondroitin-6-sulfate composite 3D scaffolds fabricated by freeze-drying and crosslinking. The use of a collagen base modified with chondroitin sulfate - an element ofthe ECM known to be required for epithelial morphogenesis [136] - is a paradigmatic example of engineering scaffolds with the goal of recapitulating the organotypic ECM. Recently, we have developed a tissue engineering approach for the generation of 3D alveolar tissue constructs with appropriate tissue morphology and cytodifferentiation using diverse biomatrices in combination with a growth factor-supplemented defined serum-free medium [133]. The goal was to recapitulate the native tissue architecture and cellular organization (Fig. 1.6A), and Matrigel™ and type I collagen hydrogels were used successfully to generate 3D alveolar tissue constructs with appropriate tissue architecture. Examples of this were branching epithelial morphogenesis and differentiation (Fig. 1.6B), as well as the establishment ofa primitive microvascular network found in apposition to alveolar epithelial cells (Fig. 1.6C).

In more recent studies we have shown that micro and nanofibrous scaffolds electrospun from natural ECM proteins, such as elastin, in cooperation with

Proteins Extracted From Lungs Tissues

Fig. 1.6 Schematic of alveolar structure and immunophenotyping of engineered distal lung tissue constructs. (A) Diagram of alveolar structure and function (adopted from the NIH (NHLI) website). (B) Cytokeratin staining (brownish-red) of alveolar-forming structure following 7 days of 3D culture of fetal pulmonary cells in Matrigel™. (C) Cytokeratin (epithelial cells, green) and isolectin B4 (endothelial cells, red) double-labeling of fetal pulmonary cell construct generated in type I collagen gel with tissue-specific growth factors.

Fig. 1.6 Schematic of alveolar structure and immunophenotyping of engineered distal lung tissue constructs. (A) Diagram of alveolar structure and function (adopted from the NIH (NHLI) website). (B) Cytokeratin staining (brownish-red) of alveolar-forming structure following 7 days of 3D culture of fetal pulmonary cells in Matrigel™. (C) Cytokeratin (epithelial cells, green) and isolectin B4 (endothelial cells, red) double-labeling of fetal pulmonary cell construct generated in type I collagen gel with tissue-specific growth factors.

tissue-specific growth factors, support histiotypic co-morphogenesis of epithelial and endothelial tissue components, as required for engineering a high-fidelity alveolar tissue model [137].

Natural ECM proteins (e.g., laminin) promote AE2 morphology and differentiation in vitro. A plethora of in-vitro studies has been conducted on the effect of the ECM substrate used for culture on alveolar epithelial cells, and from these investigations much information can be applied to the development of appropriate 3D biomatrices for lung tissue engineering. However, future studies on how these matrices affect other cell types of the alveoli, such as the microvascular ECs, will be required in order to develop a truly organotypic tissue culture model. A summary of some relevant findings on the effects of various ECM constituents on alveolar epithelial cell differentiation and morphogenesis in diverse 2D and 3D in-vitro culture systems is provided in Table 1.2.

Table 1.2 Important effects of the extracellular matrix (ECM) on alveolar cell and tissue culture models generated in vitro.

Cell source

ECM/Scaffold

Major findings

Ref.

Mixed primary mouse fetal lung cells

Matrigel™, PLGA foams, PLLA fibers

Branching morphogenesis and AE2 differentiation for up to 4 weeks in Matrigel™ with growth factor-defined medium. Synthetic polymers did not support AE2 differentiation

[131] [135]

Mixed primary rat fetal lung cells

Gelatin sponge matrix

Formation of alveolar-like strucUires for up to 6 weeks with progressive AE2 de -differentiation

[149]

-

Fibronectin or laminin

Epithelial cells and fibroblasts expressed a^Pj, a6Pj, and the 65 kDa laminin-elastin receptor. Enhanced adhesion of epithelial cells on laminin was observed

[235]

-

Collagen-GAG (chondroitin-6-sulfate) porous scaffold

Mixed primary cultures formed histiotypic alveolar-like strucUires for up to 3 weeks in vitro. Matrices contracted over time

[133]

Fetal rat lung organ explants

N/A

Chlorate disruption of chondroitin sulfate proteoglycans in the tissue ECM resulted in arrested epithelial branching morphogenesis and reduced expression of AE2 genes

[134]

Primary fetal rat AE2 cells

Laminin, fibronectin, vitronectin, collagen, or elastin

ECM proteins supported ERK activation upon mechanical stimulation; however, laminin induced surfactant protein C expression and AE2 differentiated phenotype

[236]

Primary neonatal and adult rat AE2 cells

MDCK cell-derived ECM

Combination of M DCK basement membrane, dexamethasone and cyclic AM P maintained surfactant protein expression in neonatal, but not adult AE2 cells

[132]

SV40-T2 immortalized adult rat AE2 cell line

Fibrillar collagen matrix imbibed with Matrigel™

SV40-T2 cells formed a continuous lamina densa containing laminin, collagen IV, entactin and perlecan with Matrigel™ supplementation, but not without

[137]

Primary adult rat AE2 cells

Acellularized human alveolar tissue

AE2 cells flattened and took on an AEl-like phenotype, while AE2 cells on amnionic membrane maintained differentiation and morphology

[237]

-

Pulmonary endothelial-derived ECM

Mitogenic stimulation, well spreading, loss of lamellar bodies (AEl-like phenotype)

[220]

-

Type I collagen gels

AE2 cells proliferation and alveolar-like strucUires differentiation

[150]

Cell source

ECM/Scaffold

Major findings

Ref.

-

Fibronectin or type I collagen

AEC migration enhanced on fibronectin. OyP, antibody inhibited fibronectin migration, while a, inhibited migration on type I collagen

[238]

-

Fibronectin or Matrigel™

ECM modulates gap junction expression. Fibronectin increases connexin43, while Matrigel™ promotes connexin26

[239]

-

Mixture of type I collagen and Matrigel™

KGF in combination with this ECM mixture supported maximal surfactant protein A and D secretion as compared to HGF and FGF-10 media supplementation

[152]

-

Fibronectin

FGF-1 increases DNA synthesis and AE2 attachment to fibronectin

[240]

-

Collagen I, fibronectin, and laminin-5

Both collagen type I and laminin-5 were needed for prolonged maintenance of the AE2 phenotype. In the absence of laminin-5, AE2 cells tended to take on an AEl-like phenotype

[241]

Adult rat AE2/AE1 mixed cultures

Fibronectin-collagen type I-laminin-5

Culture on this ECM combination maintained mixed cultures of AE2 and AE1 cells with ratios similar to those found in human alveoli in situ

[130]

Guinea pig AE2 cells

Acellularized human amnionic membrane

Acellularized human amnionic membrane and fibroblast co-culture or KGF supplementation maintained AE2 morphology and differentiation

[242]

Day 29 fetal rabbit AE2 cells

Matrigel™

Basement membrane ECM maintained AE2 morphology and differentiation for 3 weeks, while cells dedifferentiated on plastic by 5 days

AE1 = alveolar type I

AE2 = alveolar type II

TGF-P = transforming growth factor beta

ECM = extracellular matrix

ERK = extracellular signal-related kinase

FGF = fibroblast growth factor

GAG = glycosaminoglycan

KGF = keratinocyte growth factor (a.k.a. fibroblast growth factor-7) MDCK = Madin-Darby canine kidney

Review of this information is helpful in the selection and design of biomaterials and biomatrices, respectively, for engineering alveolar tissue models in vitro.

In the developing lung, the complex basal lamina on which the epithelium resides is composed mainly of type IV collagen, laminin and a tissue-specific mix of glycosaminoglycans [138]. The importance of the ECM composition (specifically laminin) for modulating AE2 cell structure and function has been well documented [139]. Early studies by others [140], as well as our data, have shown that AE2 cells grown two-dimensionally on Matrigel™ retain their differentiated form when compared with cells cultured on plastic surfaces. In contrast to the differentiating effects of Matrigel™, non-specific ECM produced by ECs failed to maintain an AE2 cell phenotype [141]. Results from our laboratory indicate that mixed populations of fetal pulmonary cells cultured on synthetic scaffolds in conventional serum-enriched medium do not maintain AE2 cell differentiation for extended periods [133]. Taken together, these findings indicate that the ECM composition is a key parameter in the design of an optimal 3D alveolar tissue model for pharmaceutical purposes.

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