Matrix composition and mechanics of decellularized lung scaffolds

Abstract

The utility of decellularized native tissues for tissue engineering has been widely demonstrated. Here, we examine the production of decellularized lung scaffolds from native rodent lung using two different techniques, principally defined by use of either the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or sodium dodecyl sulfate (SDS). All viable cellular material is removed, including at least 99% of DNA. Histochemical staining and mechanical testing indicate that collagen and elastin are retained in the decellularized matrices with CHAPS-based decellularization, while SDS-based decellularization leads to loss of collagen and decline in mechanical strength. Quantitative assays confirm that most collagen is retained with CHAPS treatment but that about 80% of collagen is lost with SDS treatment. In contrast, for both detergent methods, at least 60% of elastin content is lost along with about 95% of native proteoglycan content. Mechanical testing of the decellularized scaffolds indicates that they are mechanically similar to native lung using CHAPS decellularization, including retained tensile strength and elastic behavior, demonstrating the importance of collagen and elastin in lung mechanics. With SDS decellularization, the mechanical integrity of scaffolds is significantly diminished with some loss of elastic function as well. Finally, a simple theoretical model of peripheral lung matrix mechanics is consonant with our experimental findings. This work demonstrates the feasibility of producing a decellularized lung scaffold that can be used to study lung matrix biology and mechanics, independent of the effects of cellular components.

Fig. 1

Diagram of model used to study tissue mechanics. A strip of lung tissue is modeled as n elements in series. Each element is a spring (with spring constant k, modeling an elastin fiber) in parallel with a string (with length l, modeling a collagen fiber). A force F is applied to the tissue, causing extension of the springs and elongation of the strings.

Fig. 2

Native and decellularized lung. a, b Hematoxylin and eosin histological stain of native (a) and decellularized (b) lung demonstrates loss of cellular and nuclear material after decellularization. Scale bars are 50 μm. c, d DAPI stain shows DNA in blue of native (c) and decellularized (d) lung and demonstrates loss of organized nuclear material in decellularized lung, although some faint DNA is distributed in the decellularized tissue. Scale bars are 20 μm. e–f Transmission electron microscopy of decellularized lung showing retained alveolar septa. Scale bars are 20 μm in e and 10 μm in f.

Fig. 3

Histochemical staining of native and decellularized lung. a–c Verhoeff-van Gieson staining for elastin in native lung (a), CHAPS-decellularized lung (b), and SDS-decellularized lung (c) shows retained but diminished elastin fibers (black). d–f Alcian blue staining for GAGs (blue) in native lung (d), CHAPS-decellularized lung (e) and SDS-decellularized lung (f) shows loss of GAGs in both CHAPS- and SDS-decellularized lung (blue). Scale bars in a–c are 20 μm and in d–f are 50 μm.

Fig. 4

Stress-strain curves. a Elastic cycling of tissue strips demonstrates hysteresis in native (red), CHAPS-treated decellularized (green) and SDS-treated decellularized lung (blue). b Tensile strength testing indicates similar curve profiles for native and CHAPS-treated decellularized lung, but weaker tissue with lower ultimate tensile stress after SDS treatment.

Fig. 5

Modeling studies of lung tissue strips. Solid lines are native lung samples, blue × marks are CHAPS-treated decellularized lung samples, and red circles are SDS-treated decellularized lung samples (n = 5 for each). a Distribution of collagen fiber stop lengths showing similar distribution of stop lengths for native and acellular lung, with a nonsignificant trend toward shorter stop lengths in SDS-treated lung (arrow). b Distribution of elastic spring constants are similar in all tissues, though tend to lower numbers overall for SDS-treated lung.