Preservation of micro-architecture and angiogenic potential in a pulmonary acellular matrix obtained using intermittent intra-tracheal flow of detergent enzymatic treatment

Abstract

Tissue engineering of autologous lung tissue aims to become a therapeutic alternative to transplantation. Efforts published so far in creating scaffolds have used harsh decellularization techniques that damage the extracellular matrix (ECM), deplete its components and take up to 5 weeks to perform. The aim of this study was to create a lung natural acellular scaffold using a method that will reduce the time of production and better preserve scaffold architecture and ECM components. Decellularization of rat lungs via the intratracheal route removed most of the nuclear material when compared to the other entry points. An intermittent inflation approach that mimics lung respiration yielded an acellular scaffold in a shorter time with an improved preservation of pulmonary micro-architecture. Electron microscopy demonstrated the maintenance of an intact alveolar network, with no evidence of collapse or tearing. Pulsatile dye injection via the vasculature indicated an intact capillary network in the scaffold. Morphometry analysis demonstrated a significant increase in alveolar fractional volume, with alveolar size analysis confirming that alveolar dimensions were maintained. Biomechanical testing of the scaffolds indicated an increase in resistance and elastance when compared to fresh lungs. Staining and quantification for ECM components showed a presence of collagen, elastin, GAG and laminin. The intratracheal intermittent decellularization methodology could be translated to sheep lungs, demonstrating a preservation of ECM components, alveolar and vascular architecture. Decellularization treatment and methodology preserves lung architecture and ECM whilst reducing the production time to 3 h. Cell seeding and in vivo experiments are necessary to proceed towards clinical translation.

Fig. 1

Decellularization via the vascular route reduces DNA (p < 0.05), following 1 cycle of treatment (A), with histological examination demonstrating an incomplete decellularization with nuclear and cytoplasmic material present (D, G, J, M). Further cycles do not reduce DNA significantly further. Decellularization from both vascular and tracheal routes leads to a reduction in DNA (p < 0.05) after 2 cycles with no significant decrease in 3 and 4 cycles (B). Histology confirms the removal of almost the entirety of nuclear and cytoplasmic material, with weak cellular staining being present on the lobar edges (E, H). Staining with with EVG and AB confirm a preservation of elastin and GAG (K, N). Decellularization via the tracheal route reduces DNA (p < 0.05) after only 1 cycle of treatment (C). Histology staining confirms an absence of nuclear material (F, I) and maintenance of elastin and GAG (L, O); IV: Intravascular, IV/IT: intravascular/intratracheal, IT: intratracheal, H&E: hematoxylin and eosin, MT: Masson's trichrome, EVG: elastin Van Gieson, AB: alcian blue, *: P < 0.05, †: P < 0.01, all statistical comparisons to fresh tissue, scale bar: 100 μm.

Fig. 2

Decellularization using an intermittent intratracheal decellularization methodology yields an acellular scaffold that is macroscopically transparent (A) in a shorter time-interval when compared to the continous intratracheal protocol. Cellular material is removed equally well to the continuous methodology as evident by H&E staining and DNA quantification; H&E: hematoxylin and eosin, *: P < 0.05, all statistical comparisons to fresh tissue, scale bar: 100 μm.

Fig. 3

SEM demonstrates that in decellularized lung obtained with continuous inflation the bronchovascular tree (A, B) and alveolar network (D, E) are maintained but in a poor manner with weak collagen fibrils. Scaffolds obtained with intermittent intratracheal inflation show a complete preservation of bronchopulmonary structures (A, C), alveolar ducts and alveolar network (D, F) with an absence of cell nuclei. TEM of fresh lung shows the alveolar-capillary junction with red blood cells in close contact to the alveoli (asterisk, G), which following continuous decellularization, ruptures at a number of areas (black arrows, H), with exposure of the collagen fibers to the alveolus (white arrow, H). In sharp contrast, the intermittent approach leads to maintenance of the basement membrane and collagen fibers (I); SEM: scanning electron microscopy, TEM: transmission electron microscopy.

Fig. 4

Pulsatile trypan blue injection of the pulmonary artery demonstrates progressive dye distribution from the central areas of the scaffold (A, B) to the periphery where fine branching of the capillaries is visible (C, D). Morphometry analysis of fresh lung and acellular scaffold (E) shows a significant decrease in non-parenchymal fractional volume (P < 0.001) and a corresponding increase in alveolar fractional volume (P < 0.05). Quantitative analysis of alveolar size indicates no differences in alveolar dimension following decellularization (F). Decellularized rat lungs show significantly increased airway elastance (G) and elastance (H) at positive end expiratory pressures (PEEP) of 3 and 6 cm H₂O (p < 0.05); N: non-parenchymal fractional volume, S: septal fractional volume, A: alveolar space fractional volume, C: conducting airway fractional volume, AU: arbitrary units, *: P < 0.05, ‡: P < 0.001, all statistical comparisons to fresh tissue, scale bar: 100 μm.

Fig. 5

MT staining demonstrates a hierarchical alveolar network, with preservation of collagen in both central and peripheral areas (A). EVG and AB staining show preservation of elastic fibers and GAG (B, C). Immunofluorescence for collagen I, collagen III and laminin shows maintenance of the ECM components with no DAPI staining in the scaffolds when compared to fresh tissue (E–G). GAG quantification demonstrates a reduction to 19% of the fresh value (P < 0.01) (H); MT: Masson's trichrome, EVG: elastin Van Gieson, AB: alcian blue, GAG: glycosaminoglycans *: P < 0.05, †: P < 0.01, all statistical comparisons to fresh tissue, scale bar: 100 μm.

Fig. 6

The angiogenic properties of the scaffold are demonstrated in vivo following placement on top of the chicken chorioallantoic membrane. On days 7 after implantation, the number of vessels converging towards the lung matrices is significantly increased in comparison to the same samples at day 0 (P < 0.05) and to the polyester membrane that was used as a negative control (P < 0.01) (A). Representative images of lung tissue placed in ovo at 0 (B), and 7 days (C) Of incubation, indicate attraction of blood vessels that, in a cogwheel fashion, seem to penetrate the tissue. The polyester control, at the same time-points, has no effect on vascular development around it (D,E); *: P < 0.05, comparison to scaffold, day 0 §: P < 0.01, comparison to control, day 7.

Fig. 7

The decellularized sheep scaffold represents a solid mass that maintains the architecture of the original lung, whilst being transparent indicating a lack of cells (A). Decellularization is achieved at 12 cycles with H&E (B) and DNA analysis (P < 0.05) confirming the absence of nuclear material. SEM shows no visible cells and preservation of bronchovascular structures (D), alveolar ducts and network (E), as well as the capillaries that are in close contact with the alveoli (asterisk, F); *: P < 0.05, all statistical comparisons to fresh tissue, scale bar: 100 μm.

Fig. 8

TEM shows maintenance of the basement membrane and collagen fibrils (A). MT staining indicates, similarly to the rat scaffold, a hierarchical collagenous network (B). EVG and AB staining display preservation of elastin and GAG (C, D). GAG quantification demonstrates a non-significant decrease in GAG values when compared to fresh tissue (E); H&E: hematoxylin and eosin, MT: Masson's trichrome, EVG: elastin Van Gieson, AB: alcian blue, GAG: glycosaminoglycans, SEM: scanning electron microscopy, TEM: transmission electron microscopy, *: P < 0.05, †: P < 0.01, all statistical comparisons to fresh tissue, scale bar: 100 μm.