Patient-to-patient variability in autologous pericardial matrix scaffolds for cardiac repair

Abstract

The pursuit of alternate therapies for end-stage heart failure post-myocardial infarction has led to the development of a variety of in situ gelling materials to be used as cellular or acellular scaffolds for cardiac repair. Previously, a protocol was established to decellularize human and porcine pericardia and process the extracellular matrix (ECM) into an injectable form. The resulting gels were found to retain components of the native extracellular matrix; cell infiltration was facilitated in vivo, and neovascularization was observed by 2 weeks. However, the assertion that an injectable form of human pericardial tissue could be a potentially autologous scaffold for myocardial tissue engineering requires assessment of the patient-to-patient variability. With this work, seven human pericardia from a relevant patient demographic are processed into injectable matrix materials that gel when brought to physiologic conditions. The resulting materials are compared with respect to their protein composition, glycosaminoglycan content, in vitro degradation, in vivo gelation, and microstructure. It is observed that a diminished collagen content in a subset of samples prevents in vitro gelation but not in vivo gelation at lower ECM concentrations. The structure is similarly fibrous and porous across all samples, implying the cell infiltration may be similarly facilitated. The biochemical composition as characterized by tandem mass spectrometry is comparable; basic ECM components are conserved across all samples, and the presence of a wide variety of ECM proteins and glycoproteins demonstrate the retention of biochemical complexity post-processing. It is concluded that the variability within human pericardial tissue specimens does not prevent them from being processed into injectable scaffolds; therefore, pericardial tissue offers a promising source as an autologous, injectable biomaterial scaffold.

Fig. 1

Decellularization verification. Hematoxylin and eosin staining on 10 μm sections of decellularized samples reveals a lack of nucleic material (A–G) in all seven human pericardium samples (HP-A through HP-G) compared to the section of a fresh sample of HP-A (H), scale bar is 1 mm.

Fig. 2

SDS-PAGE. An Imperial Protein stain of samples run on a poly-acrylamide gel indicates variable protein content, specifically lower collagen content in HP-D and HP-F.

Fig. 3

Peptide mass distribution. Precursor mass information for all peptides was derived from the MS/MS data, here binned in 1 kDa increments to identify differences in the mass distribution, such as the left-ward shift of peptides in HP-B.

Fig. 4

Scanning electron microscopy. ESEM images reveal a fibrous network in each sample (A–G). Quantification of fiber diameter (H) reveals no statistical difference across samples – all with approximately 200 nm fibers (p = 0.19). HP-A through HP-E (A–E) were gelled at 6 mg/mL and HP-F and HP-G (F, G) were gelled at 8 mg/mL. Fiber diameter data shown as mean ± standard deviation.

Fig. 5

Storage modulus. Due to high variability within each matrix gel sample, all samples are statistically similar (p = 0.06). HP-F and HP-G were gelled at 8 mg/mL, all other samples were gelled at 6 mg/mL. Data is presented as mean ± standard deviation.

Fig. 6

Degradation. Ninhydrin reactivity after 2, 4, and 12 hours of incubation with collagenase. Absorbance values indicates a variable protein content across the gels, though the samples degrade in a similar fashion.

Fig. 7

In vivo gelation. Hematoxylin and eosin stain of the injection region identified on a short-axis cross-section. Within 45 minutes of injection, all materials form a porous gel visible with H&E. Arrows indicate injection region. Scale bar is 1 mm (A–G); scale bar is 500 μm (H).