Elastin-like protein matrix reinforced with collagen microfibers for soft tissue repair

Abstract

Artificial composites designed to mimic the structure and properties of native extracellular matrix may lead to acellular materials for soft tissue repair and replacement, which display mechanical strength, stiffness, and resilience resembling native tissue. We describe the fabrication of thin lamellae consisting of continuous collagen microfiber embedded at controlled orientations and densities in a recombinant elastin-like protein polymer matrix. Multilamellar stacking affords flexible, protein-based composite sheets whose properties are dependent upon both the elastomeric matrix and collagen content and organization. Sheets are produced with properties that range over 13-fold in elongation to break (23-314%), six-fold in Young's modulus (5.3-33.1 MPa), and more than two-fold in tensile strength (1.85-4.08 MPa), exceeding that of a number of native human tissues, including urinary bladder, pulmonary artery, and aorta. A sheet approximating the mechanical response of human abdominal wall fascia is investigated as a fascial substitute for ventral hernia repair. Protein-based composite patches prevent hernia recurrence in Wistar rats over an 8-week period with new tissue formation and sustained structural integrity.

Figure 1

Fabrication of a collagen microfiber reinforced elastin-like protein sheet. (a) Collagen microfiber is wound about rectangular frames to obtain the desired orientation and average spacing. (b) A cooled protein polymer solution is distributed over the microfiber layout and molded into a thin membrane through a temperature-drive sol gel process. (c) Stacked membranes are laminated by a temperature transition to yield multilamellar, angle-ply composites (d, e).

Figure 2

Structural analysis of a multilamellar sheet. As illustrated in (a), the fiber cross-section will appear circular or elliptical when examined by SEM after sectioning along the x- or y-plane (c and d, respectively). Sheets did not display evidence of delamination (b, c, d). Sectioning artifacts appeared in (c) as vertical microgrooves and in (d) as feathered horizontal ridges in the protein polymer, indicated by arrows. Three-dimensional reconstructions using the DVI technique display the fiber component in (e). Transmission electron microscopy sections taken in the plane of the collagen fibers demonstrated a D-periodic banded fibrillar structure, aligned with the overall fiber axis (f), while sections perpendicular to the collagen fiber revealed densely packed fibril cross sections (g). Analysis of collagen fiber by TEM has demonstrated a mean diameter of 33 ± 4 nm and a fibril density of 73 ± 6% (v/v) [19].

Figure 3

Mechanical responses. (a) Composites with fiber angles of 0° (●), 15° (•••), 25° (---), 90° (—), and without fiber (○) demonstrate microfiber alignment with loading enhances stiffness and decreased strain at fiber damage. (b) Designs with 15° orientation and average microfiber fractions of 18% (●), 7% (---), and 3% (○), and without fiber (—) indicate that increasing microfiber content elevates stiffness and strength without changing strain at fiber damage. (c) The mechanical response of the 25° orientation (—) resembled human linea alba. (Symbols represent tissue strips from the infraumbilical or supraumbilical region tested in the oblique or transverse orientations, averages of responses reported by Gräβel and coworkers [29]. (d, e) The 0° and 15° composites after microfiber staining, images oriented with the loading direction horizontal (scale 2 mm).

Figure 4

Dependence of mechanical properties on microfiber layout. Increased fiber fraction and alignment to the loading direction increased modulus (a, e). Increased fiber fraction and alignment also tended to increase the stress at which specimens displayed fiber network damage (b, f). Increased fiber fraction did not significantly enhance UTS, but alignment of fibers in the loading direction enhanced UTS compared to higher fiber angles (c, g). Resilience increased with increasing fiber fraction (d), and was also elevated when fibers were aligned or angled to the loading direction compared to the perpendicular layout (h). Significance indicated at the p<0.05 level, ns indicates non-significant differences, error bars represent standard deviations.

Figure 5

Abdominal defect repair. Appearance of the multilamellar elastin-composite immediately following implant (a) and at 8 weeks (b). None of the repaired defects demonstrated hernia formation for the duration of the study (c), as compared to unrepaired defects (d). Scale 10 mm.

Figure 6

Histology of abdominal repair materials. The appearance of non-implanted multilamellar protein composite sheets and the porcine dermis product is shown in (a) and (d), respectively (100×). After 8 weeks, the elastin-like protein component of the engineered composites appeared largely absent, except in rare areas (b, c). In contrast, collagen microfibers were observable throughout the specimens (b, 40×, elastin-like protein and collagen fiber indicated with solid and dashed arrows, respectively). In regions where cells and fibrous tissue replaced the elastin-like protein, the spacing between collagen fibers increased (c, 100×). The dense collagen of the porcine dermis product appeared to have separated, with cell and tissue ingrowth between implant fragments (right side of e, 40×, and in g, 100×). The arrows in (g) indicate implant fragments. In (e) the host-implant interface is apparent, with the abdominal wall at left in and the porcine dermis at right. Identifiable fragments of the porcine dermal product were absent from many regions of the harvested parches (f, 200×). Scale bars 200 µm.