Efficacy of engineered FVIII-producing skeletal muscle enhanced by growth factor-releasing co-axial electrospun fibers

Abstract

Co-axial electrospun fibers can offer both topographical and biochemical cues for tissue engineering applications. In this study, we demonstrate the sustained treatment of hemophilia through a non-viral, tissue engineering approach facilitated by growth factor-releasing co-axial electrospun fibers. FVIII-producing skeletal myotubes were first engineered on aligned electrospun fibers in vitro, followed by implantation in hemophilic mice with or without a layer of core-shell electrospun fibers designed to provide sustained delivery of angiogenic or lymphangiogenic growth factors, which serves to stimulate the lymphatic or vascular systems to enhance the FVIII transport from the implant site into systemic circulation. Upon subcutaneous implantation into hemophilic mice, the construct seamlessly integrated with the host tissue within one month, and specifically induced either vascular or lymphatic network infiltration in accordance with the growth factors released from the electrospun fibers. Engineered constructs that induced angiogenesis resulted in sustained elevation of plasma FVIII and significantly reduced blood coagulation time for at least 2-months. Biomaterials-assisted functional tissue engineering was shown in this study to offer protein replacement therapy for a genetic disorder such as hemophilia.

Figure 1. Core-shell electrospun fibers to provide sustained protein delivery

TEM (a) and SEM (b) images of the co-axially electrospun fibers. (c) Mono-axial fibers serve as a comparison to illustrate the core-shell structure. SEM (d) and confocal (e) images of encapsulated FITC-BSA within aligned co-axial fibers demonstrated uniform protein distribution within the fibers, compared to clusters of precipitated proteins in mono-axially electrospun fibers (f). Controlled release study of the encapsulated growth factors (PDGF and VEGFA in (g) and VEGFC in (h)) demonstrated that proteins were released in a sustained manner over a period of 30 days.

Figure 2. Skeletal myotubes engineered on electrospun fibers

GFP⁺ myoblasts were seeded on aligned fibers and cultured under differentiation medium for 0 (a), 2 (b) and 7 days (c). (d) The differentiated skeletal muscle constructs were stained with α-actinin (red) and DAPI (blue) to illustrate myotube assembly. (e) Bright field image of the skeletal muscle construct. (f) H&E cross sectional view of the multi-layered SMEC. Top view (g) and cross sectional SEM images (h) revealed that cell-produced ECM (average thickness of 20 μm) encased the underlying scaffold. (i) Prior to cell culture, the scaffold had an average thickness of 10 μm. (j) Transfected myoblasts maintained stable GFP expression over nine weeks. (k) FVIII production from the FVIII engineered skeletal muscle construct averaged around 1U per day. The seeded cells were cultured in proliferation medium between days 0–5 and switched to differentiation medium between days 5–30. The transient drop off in production on day seven is likely due to the initiation of cell differentiation.

Figure 3. Host-donor tissue integration

(a–c) Histology images of the tissue cross-section at one week (a), one month (b) and two month time points (c). (d–g) 20x histology and immunofluorescence images of the tissue cross-section at one week (d–e) and one month (f–g) time points. The tissues were labeled for GFP (green), fibroblasts (blue) and macrophages (red) to evaluate the host-donor response and integration. (h–k) As a control, the induced host response to injected GFP⁺ myoblasts was evaluated at one week (h–i) and one month (j–k). One month post implantation, the implanted tissue showed persistence of GFP signal (l), and was stained positive for Ki67 (m) and negative for cytotoxic T-cells (n). The implanted tissue was also stained positive for skeletal muscle specific markers such as fast skeletal myosin heavy chain (o), desmin (p) and α-actinin (q). In panels b–c, S = skin, M = muscle, I = implant and F = fat.

Figure 4. Angio/lymphangiogenesis induced by factors released from scaffold

(a–f) Confocal microscopy images tracking localized angio/lymphangiogenesis around the GFP⁺ SMEC at one week (a–c) and one month (d–f). In the group encapsulated with VEGFA and PDGF-bb (b & e), there were significantly more developing blood vessels (labeled red for α-smooth muscle actin) in close proximity when compared to the construct alone group (a&d). Similarly, there was a significantly higher density of lymphatic vessels (labeled blue for LYVE-1) around the group encapsulated with VEGFC (c & f). The density and diameter of the developing blood and lymphatic vessels were quantified based on 3 mice (n=3) per group and reported in (g–j). The lymphatic vessels that developed around the VEGFC group remained larger in size (g) and higher in density (h) at 1 week and 1 month. Likewise, the VEGFA group induced a higher amount of microvessels (< 35 μm) at both 1 week and 1 month time points (i & j). In panels g–h, * denotes statistical significance over VEGFA, construct alone and control groups. Similarly, in panel j * represents statistical significance over VEGFC, construct alone and control groups.

Figure 5. Effects of FVIII-producing SMECs on phenotypic correction of hemophilic A mice

(a) FVIII was injected near the implant site to evaluate protein transport efficiency from the subcutaneous space into circulation. VEGFA group showed higher amount of plasma hFVIII (8%) compared to the other experimental groups. (b) Plasma cFVIII level of hemophilic mice receiving different SMEC implants over a period of two months. (c) Bethesda assay for development of anti-cFVIII antibodies over two months. (d) Blood coagulation time of different experimental groups at two month post SMEC implantation. Normal refers to non-hemophilic mice while untreated denotes untreated hemophilic mice. The reported values are based on 5 mice per group (n = 5). * denotes statistical significance over VEGFC, construct alone and control groups, while ⁺ represents statistical significance when compared to VEGFA, construct alone and control groups.