Direct fabrication of microfibrous tubular graft with a micro structure similar to native matrix fiber
By directly electrospinning polymer fibers onto a rotating mandrel, we successfully made microfibrous tubular grafts (). This is a significant progress towards making seamless microfibrous grafts. The PLLA microfibers made by electrospinning formed a structure () similar to native matrix fibers. The average diameter of the fibers was approximately 2 microns. As seen in the image, electrospinning process resulted in a highly porous and random structure of fibers, which is excellent tissue engineering scaffold.
Figure 1 Structure and chemical modification of microfibrous vascular grafts. (A) SEM image of the electrospun, microfibrous vascular graft. Scale bar = 0.5 mm. (B) SEM of the luminal surface of the electrospun vascular graft. Scale bar = 10 µm. (C) Conjugation (more ...)
Microfibers could be modified by using PEG and hirudin
PEG is capable of creating a brush-like layer onto various surfaces and was used to create a protein and platelet repulsive surface on the PLLA microfiber scaffold. We first conjugated PEG onto microfibers, and then linked hirudin to the end of PEG molecule (). The purpose was to use PEG to resist protein adsorption and platelet adhesion and use hirudin to inactivate thrombin that reached the luminal surface of the vascular grafts. The successful conjugation of PEG and hirudin onto microfibers was confirmed by immunostaining for hirudin, which showed the coating of hirudin on individual microfibers ().
PEG and hirudin-PEG conjugated surfaces reduced the number and aggravation of adherent platelets
To determine whether the grafts modified by PEG and hirudin-PEG can reduce platelet adhesion, platelets were incubated with un-treated grafts and PEG- or hirudin-PEG-modified grafts. As shown in , both CD41 staining for platelets and SEM showed that microfibrous scaffolds with conjugated PEG and hirudin-PEG had fewer numbers of platelets on their surfaces than untreated microfibrous scaffolds. The reduction of platelet adhesion on PEG and hirudin-PEG microfibrous scaffolds was statistically significant (), which may help reduce the possibility of early in vivo graft failures due to thrombosis and platelet adhesion/activation. The similar reduction of platelet adhesion by PEG and hirudin-PEG suggested that PEG contributed to this anti-platelet adhesion property.
Figure 2 Representative images of human platelets seeded onto PLLA (A, D), PEG-PLLA (B, E) and hirudin-PEG-PLLA (C, F) surfaces. CD41 mouse anti-human antibody and Alexa Fluor 488 secondary were used to stain platelets on PLLA (A), PEG-PLLA (B) and hirudin-PEG-PLLA (more ...)
In addition, SEM demonstrated the morphological characteristics of the platelets on the microfibrous PLLA scaffolds. The adherent platelets on the untreated surfaces appeared to have “spiky” protrusions and pseudopods, indicating that they were activated and aggravated by coming into contact with the microfiber sample (). In contrast, fewer adherent platelets were found on the surfaces modified by PEG or hirudin-PEG, and these platelets did not have the same “spiky” protrusions as on the control samples ().
PEG and hirudin-PEG modified grafts improved patency rates in vivo
To compare the anti-thrombogenic property of the vascular grafts in vivo, untreated microfibrous grafts and the grafts modified with PEG or hirudin-PEG were implanted into the common carotid artery of rats for 1 month. Patency was determined by ultrasound and necropsy. Graft patency was determined by the unobstructed flow of blood through the graft. At 1 month, 6 out of 12 (50%) untreated grafts were patent, 9 out of 12 (75%) PEG grafts were patent, and 10 of 12 (83%) hirudin-PEG grafts were patent. These results suggest that both PEG and hirudin improved the patency rate.
To further determine the long-term remodeling of the patent grafts, hirudin-PEG-modified grafts were implanted for 6 months. At 6 months, 6 of the 7 (86%) implanted hirudin-PEG grafts were patent. Based on histological analysis, the failed grafts were clogged with thrombus, indicating the imperfect patency rate was related to easiness of clotting in 1-mm grafts.
The patent grafts from hirudin-PEG group are shown in as representatives. shows a stereomicrograph image of an implanted graft moments after the anastomosis was completed. The porous structure of the graft was immediately filled with red blood cells and other cellular components, and the color of the graft changed from milky white to a red. The interrupted suture technique did not result in any of bleeding or leakage at the anastomotic sites. Good blood flow was observed at both the proximal and distal ends of the graft.
Figure 3 Images of 1-mm internal diameter hirudin-PEG-PLLA grafts in vivo. (A) Image taken immediately after implantation of the graft. (B) After 1 month. (C) After 6 months. (D) Stereomicrograph image of the luminal surface of a freshly explanted 1-month sample. (more ...)
After 1 month, there was visible angiogenesis in the wall of the graft (). The presence of newly formed micro-vessels indicates the integration of the graft into the host’s vasculature. In addition, it suggests that the graft is becoming a living part of the host and that angiogenesis is necessary to supply nutrients, oxygen and other diffusible chemicals to the local cells that reside within and around the graft. It was observed that the amount of angiogenesis was slightly less in the 6-month grafts (), suggesting that the angiogenesis was part of an acute wound healing process in the graft.
A suture site, as well as the characteristics of the graft after 1 month was captured by a stereomicroscope (). The luminal surface was presented by transecting the graft along the longitudinal direction. The anastomotic sites were free of thrombosis and intimal hyperplasia.
Graft patency was also monitored by using Doppler Duplex () and magnetic resonance imaging (MRI) (), showing normal flow rate in the patent grafts.
Endothelialization, cellular infiltration and organization
The patent grafts (either in the untreated group or hirudin-treated group) showed similar histological results. Therefore, only hirudin-treated grafts are shown as representatives. Patent grafts (exemplified by hirudin-PEG grafts in ) showed very little signs of thrombosis and/or intimal hyperplasia on the luminal walls of the graft at either the 1 () or 6-month () time point. It is evident that the neo- tissue formation on the outer surface of the grafts was significant at both time points. The neo- tissue in the 1-month samples had a highly porous and loose tissue structure on the outside of the graft (). On the other hand, the 6-month sample had dense neo-tissue with extracellular matrix alignment in the circumferential direction (). The nucleus staining revealed that there were cells within the walls of the graft and the neo-tissue in the outer layer (), suggesting that the graft is capable of supporting cellular ingrowth.
Figure 4 Cross sections of the explanted hirudin-PEG-PLLA grafts at 1 month (A, C, E) and 6 month (B, D, F) post implantation. Hematoxylin staining (A, B, C, D) showed the patency and structure of the grafts. Nucleus staining by DAPI (E, F) showed the distribution (more ...)
Endothelialization on the luminal surface is important to maintain the long-term patency of vascular grafts. All patent samples had complete endothelial coverage at the 1 () and 6-month () time points. The newly formed capillaries and micro-vessels were evident in the neo-tissue of the graft (Figure A, C, D), which suggests the remodeling of the grafts. Immunofluorescent en face staining revealed that ECs had aligned in the flow direction and had morphological appearance similar to ECs in native arteries ().
Figure 5 Endothelialization, SMCs and inflammatory cells in hirudin-PEG-PLLA grafts. ECs were stained by using CD31 antibody (A–D), SMCs were stained by using SM-MHC antibody (E–F), and monocytes and macrophages were stained by using CD68 antibody (more ...)
Smooth muscle cell (SMC) presence and organization are also important for the long-term stability of vascular grafts. Interestingly, SMC staining showed that SMCs were mostly in the neo-tissue surrounding the grafts after 1 month () and 6 months () post-implantation. The 6-month sample () had a clearly defined band of SMCs that are highly organized and aligned in the circumferential direction. There was no sign of a neo-intima or intimal hyperplasia in these patent grafts.
The inflammatory responses to the PLLA vascular graft was monitored via immunostaining of CD68, a cell surface marker for monocytes and macrophages (). There were few CD68 positive cells within the walls of the graft, suggesting minimal inflammatory responses to the grafts.
Mechanical strength increased after in vivo remodeling
Mechanical strength is critical for the long-term stability of the grafts. We performed mechanical tests by using rings of the non-implanted grafts and explanted grafts (). Representative stress-strain curve of explanted grafts at 1- and 6-month time points are shown in . The elastic modulus of the grafts before implantation was about 3.5 MPa (). After 1 month, a slight increase of elastic modulus (5.5 MPa) of the grafts was observed. After 6 months of implantation, the grafts had a significant increase of elastic modulus (11.1 MPa), suggesting a significant remodeling of the grafts in vivo.
Figure 6 Mechanical characterization of the tensile strength of hirudin-PEG-PLLA vascular grafts. (A) Two stainless steel rods were placed through the lumen of the ring segment of the graft. The sample was then loaded onto the mechanical tester, and the applied (more ...)