Here we developed small diameter microfibrous vascular grafts that could harness the endogenous regeneration potential by recruiting both endothelial and smooth muscle progenitor cells, thereby addressing two critical issues of vascular grafts: endothelialization and the remodeling of the vascular wall. The accelerated endothelialization and the increase of SMPC recruitment resulted in the improvement of long-term patency and the increase of the mechanical strength of the vascular grafts respectively. These results demonstrate that endogenous progenitor cells are capable of regenerating vascular tissues if vascular grafts are engineered to harness this potential. This in situ tissue engineering approach by recruiting endogenous progenitor cells breaks new grounds in vascular tissue engineering, and demonstrates the feasibility of making bioactive vascular grafts available off-the-shelf. On the other hand, cell-seeded grafts can also enhance the biocompatibility and non-thrombogenic property of the grafts, and possibly promote cell recruitment by paracrine signaling. However, there are still several barriers to overcome to scale up the clinical treatment, e.g., cell characterization and manipulation, cell survival in the grafts during the surgery, and potential cell detachment under flow.
Despite extensive research on engineering the luminal surfaces, prosthetic vascular grafts still lack endothelialization in human. Pre-seeding ECs on luminal surfaces with sufficient pre-implant culture can improve clinical outcomes [32
], but the procedure still requires extensive graft preparation before the surgery.. Heparin possesses excellent anticoagulant and antithrombogenic properties and it has been widely used as a coating reagent for blood contacting surfaces. Immobilized heparin may decrease the fibrin formation on the scaffold. It has been reported that fibrin may facilitate remodeling of vascular grafts [33
]. However, EC seeding is required to cover the luminal surface of the grafts with fibrin to avoid coagulation and clogging, and thus autologous fibrin treatment may only be used for cell-seeded grafts. Although heparin coating could suppress acute thrombus formation and improve short-term patency, oral anticoagulant and anti-platelet therapies might be needed to maintain the long-term patency of vascular grafts. Therefore, accelerated in situ
EC regeneration is desirable for vascular grafts.
It has also been shown that heparin binds to SDF-1α and stabilizes SDF-1α while maintaining its binding capability to its receptor [34
]. Our data indicated that heparin-SDF-1α binding was more stable than passively adsorbed SDF-1α under both static and flow conditions. A recent work showed that adsorbed SDF-1α could also enhance endothelialization [38
], but the stability of SDF-1α might not be optimal; in addition, the effect of immobilized SDF-1α on SMPCs is not known. We showed that EPC recruitment by heparin-bound SDF-1α was highly effective, with the majority of attached cells positive for EPC markers. This result demonstrates the feasibility of recruiting circulating EPCs under the flow condition in arteries. Besides chemotactic recruitment of EPCs, SDF-1α plays an important role in many aspects of EPC functions. For example, SDF-1α is a mediator of CD34+
EPCs trafficking between peripheral circulation and bone marrow through its receptor on EPC surface [39
]. SDF-1α could also induce EC proliferation and differentiation [30
]. It is likely that immobilized SDF-1α plays an important role in EPC recruitment and subsequent cell proliferation and EPC differentiation.
Our results suggest that the transanastomotic migration of ECs is the major mechanism of endothelialization for untreated and heparin-treated grafts. In contrast, endothelialization in SDF-1a-treated grafts involves both the transanatomotic migration of ECs at the two ends and the recruitment of EPCs in the middle portion of the grafts. The time course studies showed that SDF-1α-recruited EPCs differentiated into ECs and accelerated the endothelialization process, which was correlated with the maintenance of long-term patency of heparin-SDF-1α-treated vascular grafts between 4 and 12 weeks. In contrast, the patency of untreated and heparin-treated grafts still decreased after 4 weeks, possibly due to the incomplete endothelialization. Our results also suggest that endothelialization by the proliferation and migration of ECs from anastomotic sites at the two ends of vascular grafts was not efficient, which took more than 4 weeks to cover the 6-mm long grafts in untreated and heparin-treated grafts. Because human ECs have slower proliferation and migration rate than rat ECs and the vascular grafts used in human are generally longer, it would take more time to achieve endothelialization in the vascular grafts, and thus EPC recruitment is necessary.
Another important finding is that SDF-1α increased the recruitment of SMPCs and improved the mechanical property of the grafts. The recruited SMPCs were capable of differentiating into SMCs in vivo
and in vitro
. Interestingly, these SMPCs were negative for the markers previously identified for SMPCs. Our preliminary studies suggest that these SMPCs express Sox10, an important marker for multipotent vascular stem cells [41
] and can differentiate into SMCs, implicating a novel cell type and a new mechanism involved in vascular regeneration. These SMPCs are positive for SDF-1α receptors CXCR7 and CXCR4. It has been shown that during the wound-healing process of skin, CXCR4+
cells migrating around vessels release MMP-9 to enhance angiogenesis [42
]. Whether SDF-1α regulates the differentiation and matrix synthesis of SMPCs remains to be determined. Nevertheless, we have shown that SDF-1α has dual functions to recruit the progenitor cells of both ECs and SMCs and that endogenous progenitor cells are sufficient to regenerate blood vessels. These findings lay down a foundation for in situ
tissue engineering of blood vessels. It is also noted that SMPCs formed tissues similar to the media layer of native arteries on the outer surface of grafts, including SMCs and extensive matrix synthesis, suggesting an important role of SMPCs in vascular remodeling. These SMPCs are likely recruited from the surrounding tissues. Therefore, one could also fabricate non-degradable vascular grafts with similar mechanical property to the native arteries, and achieve the maturation and integration of the grafts in situ
effectively. Since vascular remodeling in small animals does not recapitulate all aspects in human [43
], preclinical and clinical studies will be performed to further translate this technology into clinical treatment.