Qianqian Han

Transplantation of human mesenchymal stem cells loaded on collagen scaffolds for the treatment of traumatic brain injury in rats

Studies have suggested that mesenchymal stem cells (MSCs) have therapeutic effects following traumatic brain injury (TBI). However, cell distribution and survival rate are two major barriers to their success as therapeutic treatment. The improvement of cell therapy using collagen delivery matrices had been reported. However, we know very little about the mechanisms. We labeled human bone marrow-derived mesenchymal stem cells (hMSCs) with a positron emission tomography (PET) tracer, 18F-fluoro-2-deoxy-D-glucose (FDG). hMSCs were transplanted with or without collagen scaffolds into rats with experimental TBI and the whole-body nuclear images were compared. Collagen scaffolds increased the retention of hBMSC in the lesion site and limited its distribution at the transplanted region. Significantly more hMSCs were detected in the brain when transplanted with collagen scaffolds. The results showed collagen scaffolds also efficiently improved cell survival and neurite outgrowth in vivo, resulting in better neural functional recovery. In addition, brain metabolism also improved in the collagen scaffold implanted group, as evaluated by PET. We speculated that collagen scaffolds would improve early engraftment and support the survival of grafted cells post-transplantation.

The promotion of neurological recovery in the rat spinal cord crushed injury model by collagen-binding BDNF.

Spinal cord crushed injury is clinically common. Promoting targeted neural regeneration at the crushed site of spinal cord could be important for the repair. It has been demonstrated in our previous work that native human BDNF fused with a collagen-binding domain (CBD-BDNF) can bind to collagen specifically to exert the neurotrophic effect on promoting axonal regeneration. After injury, collagen is highly accumulated at the injury site. We thus speculate that CBD-BDNF will bind to the extracellular matrix collagen and concentrate at the injury site to improve the therapy. Using the rat spinal cord crushed injury model, we have found that CBD-BDNF by one-time intrathecally injection could be retained and concentrated at the injury site for a longer time than native BDNF without collagen-binding domain. CBD-BDNF could promote better neural regeneration and locomotion recovery.

Collagen membranes loaded with collagen-binding human PDGF-BB accelerate wound healing in a rabbit dermal ischemic ulcer model

Studies have shown that exogenous platelet-derived growth factor-BB (PDGF-BB) could accelerate the ulcer healing, but the lack of efficient growth factor delivery system limits its clinical application. Our previous work has demonstrated that the native human PDGF-BB was added a collagen-binding domain (CBD), TKKTLRT, to develop a collagen-based PDGF targeting delivery system. Here, we showed that this CBD-fused PDGF-BB (CBD-PDGF) could bind to collagen membrane efficiently. We used the rabbit dermal ischemic ulcer model to study the effects of CBD-PDGF loaded on collagen membranes. Results revealed that this system maintained a higher concentration and stronger bioactivity of PDGF-BB on the collagen membranes and promoted the re-epithelialization of dermal ulcer wounds, the collagen deposition, and the formation of capillary lumens within the newly formed tissue area. It demonstrated that collagen membranes loaded with collagen-targeting human PDGF-BB could effectively promote ulcer healing.

Improvement of Sciatic Nerve Regeneration Using Laminin-Binding Human NGF-β

Background Sciatic nerve injuries often cause partial or total loss of motor, sensory and autonomic functions due to the axon discontinuity, degeneration, and eventual death which finally result in substantial functional loss and decreased quality of life. Nerve growth factor (NGF) plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical applications. We reported here by fusing with the N-terminal domain of agrin (NtA), NGF-β could target to nerve cells and improve nerve regeneration. Methods Laminin-binding assay and sustained release assay of NGF-β fused with NtA (LBD-NGF) from laminin in vitro were carried out. The bioactivity of LBD-NGF on laminin in vitro was also measured. Using the rat sciatic nerve crush injury model, the nerve repair and functional restoration by utilizing LBD-NGF were tested. Findings LBD-NGF could specifically bind to laminin and maintain NGF activity both in vitro and in vivo. In the rat sciatic nerve crush injury model, we found that LBD-NGF could be retained and concentrated at the nerve injury sites to promote nerve repair and enhance functional restoration following nerve damages. Conclusion Fused with NtA, NGF-β could bind to laminin specifically. Since laminin is the major component of nerve extracellular matrix, laminin binding NGF could target to nerve cells and improve the repair of peripheral nerve injuries.

Improved neovascularization and wound repair by targeting human basic fibroblast growth factor (bFGF) to fibrin

Targeted therapy is a new generation of therapeutics, where two critical factors are involved. One is the particular molecular target, and the other is the specific target-binding drug. In this work, the fibrin, a main component of plasma clot at wound sites, was used as the target for human bFGF, aiming to improve therapeutic neovascularization and wound repair. To endow bFGF with fibrin-targeting ability, a fibrin-binding peptide Kringle1 (K1), derived from human plasminogen, was fused to human bFGF. The recombinant K1bFGF showed high fibrin and plasma-clot-binding ability. When applied to the wound sites with plasma clots, K1bFGF induced robust neovascularization and improved wound healing. To extend the application of K1bFGF to other cases where no plasma clots exist, we developed a fibrin-scaffold/K1bFGF system. This system could induce localized neovascularization by delivery of K1bFGF in a sustained and site-targeting manner, and provide a microenvironment promoting cell growth and tissue regeneration. In summary, we successfully used the pathologic environment fibrin clot as the target for bFGF, and based on which bFGF was designed into a targeting agent by introduction of a fibrin-binding peptide. This provides a potential approach to improve therapeutic neovascularization and wound repair.

Human Basic Fibroblast Growth Factor Fused with Kringle4 Peptide Binds to a Fibrin Scaffold and Enhances Angiogenesis

Appropriate three-dimensional (3D) scaffolds and signal molecules could accelerate tissue regeneration and wound repair. In this work, we targeted human basic fibroblast growth factor (bFGF), a potent angiogenic factor, to a fibrin scaffold to improve therapeutic angiogenesis. We fused bFGF to the Kringle4 domain (K4), a fibrin-binding peptide from human plasminogen, to endow bFGF with specific fibrin-binding ability. The recombinant K4bFGF bound specifically to the fibrin scaffold so that K4bFGF was delivered in a site-specific manner, and the fibrin scaffold provided 3D support for cell migration and proliferation. Subcutaneous implantation of the fibrin scaffolds bound with K4bFGF but not with bFGF induced neovascularization. Immunohistochemical analysis showed significantly more proliferation cells in the fibrin scaffolds incorporated with K4bFGF than in those with bFGF. Moreover, the regenerative tissues were integrated well with the fibrin scaffolds, suggesting its good biocompatibility. In summary, targeted delivery of K4bFGF could potentially improve therapeutic angiogenesis.

Neuronal regeneration and protection by collagen-binding BDNF in the rat middle cerebral artery occlusion model

It has been well confirmed that brain-derived neurotrophic factor (BDNF) has therapeutic effects following stroke. However, it is difficult to be maintained at a sufficient concentration of BDNF in the infarcted hemisphere. We have shown in our previous work that BDNF fused with a collagen-binding domain (CBD-BDNF) could specifically bind to collagen. The ventricular ependyma of the brain is rich in collagen. Therefore, we have speculated that in the infarcted hemisphere, CBD-BDNF will bind to the collagen of the ventricular ependyma and stimulate the cell proliferation in the subventricular zone (SVZ). Using a rat middle cerebral artery occlusion model (MCAO), we injected CBD-BDNF into the lateral ventricle of MCAO rats. The results demonstrated that CBD-BDNF was retained at high levels in the infarcted hemisphere, promoted neural regeneration and angiogenesis, reduced cell loss, decreased apoptosis, and improved functional recovery. In addition, brain perfusion and metabolism, as evaluated by SPECT and PET, were improved in the CBD-BDNF treated group.

The promotion of cerebral ischemia recovery in rats by laminin-binding BDNF

Brain-derived neurotrophic factor (BDNF) has been shown to have therapeutic effects on cerebral ischemia. However, the delivery approach limits its application. Laminin is a rich extra cellular matrix in the central nervous system, and is highly expressed in the ischemic region after cerebral ischemia. We reported here by fusing with laminin-binding domain (LBD) to BDNF to construct laminin-binding BDNF (LBD-BDNF). LBD-BDNF could target accumulated laminin in the ischemic region and exert targeting therapy of injured neurons after ischemia. We examined the laminin-binding ability and neurotrophic bioactivity of LBD-BDNF in vitro, and assessed its targeting therapy using a rat permanent middle cerebral artery occlusion (MCAO) model in vivo. It was found that LBD-BDNF could specifically bind to laminin and maintain BDNF activity both in vitro and in vivo. LBD-BDNF treatment attenuated neural-degeneration after MCAO, and also resulted in a reduction of infarct volume that is associated with a parallel improvement in neurological functional outcome and neurogenesis in the dentate gyrus of hippocamp.

Nerve regeneration and functional recovery by collagen-binding brain-derived neurotrophic factor in an intracerebral hemorrhage model.

Brain-derived neurotrophic factor (BDNF) exerts therapeutic effects following intracerebral hemorrhage (ICH). However, it is difficult to maintain sufficient concentrations in the hemorrhage hemisphere. We demonstrated previously that BDNF fused to a collagen-binding domain (CBD) could bind to collagen in the ventricular ependyma and stimulate cell proliferation in the subventricular zone (SVZ). In this study, we verified the therapeutic effects of CBD-BDNF in the rat ICH model induced by bacterial collagenase by injecting CBD-BDNF into the lateral ventricle of ICH rats. The results demonstrated that CBD-BDNF was retained at high levels in the hemorrhage hemisphere, where it promoted neural regeneration and angiogenesis, reduced tissue loss, and improved functional recovery.

Evaluation of a bioactive bone-inducing material consisting of collagen scaffolds and collagen-binding bone morphogenetic protein 2

Bioactive bone-inducing material (BBIM) is a collagen-based scaffold composed of demineralized bone matrix and collagen-binding domain bone morphogenetic protein 2 (BMP-2). BBIM is regarded as a promising bone-inducing scaffold to repair bone defects. In this work, we evaluated the biocompatibility and osteogenecity of BBIM. Using enzyme-linked immunosorbent assay, the level of BMP-2 on BBIM was detected and considered adequate. Kunming mice were used as the animal model to investigate the acute systemic toxicity, long-term bone regeneration, ectopic bone formation, and chronic systemic toxicity. Results show that BBIM induced no serious inflammatory reaction or acute and chronic systemic toxicity. Our analyses also demonstrated significant homogeneous ectopic bone formation as well as significantly high numbers of erythrocytes in the BBIM groups in the chronic systemic toxicity study, a phenomenon which may provide indirect proof of the bone regeneration capacity of BBIM, which may be considered as a bioactivity indicator in future studies.