Zhaobo Fan

A prosurvival and proangiogenic stem cell delivery system to promote ischemic limb regeneration.

UNLABELLED Stem cell therapy is one of the most promising strategies to restore blood perfusion and promote muscle regeneration in ischemic limbs. Yet its therapeutic efficacy remains low owing to the inferior cell survival under the low oxygen and nutrient environment of the injured limbs. To increase therapeutic efficacy, high rates of both short- and long-term cell survival are essential, which current approaches do not support. In this work, we hypothesized that a high rate of short-term cell survival can be achieved by introducing a prosurvival environment into the stem cell delivery system to enhance cell survival before vascularization is established; and that a high rate of long-term cell survival can be attained by building a proangiogenic environment in the system to quickly vascularize the limbs. The system was based on a biodegradable and thermosensitive poly(N-Isopropylacrylamide)-based hydrogel, a prosurvival and proangiogenic growth factor bFGF, and bone marrow-derived mesenchymal stem cells (MSCs). bFGF can be continuously released from the system for 4weeks. The released bFGF significantly improved MSC survival and paracrine effects under low nutrient and oxygen conditions (0% FBS and 1% O2) in vitro. The prosurvival effect of the bFGF on MSCs was resulted from activating cell Kruppel-like factor 4 (KLF4) pathway. When transplanted into the ischemic limbs, the system dramatically improved MSC survival. Some of the engrafted cells were differentiated into skeletal muscle and endothelial cells, respectively. The system also promoted the proliferation of host cells. After only 2weeks of implantation, tissue blood perfusion was completely recovered; and after 4weeks, the muscle fiber diameter was restored similarly to that of the normal limbs. These pronounced results demonstrate that the developed stem cell delivery system has a potential for ischemic limb regeneration. STATEMENT OF SIGNIFICANCE Stem cell therapy is a promising strategy to restore blood perfusion and promote muscle regeneration in ischemic limbs. Yet its therapeutic efficacy remains low owing to the inferior cell survival under the ischemic environment of the injured limbs. To increase therapeutic efficacy, high rate of cell survival is essential, which current approaches do not support. In this work, we tested the hypothesis that a stem cell delivery system that can continuously release a prosurvival and proangiogenic growth factor will promote high rates of cell survival in the ischemic limbs. The prosurvival effect could augment cell survival before vascularization is established, while the proangiogenic effect could stimulate quick angiogenesis to achieve long-term cell survival. Meanwhile, the differentiation of stem cells into endothelial and myogenic lineages, and cell paracrine effects will enhance vascularization and muscle regeneration.

Regulating myogenic differentiation of mesenchymal stem cells using thermosensitive hydrogels

UNLABELLED Stem cell therapy has potential to regenerate skeletal muscle tissue in ischemic limb. However, the delivered stem cells experience low rate of myogenic differentiation. Employing injectable hydrogels as stem cell carriers may enhance the myogenic differentiation as their modulus may be tailored to induce the differentiation. Yet current approaches used to manipulate hydrogel modulus often simultaneously vary other properties that also affect stem cell differentiation, such as chemical structure, composition and water content. Thus it is challenging to demonstrate the decoupled effect of hydrogel modulus on stem cell differentiation. In this report, we decoupled the hydrogel modulus from chemical structure, composition, and water content using injectable and thermosensitive hydrogels. The hydrogels were synthesized from N-isopropylacrylamide (NIPAAm), acrylic acid (AAc), and degradable macromer 2-hydroxyethyl methacrylate-oligomer [oligolatide, oligohydroxybutyrate, or oligo(trimethylene carbonate)]. We found that using the same monomer composition and oligomer chemical structure but different oligomer length can independently vary hydrogel modulus. Rat bone marrow mesenchymal stem cells (MSCs) were encapsulated in the hydrogels with elastic expansion moduli of 11, 20, and 40 kPa, respectively. After 14 days of culture, significant myogenic differentiation was achieved for the hydrogel with elastic expansion modulus of 20 kPa, as judged from both the gene and protein expression. In addition, MSCs exhibited an elastic expansion modulus-dependent proliferation rate. The most significant proliferation was observed in the hydrogel with elastic expansion modulus of 40 kPa. These results demonstrate that the developed injectable and thermosensitive hydrogels with suitable modulus has the potential to deliver stem cells into ischemic limb for enhanced myogenic differentiation and muscle regeneration. STATEMENT OF SIGNIFICANCE Stem cell therapy for skeletal muscle regeneration in ischemic limb experiences low rate of myogenic differentiation. Employing injectable hydrogels as stem cell carriers may enhance the myogenic differentiation as hydrogel modulus may be modulated to induce the differentiation. Yet current approaches used to modulate hydrogel modulus may simultaneously vary other properties that also affect stem cell myogenic differentiation, such as chemistry, composition and water content. In this report, we decoupled the hydrogel modulus from chemistry, composition, and water content using injectable and thermosensitive hydrogels. We found that mesenchymal stem cells best differentiated into myogenic lineage in the hydrogel with elastic modulus of 20 kPa.

An Injectable Oxygen Release System to Augment Cell Survival and Promote Cardiac Repair Following Myocardial Infarction

Oxygen deficiency after myocardial infarction (MI) leads to massive cardiac cell death. Protection of cardiac cells and promotion of cardiac repair are key therapeutic goals. These goals may be achieved by re-introducing oxygen into the infarcted area. Yet current systemic oxygen delivery approaches cannot efficiently diffuse oxygen into the infarcted area that has extremely low blood flow. In this work, we developed a new oxygen delivery system that can be delivered specifically to the infarcted tissue, and continuously release oxygen to protect the cardiac cells. The system was based on a thermosensitive, injectable and fast gelation hydrogel, and oxygen releasing microspheres. The fast gelation hydrogel was used to increase microsphere retention in the heart tissue. The system was able to continuously release oxygen for 4 weeks. The released oxygen significantly increased survival of cardiac cells under the hypoxic condition (1% O2) mimicking that of the infarcted hearts. It also reduced myofibroblast formation under hypoxic condition (1% O2). After implanting into infarcted hearts for 4 weeks, the released oxygen significantly augmented cell survival, decreased macrophage density, reduced collagen deposition and myofibroblast density, and stimulated tissue angiogenesis, leading to a significant increase in cardiac function.

Modulation of Wound Healing and Scar Formation by MG53 Protein-mediated Cell Membrane Repair

Background: MG53 is a membrane repair gene whose role in wound healing has not been studied. Results: Topical administration of MG53 protein facilitates wound healing and reduces scar formation. Conclusion: This study establishes MG53 as facilitator of injury repair and inhibitor of myofibroblast differentiation during wound healing. Significance: MG53 has therapeutic benefits in treating wounds and fibrotic diseases. Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53−/− mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing.

Sustained Release of a Peptide-based Matrix Metalloproteinase-2 Inhibitor to Attenuate Adverse Cardiac Remodeling and Improve Cardiac Function Following Myocardial Infarction

Following myocardial infarction (MI), degradation of extracellular matrix (ECM) by upregulated matrix metalloproteinases (MMPs) especially MMP-2 decreases tissue mechanical properties, leading to cardiac function deterioration. Attenuation of cardiac ECM degradation at the early stage of MI has the potential to preserve tissue mechanical properties, resulting in cardiac function increase. Yet the strategy for efficiently preventing cardiac ECM degradation remains to be established. Current preclinical approaches have shown limited efficacy because of low drug dosage allocated to the heart tissue, dose-limiting side effects, and cardiac fibrosis. To address these limitations, we have developed a MMP-2 inhibitor delivery system that can be specifically delivered into infarcted hearts at early stage of MI to efficiently prevent MMP-2-mediated ECM degradation. The system was based on an injectable, degradable, fast gelation, and thermosensitive hydrogel, and a MMP-2 specific inhibitor, peptide CTTHWGFTLC (CTT). The use of fast gelation hydrogel allowed to completely retain CTT in the heart tissue. The system was able to release low molecular weight CTT over 4 weeks possibly due to the strong hydrogen bonding between the hydrogel and CTT. The release kinetics was modulated by amount of CTT loaded into the hydrogel, and using chondroitin sulfate and heparin that can interact with CTT and the hydrogel. Both glycosaminoglycans augmented CTT release, while heparin more greatly accelerated the release. After it was injected into the infarcted hearts for 4 weeks, the released CTT efficiently prevented cardiac ECM degradation as it not only increased tissue thickness but also preserved collagen composition similar to that in the normal heart tissue. In addition, the delivery system significantly improved cardiac function. Importantly, the delivery system did not induce cardiac fibrosis. These results demonstrate that the developed MMP-2 inhibitor delivery system has potential to efficiently reduce adverse myocardial remodeling and improve cardiac function.

pH-Sensitive and Thermosensitive Hydrogels as Stem-Cell Carriers for Cardiac Therapy.

Stem-cell therapy has the potential to regenerate damaged heart tissue after a heart attack. Injectable hydrogels may be used as stem-cell carriers to improve cell retention in the heart tissue. However, current hydrogels are not ideal to serve as cell carriers because most of them block blood vessels after solidification. In addition, these hydrogels have a relatively slow gelation rate (typically >60 s), which does not allow them to quickly solidify upon injection, so as to efficiently hold cells in the heart tissue. As a result, the hydrogels and cells are squeezed out of the tissue, leading to low cell retention. To address these issues, we have developed hydrogels that can quickly solidify at the pH of an infarcted heart (6-7) at 37 °C but cannot solidify at the pH of blood (7.4) at 37 °C. These hydrogels are also clinically attractive because they can be injected through catheters commonly used for minimally invasive surgeries. The hydrogels were synthesized by free-radical polymerization of N-isopropylacrylamide, propylacrylic acid, hydroxyethyl methacrylate-co-oligo(trimethylene carbonate), and methacrylate poly(ethylene oxide) methoxy ester. Hydrogel solutions were injectable through 0.2-mm-diameter catheters at pH 8.0 at 37 °C, and they can quickly form solid gels under pH 6.5 at 37 °C. All of the hydrogels showed pH-dependent degradation and mechanical properties with less mass loss and greater complex shear modulus at pH 6.5 than at pH 7.4. When cardiosphere-derived cells (CDCs) were encapsulated in the hydrogels, the cells were able to survive during a 7-day culture period. The surviving cells were differentiated into cardiac cells, as evidenced by the expression of cardiac markers at both the gene and protein levels, such as cardiac troponin T, myosin heavy chain α, calcium channel CACNA1c, cardiac troponin I, and connexin 43. The gel integrity was found to largely affect CDC cardiac differentiation. These results suggest that the developed dual-sensitive hydrogels may be promising carriers for cardiac cell therapy.

Thermosensitive and Highly Flexible Hydrogels Capable of Stimulating Cardiac Differentiation of Cardiosphere-Derived Cells under Static and Dynamic Mechanical Training Conditions.

Cardiac stem cell therapy has been considered as a promising strategy for heart tissue regeneration. Yet achieving cardiac differentiation after stem cell transplantation remains challenging. This compromises the efficacy of current stem cell therapy. Delivery of cells using matrices that stimulate the cardiac differentiation may improve the degree of cardiac differentiation in the heart tissue. In this report, we investigated whether elastic modulus of highly flexible poly(N-isopropylamide) (PNIPAAm)-based hydrogels can be modulated to stimulate the encapsulated cardiosphere derived cells (CDCs) to differentiate into cardiac lineage under static condition and dynamic stretching that mimics the heart beating condition. We have developed hydrogels whose moduli do not change under both dynamic stretching and static conditions for 14 days. The hydrogels had the same chemical structure but different elastic moduli (11, 21, and 40 kPa). CDCs were encapsulated into these hydrogels and cultured under either native heart-mimicking dynamic stretching environment (12% strain and 1 Hz frequency) or static culture condition. CDCs were able to grow in all three hydrogels. The greatest growth was found in the hydrogel with elastic modulus of 40 kPa. The dynamic stretching condition stimulated CDC growth. The CDCs demonstrated elastic modulus-dependent cardiac differentiation under both static and dynamic stretching conditions as evidenced by gene and protein expressions of cardiac markers such as MYH6, CACNA1c, cTnI, and Connexin 43. The highest differentiation was found in the 40 kPa hydrogel. These results suggest that delivery of CDCs with the 40 kPa hydrogel may enhance cardiac differentiation in the infarct hearts.

Biomimetic polyurethane/TiO 2 nanocomposite scaffolds capable of promoting biomineralization and mesenchymal stem cell proliferation

Scaffolds with extracellular matrix-like fibrous morphology, suitable mechanical properties, biomineralization capability, and excellent cytocompatibility are desired for bone regeneration. In this work, fibrous and degradable poly(ester urethane)urea (PEUU) scaffolds reinforced with titanium dioxide nanoparticles (nTiO2) were fabricated to possess these properties. To increase the interfacial interaction between PEUU and nTiO2, poly(ester urethane) (PEU) was grafted onto the nTiO2. The scaffolds were fabricated by electrospinning and exhibited fiber diameter of <1μm. SEM and EDX mapping results demonstrated that the PEU modified nTiO2 was homogeneously distributed in the fibers. In contrast, severe agglomeration was found in the scaffolds with unmodified nTiO2. PEU modified nTiO2 significantly increased Youngs modulus and tensile stress of the PEUU scaffolds while unmodified nTiO2 significantly decreased Youngs modulus and tensile stress. The greatest reinforcement effect was observed for the scaffold with 1:1 ratio of PEUU and PEU modified nTiO2. When incubating in the simulated body fluid over an 8-week period, biomineralization was occurred on the fibers. The scaffolds with PEU modified nTiO2 showed the highest Ca and P deposition than pure PEUU scaffold and PEUU scaffold with unmodified nTiO2. To examine scaffold cytocompatibility, bone marrow-derived mesenchymal stem cells were cultured on the scaffold. The PEUU scaffold with PEU modified nTiO2 demonstrated significantly higher cell proliferation compared to pure PEUU scaffold and PEUU scaffold with unmodified nTiO2. The above results demonstrate that the developed fibrous nanocomposite scaffolds have potential for bone tissue regeneration.

Myocardial Regenerative Medicine

Myocardial infarction (MI) or heart attack has high mortality rate. It is characterized by massive cardiomyocyte death and reduced cardiac function. MI is a major cause of heart failure. Effective therapies are critical to prevent infarcted heart from progressing into heart failure. Current clinical intervention after MI is mainly focused on coronary reperfusion with the purpose of reintroducing nutrient and oxygen into the damaged area. Yet reperfusion therapy cannot induce the regeneration of new cardiac muscle, and the damaged heart tissue cannot self-regenerate to restore normal tissue features and function. To fully restore cardiac function, transplantation of cells to compensate the lost cardiac cells is necessary. Therapies that improve the function of damaged heart tissue other than fully regeneration have also been explored. These include using acellular biomaterials and control of cardiac fibrosis. This chapter summarizes current approaches for cardiac regeneration and cardiac function improvement.

Thermosensitive, Fast Gelling, Photoluminescent, Highly Flexible, and Degradable Hydrogels for Stem Cell Delivery

Stem cell therapy is a promising approach to regenerate ischemic cardiovascular tissues yet experiences low efficacy. One of the major causes is inferior cell retention in tissues. Injectable cell carriers that can quickly solidify upon injection into tissues so as to immediately increase viscosity have potential to largely improve cell retention. A family of injectable, fast gelling, and thermosensitive hydrogels were developed for delivering stem cells into heart and skeletal muscle tissues. The hydrogels were also photoluminescent with low photobleaching, allowing for non-invasively tracking hydrogel biodistribution and retention by fluorescent imaging. The hydrogels were polymerized by N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacrylate (HEMA), 1-vinyl-2-pyrrolidinone (VP), and acrylate-oligolactide (AOLA), followed by conjugation with hypericin (HYP). The hydrogel solutions had thermal transition temperatures around room temperature, and were readily injectable at 4 °C. The solutions were able to quickly solidify within 7 s at 37 °C. The formed gels were highly flexible possessing similar moduli as the heart and skeletal muscle tissues. In vitro, hydrogel fluorescence intensity decreased proportionally to weight loss. After being injected into thigh muscles, the hydrogel can be detected by an in vivo imaging system for 4 weeks. The hydrogels showed excellent biocompatibility in vitro and in vivo, and can stimulate mesenchymal stem cell (MSC) proliferation and paracrine effects. The fast gelling hydrogel remarkably increased MSC retention in thigh muscles compared to slow gelling collagen, and non-gelling PBS. These hydrogels have potential to efficiently deliver stem cells into tissues. Hydrogel degradation can be non-invasively and real-time tracked. STATEMENT OF SIGNIFICANCE: Low cell retention in tissues represents one of the major causes for limited therapeutic efficacy in stem cell therapy. A family of injectable, fast gelling, and thermosensitive hydrogels that can quickly solidify upon injection into tissues were developed to improve cell retention. The hydrogels were also photoluminescent, allowing for non-invasively and real-time tracking hydrogel biodistribution and retention by fluorescent imaging.