Jianjun Guan

Injectable, rapid gelling and highly flexible hydrogel composites as growth factor and cell carriers.

A family of injectable, rapid gelling and highly flexible hydrogel composites capable of releasing insulin-like growth factor (IGF-1) and delivering mesenchymal stromal cell (MSC) were developed. Hydrogel composites were fabricated from Type I collagen, chondroitin sulfate (CS) and a thermosensitive and degradable hydrogel copolymer based on N-isopropylacrylamide, acrylic acid, N-acryloxysuccinimide and a macromer poly(trimethylene carbonate)-hydroxyethyl methacrylate. The hydrogel copolymer was gellable at body temperature before degradation and soluble at body temperature after degradation. Hydrogel composites exhibited LCSTs around room temperature. They could easily be injected through a 26-gauge needle at 4 degrees C, and were capable of gelling within 6s at 37 degrees C to form highly flexible gels with moduli matching those of the rat and human myocardium. The hydrogel composites showed good oxygen permeability; the oxygen pressure within the hydrogel composites was similar to that in the air. The effects of collagen and CS contents on LCST, gelation time, injectability, mechanical properties and degradation properties were investigated. IGF-1 was loaded into the hydrogel composites for enhanced cell survival/growth. The released IGF-1 remained bioactive during a 2-week release period. Small fraction of CS in the hydrogel composites significantly decreased IGF-1 release rate. The release kinetics appeared to be controlled mainly by hydrogel composite water content, degradation and interaction with IGF-1. Human MSC adhesion on the hydrogel composites was comparable to that on the tissue culture plate. MSCs were encapsulated in the hydrogel composites and were found to grow inside during a 7-day culture period. IGF-1 loading significantly accelerated MSC growth. RT-PCR analysis demonstrated that MSCs maintained their multipotent differentiation potential in hydrogel composites with and without IGF-1. These injectable and rapid gelling hydrogel composites demonstrated attractive properties for serving as growth factor and cell carriers for cardiovascular tissue engineering applications.

Protein-Reactive, Thermoresponsive Copolymers with High Flexibility and Biodegradability

A family of injectable, biodegradable, and thermosensitive copolymers based on N-isopropylacrylamide, acrylic acid, N-acryloxysuccinimide, and a macromer polylactide-hydroxyethyl methacrylate were synthesized by free radical polymerization. Copolymers were injectable at or below room temperature and formed robust hydrogels at 37 degrees C. The effects of monomer ratio, polylactide length, and AAc content on the chemical and physical properties of the hydrogel were investigated. Copolymers exhibited lower critical solution temperatures (LCSTs) from 18 to 26 degrees C. After complete hydrolysis, hydrogels were soluble in phosphate buffered saline at 37 degrees C with LCSTs above 40.8 degrees C. Incorporation of type I collagen at varying mass fractions by covalent reaction with the copolymer backbone slightly increased LCSTs. Water content was 32-80% without collagen and increased to 230% with collagen at 37 degrees C. Hydrogels were highly flexible and relatively strong at 37 degrees C, with tensile strengths from 0.3 to 1.1 MPa and elongations at break from 344 to 1841% depending on NIPAAm/HEMAPLA ratio, AAc content, and polylactide length. Increasing the collagen content decreased both elongation at break and tensile strength. Hydrogel weight loss at 37 degrees C was 85-96% over 21 days and varied with polylactide content. Hydrogel weight loss at 37 degrees C was 85-96% over 21 days and varied with polylactide content. Degradation products were shown to be noncytotoxic. Cell adhesion on the hydrogels was 30% of that for tissue culture polystyrene but increased to statistically approximate this control surface after collagen incorporation. These newly described thermoresponsive copolymers demonstrated attractive properties to serve as cell or pharmaceutical delivery vehicles for a variety of tissue engineering applications.

Periostin modulates myofibroblast differentiation during full-thickness cutaneous wound repair

The matricellular protein periostin is expressed in the skin. Although periostin has been hypothesized to contribute to dermal homeostasis and repair, this has not been directly tested. To assess the contribution of periostin to dermal healing, 6 mm full-thickness excisional wounds were created in the skin of periostin-knockout and wild-type, sex-matched control mice. In wild-type mice, periostin was potently induced 5–7 days after wounding. In the absence of periostin, day 7 wounds showed a significant reduction in myofibroblasts, as visualized by expression of α-smooth muscle actin (α-SMA) within the granulation tissue. Delivery of recombinant human periostin by electrospun collagen scaffolds restored α-SMA expression. Isolated wild-type and knockout dermal fibroblasts did not differ in in vitro assays of adhesion or migration; however, in 3D culture, periostin-knockout fibroblasts showed a significantly reduced ability to contract a collagen matrix, and adopted a dendritic phenotype. Recombinant periostin restored the defects in cell morphology and matrix contraction displayed by periostin-deficient fibroblasts in a manner that was sensitive to a neutralizing anti-β1-integrin and to the FAK and Src inhibitor PP2. We propose that periostin promotes wound contraction by facilitating myofibroblast differentiation and contraction.

Injectable, highly flexible, and thermosensitive hydrogels capable of delivering superoxide dismutase.

Injectable hydrogels are attractive for cell and drug delivery. In this work, we synthesized a family of injectable, biodegradable, fast gelling and thermosensitive hydrogels based on N-isopropylacrylamide (NIPAAm), acrylic acid (AAc), dimethyl-gamma-butyrolactone acrylate (DBA), and 2-hydroxyethyl methacrylate-poly(trimethylene carbontate) (HEMAPTMC) macromer. Type I collagen was composited with the hydrogels to improve their biocompatibility. The hydrogel copolymer solutions were readily injectable at 4 degrees C. The solutions exhibited thermal transition temperatures ranging from 23.6 to 24.5 degrees C and were capable of gelation within 7 s at 37 degrees C to form highly flexible and soft hydrogels with moduli from 39 to 119 KPa and breaking strains >1000%, depending on the copolymer composition and collagen addition. After 2 weeks incubation in PBS, the hydrogels demonstrated weight losses ranging from 10-20%. The completely degraded hydrogels had thermal transition temperatures >40 degrees C and were soluble at body temperature. Superoxide dismutase (SOD) was encapsulated in the hydrogels for the purpose of capturing superoxide within the inflammatory tissue after being delivered in vivo. The hydrogels demonstrated a sustained release profile during a 21-day release period. The release kinetics was dependent on the SOD loading, collagen addition, hydrogel degradation and water content. The released SOD remained bioactive during the entire release period. To test in vitro if the loaded SOD could protect cells encapsulated within the hydrogel from attack by superoxide, human mesenchymal stem cells (MSC) were encapsulated in SOD-loaded hydrogels and cultured in medium containing superoxide generated by activated macrophages. It was found that SOD loading largely suppressed superoxide penetration into the hydrogel and cell membrane. Under normal culture conditions, SOD loading stimulated MSC growth. The SOD-loaded hydrogel exhibited significantly higher cell numbers than the non-SOD loaded hydrogel during a 7-day culture period. These results demonstrated that the developed hydrogels could be used as delivery vehicles for stem cell therapy and drug delivery.

Differential roles of hypoxia inducible factor subunits in multipotential stromal cells under hypoxic condition

Cell therapy with bone marrow multipotential stromal cells (MSCs) represents a promising approach to promote wound healing and tissue regeneration. MSCs expanded in vitro lose early progenitors with differentiation and therapeutic potentials under normoxic condition, whereas hypoxic condition promotes MSC self‐renewal through preserving colony forming early progenitors and maintaining undifferentiated phenotypes. Hypoxia inducible factor (HIF) pathway is a crucial signaling pathway activated in hypoxic condition. We evaluated the roles of HIFs in MSC differentiation, colony formation, and paracrine activity under hypoxic condition. Hypoxic condition reversibly decreased osteogenic and adipogenic differentiation. Decrease of osteogenic differentiation depended on HIF pathway; whereas decrease of adipogenic differentiation depended on the activation of unfolded protein response (UPR), but not HIFs. Hypoxia‐mediated increase of MSC colony formation was not HIF‐dependent also. Hypoxic exposure increased secretion of VEGF, HGF, and basic FGF in a HIF‐dependent manner. These findings suggest that HIF has a limited, but pivotal role in enhancing MSC self‐renewal and growth factor secretions under hypoxic condition. J. Cell. Biochem. 112: 804–817, 2011.

Thermosensitive hydrogels for drug delivery

Introduction: Controlled drug delivery has been widely applied in areas such as cancer therapy and tissue regeneration. Thermosensitive hydrogel-based drug delivery systems have increasingly attracted the attention of the drug delivery community, as the drugs can be readily encapsulated and released by the hydrogels. Areas covered: Thermosensitive hydrogels that can serve as drug carriers are discussed in this paper. Strategies used to control hydrogel properties, in order to tailor drug release kinetics, are also reviewed. This paper also introduces applications of the thermosensitive hydrogel-based drug delivery systems in cancer therapy and tissue regeneration. Expert opinion: When designing a drug delivery system using thermosensitive hydrogels, one needs to consider what type of thermosensitive hydrogel needs to be used, and how to manipulate its properties to meet the desired drug release kinetics. For material selection, both naturally derived and synthetic thermosensitive polymers can be used. Various methods can be used to tailor thermosensitive hydrogel properties in order to achieve the desired drug release profile.

Fabrication and characterization of prosurvival growth factor releasing, anisotropic scaffolds for enhanced mesenchymal stem cell survival/growth and orientation.

Scaffolds that not only mimic the mechanical and structural properties of the target tissue but also support cell survival/growth are likely necessary for the development of mechanically functional cardiovascular tissues. To reach these goals, we have generated scaffolds that are elastic to approximate soft tissue mechanical properties, are nanofibrous to mimic fibrous nature of extracellular matrix (ECM), have aligned structure to guide cellular alignment, and are capable of releasing insulin-like growth factor (IGF-1) to administrate cellular growth and survival. We have developed a technique that can quickly fabricate (<3 h) such scaffolds by simultaneously electrospinning elastase-sensitive polyurethaneurea nanofibers, encapsulating IGF-1 into poly(lactide-co-glycolide) (PLGA) microspheres and assembling them into scaffolds. Scaffold morphology, mechanical properties, degradation with or without elastase, and bioactivity of the released IGF-1 were assessed. The scaffolds had degree of alignment approximately 70%. They were flexible and relatively strong, with tensile strengths of 3.4-11.1 MPa, elongations at break of 71-88%, and moduli of 2.3-7.9 MPa at the alignment direction. IGF-1 release profile and bioactivity were dependent on PLGA content and molecular weight and IGF-1 loading. The released IGF-1 remained bioactive for 4 weeks. The fabricated nanofibers were elastase-sensitive with weight remaining <59% after a 4-week degradation in the presence of elastase. Mesenchymal stem cells (MSCs) were seeded on the scaffolds and cultured either under normal culture conditions (21% O(2), 5% CO(2), and 20% fetal bovine serum (FBS)) or hypoxia/nutrient starvation conditions (5% O(2), 5% CO(2), and 1% FBS) to evaluate the effect of IGF-1 loading on cell growth and survival. Under normal culture conditions, MSCs were found to align on the scaffolds with a degree of alignment matching that of the scaffold. The IGF-1 loaded scaffolds enhanced MSC growth during a 7-day culture period, with higher IGF-1 content showing better stimulus effect. Under hypoxia/nutrient starvation conditions, the IGF-1 loaded scaffolds were found to significantly improve MSC survival.

An oxygen release system to augment cardiac progenitor cell survival and differentiation under hypoxic condition.

Stem cell therapy has the potential to regenerate heart tissue damaged by myocardial infarction (MI), but it experiences extremely low efficacy. One of the major causes is the inferior cell survival under hypoxic condition of the infarcted hearts. We examined whether an oxygen-releasing system capable of sustainedly supplying oxygen to stem cells would augment cell survival and cardiac differentiation under hypoxic condition mimicking that of the infarcted hearts. The oxygen-releasing system consisted of hydrogen peroxide (H(2)O(2))-releasing microspheres, catalase and an injectable, thermosensitive hydrogel. The microspheres were based on poly(lactide-co-glycolide) (PLGA) and a complex of H(2)O(2) and poly(2-vinlypyrridione) (PVP). The oxygen was generated after the released H(2)O(2) was decomposed by catalase. The hydrogel was designed to improve the retention of microspheres and stem cells in the beating heart tissue during myocardial injection. The oxygen-releasing system was capable of sustainedly releasing oxygen for at least two weeks. The release kinetics was dependent on the ratio of H(2)O(2)/VP. The hydrogel was based on N-isopropylacrylamide (NIPAAm), acrylic acid (AAc), and a macromer hydroxyethyl methacrylate-oligo(hydroxybutyrate) (HEMA-oHB). The hydrogel had a stiffness matching that of the heart tissue and was able to stimulate the cardiosphere-derived cells (CDCs) to differentiate into cardiomyocytes. Under hypoxic condition mimicking that of the infarcted hearts (1% O(2)), CDCs encapsulated in the hydrogel experienced massive cell death. Introduction of oxygen release in the hydrogel significantly augmented cell survival; no cell death was found after seven days of culture, and cells even grew after seven days. Under hypoxic condition, cardiac differentiation of CDCs was completely silenced in the hydrogel, as confirmed at both mRNA and protein levels. However, introduction of oxygen release restored the differentiation. These results demonstrate that the developed oxygen-releasing system has great potential to improve the efficacy of cardiac stem cell therapy.

High-efficiency matrix modulus-induced cardiac differentiation of human mesenchymal stem cells inside a thermosensitive hydrogel

Mesenchymal stem cells (MSCs) experience an extremely low rate of cardiac differentiation after transplantation into infarcted hearts, in part due to the inability of stiff scar tissue to support differentiation. We hypothesized that delivering MSCs in a hydrogel with a modulus matched to that of native heart tissue should stimulate MSC differentiation into cardiac cells. We have developed a thermosensitive and injectable hydrogel suitable for the delivery of cells into the heart, and found that the appropriate gel modulus can differentiate MSCs into cardiac cells with high efficiency. The hydrogel was based on N-isopropylacrylamide, N-acryloxysuccinimide, acrylic acid and poly(trimethylene carbonate)-hydroxyethyl methacrylate. The hydrogel solution can be readily injected through needles commonly used for heart injection, and is capable of gelling within 7s at 37°C. The formed gels were highly flexible, with breaking strains (>300%) higher than that of native heart tissue and moduli within the range of native heart tissue (1-140 kPa). Controlling the concentration of the hydrogel solution resulted in hydrogels with three different moduli: 16, 45 and 65 kPa. The moduli were decoupled from the gel water content and oxygen diffusion, parameters that can also influence cell differentiation. MSCs survived in the hydrogels throughout the entire culture period, and it was observed that gel stiffness did not affect cell survival. After 14 days of culture, more than 76% of MSCs had differentiated into cardiac cells in the 45 and 65 kPa gels, as confirmed by the expression of cardiac markers at both the gene and protein levels. MSCs in the hydrogel with the 65 kPa modulus had the highest differentiation efficiency. The differentiated cells also developed calcium channels that imparted an electrophysiological property, and gap junctions for cell-cell communication. The efficiency of differentiation reported in this study was much higher than for the differentiation approaches described in the literature, such as chemical induction and co-culture of MSCs and cardiomyocytes. These results indicate that the novel hydrogel holds great promise for delivering MSCs into an infarcted heart for the generation of new heart tissue.

Synthesis, characterization and surface modification of low moduli poly(ether carbonate urethane)ureas for soft tissue engineering

Flexible scaffolds are of great interest in engineering functional and mechano-active soft tissues as such scaffolds might allow mechanical stimuli to transfer effectively from the scaffolds to cells during tissue development. Towards this end, we have developed a family of flexible poly(ether carbonate urethane)ureas (PECUUs) with a triblock copolymer poly(trimethylene carbonate)-poly(ethylene oxide)-poly(trimethylene carbonate) (PTMC-PEO-PTMC) or pentablock copolymers PTMC-PEO-PPO-PEO-PTMC (PPO, polypropylene oxide) as soft segments, linked by 1,4-diisocyanatobutane and putrescine. All of the PECUUs had low glass transition temperatures (<-46 degrees C). The PTMC-PEO-PTMC-containing PECUUs had low tensile strength and breaking strain. Replacing PEO with the similar length PEO-PPO-PEO resulted in highly flexible and soft PECUUs possessing breaking strains of 362-711%, tensile strengths of 8-18MPa and moduli of 5.5-7.4MPa at room temperature in air. Under aqueous conditions at 37 degrees C, these polymers remained flexible while their moduli were decreased to 3.4-4.0MPa. PECUUs based on PTMC-PEO-PPO-PEO-PTMC were thermosensitive as the water content at 37 degrees C was lower than that at 4 degrees C. PECUU using PTMC-PEO-PTMC as a soft segment showed 30% weight loss over 6weeks in PBS at 37 degrees C, while that using PTMC-PEO-PPO-PEO-PTMC as a soft segment had weight loss <6%. Degradation products were found to lack cytotoxicity. The mechanical stresses and moduli of PECUUs based on PTMC-PEO-PPO-PEO-PTMC were unchanged during the degradation. To enhance cell adhesion, PECUUs were surface modified with Arg-Gly-Asp-Ser (RGDS). Smooth muscle cell adhesion was 114% of tissue culture polystyrene for unmodified PECUU and >180% for RGDS-modified PECUUs, with cell viability on both surfaces increasing during culture. These low moduli polyurethanes may find applications in engineering cardiovascular or other soft tissues.