Guifei Li

Self-Healing Supramolecular Self-Assembled Hydrogels Based on Poly(l-glutamic acid)

Self-healing polymeric hydrogels have the capability to recover their structures and functionalities upon injury, which are extremely attractive in emerging biomedical applications. This research reports a new kind of self-healing polypeptide hydrogels based on self-assembly between cholesterol (Chol)-modified triblock poly(L-glutamic acid)-block-poly(ethylene glycol)-block-poly(L-glutamic acid) ((PLGA-b-PEG-b-PLGA)-g-Chol) and β-cyclodextrin (β-CD)-modified poly(L-glutamic acid) (PLGA-g-β-CD). The hydrogel formation relied on the host and guest linkage between β-CD and Chol. This study demonstrates the influences of polymer concentration and β-CD/Chol molar ratio on viscoelastic behavior of the hydrogels. The results showed that storage modulus was highest at polymer concentration of 15% w/v and β-CD/Chol molar ratio of 1:1. The effect of the PLGA molecular weight in (PLGA-b-PEG-b-PLGA)-g-Chol on viscoelastic behavior, mechanical properties and in vitro degradation of the supramolecular hydrogels was also studied. The hydrogels showed outstanding self-healing capability and good cytocompatibility. The multilayer structure was constructed using hydrogels with self-healing ability. The developed hydrogels provide a fascinating glimpse for the applications in tissue engineering.

Injectable in situ forming poly(L-glutamic acid) hydrogels for cartilage tissue engineering

Injectable, in situ forming hydrogels have exhibited many advantages in regenerative medicine. Herein, we present the novel design of poly(l-glutamic acid) injectable hydrogels via the self-crosslinking of adipic dihydrazide (ADH)-modified poly(l-glutamic acid) (PLGA-ADH) and aldehyde-modified poly(l-glutamic acid) (PLGA-CHO), and investigate their potential in cartilage tissue engineering. Both the hydrazide modification degree of PLGA-ADH and oxidation degree of PLGA-CHO can be adjusted by the amount of activators and sodium periodate, respectively. Experiments reveal that the solid content of the hydrogels, -NH2/-CHO molar ratio, and oxidation degree of PLGA-CHO have a great effect on the gelation time, equilibrium swelling, mechanical properties, microscopic morphology, and in vitro degradation of the hydrogels. Encapsulation of rabbit chondrocytes within the hydrogels showed viability of the entrapped cells and cytocompatibility of the injectable hydrogels. A preliminary study exhibits injectability and rapid in vivo gel formation, as well as mechanical stability, cell ingrowth, and ectopic cartilage formation. These results suggest that the PLGA hydrogel has potential as an injectable cell delivery carrier for cartilage regeneration and could serve as a new biomaterial for tissue engineering.

Strategy for constructing vascularized adipose units in poly(l-glutamic acid) hydrogel porous scaffold through inducing in-situ formation of ASCs spheroids

Vascularization is of great importance to adipose tissue regeneration. Here we introduced a paradigm that using scaffold to induce ASC spheroids, so to promote vascularized adipose tissue regeneration. Poly (l-glutamic acid) (PLGA) was activated by EDC, followed by being cross-linked by Adipic dihydrazide (ADH) to form a homogeneous hydrogel. Lyophilization was then carried out to create porous structure. The PLGA hydrogel scaffold possessed a significant swollen hydrophilic network to weaken cell-scaffold adhesion but drive ASCs to aggregate to form spheroids. Increase of seeding cell density was proved to result in the increase of spheroid size, upregulating angiogenic genes (VEGF and FGF-2) expression by enhancing the hypoxia-induced paracrine secretion. Also, the adipogenic differentiation of ASCs was achieved in spheroids in vitro. Moreover, the in vivo vascularized adipose tissue regeneration was evaluated in the dorsum of nude mice. After 12weeks post-implantation, the significant angiogenesis was found in both adipogenic induced and non-induced engineered tissue. In adipogenic induced group, the clear ring-like morphology, the large vacuole in the middle of the cell and the Oil red O staining demonstrated adipose tissue formation. STATEMENT OF SIGNIFICANCE Vascularization is of great importance to adipose tissue regeneration. Adipose derived stem cell (ASC) spheroids possessed not only the high efficiency of vascularization, but also the improved differentiation ability. Several research works have illustrated the advantage of ASC spheroids in vascularization. However, in adipose regeneration, ASC spheroid was rarely used. Even so, it is reasonable to believe that ASC spheroids hold a great promise in vascularized adipose tissue engineering. Thus in the present study, we introduced a method to create lots of ASC spheroids that acted as lots of individual adipogenesis and angiogenesis units inside of a porous hydrogel scaffold. Then, the scaffold carrying ASC spheroids was implanted subcutaneously in nude mice to preliminarily evaluate the adipose tissue generation and blood vessel formation.

Regeneration of hyaline-like cartilage and subchondral bone simultaneously by poly(L-glutamic acid) based osteochondral scaffolds with induced autologous adipose derived stem cells

Osteochondral tissue engineering is challenged by the difficulty in the regeneration of hyaline cartilage and the simultaneous regeneration of subchondral bone. In the present study, nhydroxyapatite-graft-poly(l-glutamic acid) (nHA-g-PLGA) was prepared by surface-initiated ring-opening polymerization, which was then used to fabricate an osteogenic scaffold (scaffold O) instead of nHA to achieve better mechanical performance. Then, a single osteochondral scaffold was fabricated by combining the poly(l-glutamic acid) (PLGA)/chitosan (CS) amide bonded hydrogel and the PLGA/CS/nHA-g-PLGA polyelectrolyte complex (PEC), possessing two different regions to support both hyaline cartilage and underlying bone regeneration, respectively. Autologous adipose derived stem cells (ASCs) were seeded into the osteochondral scaffold. The chondrogenesis of ASCs in the scaffold was triggered in vitro by TGF-β1 and IGF-1 for 7 days. In vitro, a chondrogenic scaffold (scaffold C) exhibited the ability to drive adipose derived stem cell (ASC) aggregates to form multicellular spheroids with a diameter of 80-110 μm in situ, thus promoting the chondrogenesis while limiting COL I deposition when compared to ASCs adhered in scaffold O. Scaffold O showed the ability to bind abundant BMP-2. Osteochondral scaffolds with induced ASC spheroids in scaffold C and bonded BMP-2 in scaffold O were transplanted into rabbit osteochondral defects as group I for in vivo regeneration. At the same time, osteochondral scaffolds with only bonded BMP-2 in scaffold O and bare osteochondral scaffolds were filled into rabbit osteochondral defects to serve as group II and group III, respectively. After 12 weeks post-implantation, cartilage and subchondral bone tissues were both regenerated with the support of induced ASC spheroids and bonded BMP-2 in group I. However, in group II, cartilage was not repaired while subchondral bone was regenerated. In group III, the regeneration of both cartilage and subchondral bone was limited.

Fabrication of injectable hydrogels based on poly(L-glutamic acid) and chitosan

Injectable hydrogels as an important biomaterial class have been widely used in regenerative medicine. A series of injectable poly(L-glutamic acid)/chitosan (PLGA/CS) hydrogels were fabricated by self-crosslinking aldehyde-modified PLGA (PLGA–CHO) and lactic acid-modified chitosan (CS–LA). The oxidation degree of PLGA–CHO and degree of substitution (DS) of CS–LA could be adjusted by the amount of sodium periodate and lactic acid, respectively. The effect of the solid content of the hydrogels, oxidation degree of PLGA–CHO and CS–LA/PLGA–CHO mass ratio on the gelation time, gel content, water uptake, mechanical properties, microscopic morphology, and in vitro degradation of the hydrogels was examined. The pH and ion sensitivity of PLGA/CS hydrogels was also examined. Encapsulation of rabbit chondrocytes within the hydrogels showed viability of the entrapped cells and cytocompatibility of the injectable hydrogels. The injectable PLGA/CS hydrogels demonstrated attractive properties for future application in pharmaceutical delivery and tissue engineering.

Injectable Stem Cell Laden Open Porous Microgels That Favor Adipogenesis: In Vitro and in Vivo Evaluation

Microgels, with large surface area per volume, show great advantages in adipose tissue engineering due to their injectability and similarity with natural extracellular matrix. However, to date, no studies have tried applying microgels to adipose tissue regeneration. Herein, based on double-bonded poly(l-glutamic acid)-g-2-hydroxyethyl methacrylate (PLGA-g-HEMA) and maleic anhydride-modified chitosan (MCS), an open porous microgel with high hydrophilicity and great injectability is successfully prepared (microgels diameters of 200-300 μm, pore diameter of 38 μm, and porosity of 88.3%). The storage modulus of 30 mg/mL of the microgel dispersions is 2000 Pa, which is similar to that of the native adipose tissue. The spheroidal stem cell shape and extensive cell-cell connections can be formed in the present microgels to promote adipogenic differentiation and realize adipose tissue regeneration. After injection in vitro, the microgels can maintain high stem cell viability up to 14 days. The extensive Oil red O staining is observed after adipogenic induction for 14 days. After 12 weeks postimplantation, adipose tissues can be regenerated well. Blood vessels are formed in the neogenerated tissues. The degradation rate of microgels roughly matches with the adipose tissue formation rate. The study offers an applicable microgel system to boost the adipose tissue regeneration.

Templated fabrication of pH-responsive poly( l -glutamic acid) based nanogels via surface-grafting and macromolecular crosslinking

A novel class of pH-responsive hollow poly(L-glutamic acid)/chitosan (PLGA/CS) nanogels was fabricated by a templating approach, which was mild and surfactant free, and combined with a “grafting from” method and intermacromolecular crosslinking technique. The surface grafting, crosslinking reaction, nanogel fabrication and microstructure were investigated by FTIR, 1H NMR, XRD, TGA, light scattering, and electron microscopy. The size of the resultant PLGA/CS nanogels could be accurately controlled by simply changing the size of the silica template. The nanogels responded to changes in environmental pH, elucidated according to the variation of the size of the nanogels and zeta potential at different pH values. Taking water-soluble antineoplastic agent mitoxantrone (MTX) as a model drug, the nanogels presented high loading ability at high-pH environment and rapid MTX release behavior under acidic conditions. MTT assays used to study the in vitro cytotoxicity of PLGA/CS nanogels showed a negligible cytotoxicity in mouse fibroblast L929 cells. Compared with bare MTX, MTX loaded PLGA/CS nanogels exhibited an enhanced inhibition effect to human gastric carcinoma SGC7901 cells. Fluorescence microscopy and flow cytometry analysis results demonstrated efficient cellular uptake of the PLGA/CS nanogels into the cells. These studies suggest that such pH-responsive PLGA/CS hollow nanogels might have great potential in controlled drug delivery systems.

Preparation of mussel-inspired injectable hydrogels based on dual-functionalized alginate with improved adhesive, self-healing, and mechanical properties

Injectable hydrogels have aroused much attention for the advantages such as minimally invasive surgery, avoidance of surgical trauma, and filling and repairing irregularly shaped tissue defects. Mussel-inspired injectable hydrogels can be immobilized on the surface of tissues, resulting in stable biomaterial-tissue integration. However, the commonly used biomimetic mussel-inspired hydrogels are prepared by the oxidation of catechol groups, which involves the introduction or production of cytotoxic substances. Moreover, mussel-inspired hydrogels generally display weak mechanical strength and poor adhesiveness because of the consumption of catechol groups during oxidation. Herein, we described a strategy to prepare mussel-inspired injectable hydrogels via the Schiff base reaction. We grafted dopamine, an adhesive motif discovered in the holdfast pads of mussels, to aldehyde-modified alginate backbones. A series of injectable mussel-inspired adhesive, self-healing hydrogels were fabricated by in situ crosslinking of hydrazide-modified poly(l-glutamic acid) (PLGA-ADH) and dual-functionalized alginate (catechol- and aldehyde-modified alginate, ALG-CHO-Catechol). Also, oxidized ALG-CHO-Catechol hydrogels and PLGA/ALG-CHO hydrogels were prepared for comparison. The effects of the crosslinking method, catechol grafting ratio and solid content on the mechanical properties, self-healing behavior, adhesive properties, and hemostatic ability were investigated. Compared with the observations for oxidized ALG-CHO-Catechol hydrogels, more reasonable gelation time and notably enhanced mechanical properties and adhesive behavior were detected in the PLGA/ALG-CHO-Catechol hydrogel system. The PLGA/ALG-CHO-Catechol hydrogels also displayed clear self-healing ability and good cytocompatibility. The strong bioadhesion endowed the PLGA/ALG-CHO-Catechol hydrogels with superior hemostatic performance. These results suggested that PLGA/ALG-CHO-Catechol hydrogel might have great potential as an antibleeding and tissue repair material.

Nanocomposite Porous Microcarriers Based on Strontium-Substituted HA-g-Poly(γ-benzyl-l-glutamate) for Bone Tissue Engineering

Porous microcarriers have aroused increasing attention recently, which can create a protected environment for sufficient cell seeding density, facilitate oxygen and nutrient transfer, and well support the cell attachment and growth. In this study, porous microcarriers fabricated from the strontium-substituted hydroxyapatite- graft-poly(γ-benzyl-l-glutamate) (Sr10-HA- g-PBLG) hybrid nanocomposite were developed. The surface grating of PBLG, the micromorphology and element distribution, mechanical strength, in vitro degradation, and Sr2+ ion release of the obtained Sr10-HA- g-PBLG porous microcarriers were investigated, respectively. The grafting ratio and the molecular weight of the grafted PBLG of Sr10-HA- g-PBLG could be effectively controlled by varying the initial ratio of BLG-NCA to Sr10-HA-NH2. The microcarriers exhibited a highly porous and interconnected microstructure with the porosity of about 90% and overall density of 1.03-1.06 g/cm3. Also, the degradation rate of Sr10-HA-PBLG microcarriers could be effectively controlled and long-term Sr2+ release was obtained. The Sr10-HA-PBLG microcarriers allowed cells adhesion, infiltration, and proliferation and promoted the osteogenic differentiation of rabbit adipose-derived stem cells (ADSCs). Successful healing of femoral bone defect was proved by injection of the ADSCs-seeded Sr10-HA-PBLG microcarriers in a rabbit model.

Sr-HA-graft-Poly(γ-benzyl-l-glutamate) Nanocomposite Microcarriers: Controllable Sr2+ Release for Accelerating Osteogenenisis and Bony Nonunion Repair

The microcarrier system offers an attractive method for cellular amplification and phenotype enhancement in the field of bone tissue engineering. However, it remains a challenge to fabricate porous microcarriers with osteoinductive activity for speedy and high-quality osseointegration in regeneration of serious complication of bone fracture, like nonunion. Here, we present a facile method for the first time manufacture microcarriers with osteogenic effects and properties based on well controlled and long-term Sr2+ release. At first, strontium-substituted hydroxyapatite was prepared (Sr-HA) and a novel Sr-HA-graft-poly(γ-benzyl-l-glutamate) (Sr-HA-PBLG) nanocomposite was synthesized. Then, the microcarriers with highly interconnected macropores were fabricated by a double emulsion method, which allowed cells to adhere and proliferate and secrete extracellular matrix. Besides, the microcarriers with a relatively uniform diameter of 271.5 ± 45.0 μm are feasible for injection. The Sr-HA-PBLG microcarriers efficiently promoted osteogenic gene expression in vitro. With injection of the Sr-HA-PBLG microcarriers loading adipose derived stem cells (ADSCs) into the nonunion sites, bone regeneration was observed at 8 weeks after injection in a mice model.