Kunxi Zhang

Injectable In Situ Self-Cross-Linking Hydrogels Based on Poly(l-glutamic acid) and Alginate for Cartilage Tissue Engineering

Injectable hydrogels as an important biomaterial class have been widely used in regenerative medicine. A series of injectable poly(l-glutamic acid)/alginate (PLGA/ALG) hydrogels were fabricated by self-cross-linking of hydrazide-modified poly(l-glutamic acid) (PLGA-ADH) and aldehyde-modified alginate (ALG-CHO). Both the degree of PLGA modification and the oxidation degree of ALG-CHO could be adjusted by the amount of activators and sodium periodate, respectively. The effect of the solid content of the hydrogels and oxidation degree of ALG-CHO on the gelation time, equilibrium swelling, mechanical properties, microscopic morphology, and in vitro degradation of the hydrogels was examined. Encapsulation of rabbit chondrocytes within hydrogels showed viability of the entrapped cells and good biocompatibility of the injectable hydrogels. A preliminary study exhibited injectability and rapid in vivo gel formation, as well as mechanical stability, cell ingrowth, and ectopic cartilage formation. The injectable PLGA/ALG hydrogels demonstrated attractive properties for future application in a variety of pharmaceutical delivery and tissue engineering, especially in cartilage tissue engineering.

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.

Repair of an articular cartilage defect using adipose-derived stem cells loaded on a polyelectrolyte complex scaffold based on poly(L-glutamic acid) and chitosan

As a synthetic polypeptide water-soluble poly(l-glutamic acid) (PLGA) was designed to fabricate scaffolds for cartilage tissue engineering. Chitosan (CHI) has been employed as a physical cross-linking component in the construction of scaffolds. PLGA/CHI scaffolds act as sponges with a swelling ratio of 760±45% (mass%), showing promising biocompatibility and biodegradation. Autologous adipose-derived stem cells (ASCs) were expanded and seeded on PLGA/CHI scaffolds, ASC/scaffold constructs were then subjected to chondrogenic induction in vitro for 2weeks. The results showed that PLGA/CHI scaffolds could effectively support ASC adherence, proliferation and chondrogenic differentiation. The ASCs/scaffold constructs were then transplanted to repair full thickness articular cartilage defects (4mm in diameter, to the depth of subchondral bone) created in rabbit femur trochlea. Histological observations found that articular defects were covered with newly formed cartilage 6weeks post-implantation. After 12weeks the regenerated cartilage had integrated well with the surrounding native cartilage and subchondral bone. Toluidine blue and immunohistochemical staining confirmed similar accumulation of glycosaminoglycans and type II collagen in engineered cartilage as in native cartilage 12weeks post-implantation. The result was further supported by quantitative analysis of extracellular matrix deposition. The compressive modulus of the engineered cartilage increased significantly from 30% of that of normal cartilage at 6weeks to 83% at 12weeks. Cyto-nanoindentation also showed analogous biomechanical behavior of the engineered cartilage to that of native cartilage. The results of the present study thus demonstrate the potentiality of PLGA/CHI scaffolds in cartilage tissue engineering.

Fabrication of poly(L-glutamic acid)/chitosan polyelectrolyte complex porous scaffolds for tissue engineering

Porous scaffolds composed of polypeptides and polysaccharides have remarkable biocompatibility and potential to mimic an extracellular matrix for tissue engineering. This study presented a novel design of polyelectrolyte complex porous scaffolds of a synthetic polypeptide poly(l-glutamic acid) (PLGA) and a natural polysaccharide chitosan (CS) using a freeze drying method. The microstructure of the porous scaffolds could be adjusted by changing the freezing temperature and solid content of the reacting polymer. PLGA/CS scaffolds fabricated from 2% solid content and at a freezing temperature of -20 °C exhibited an interconnected porous structure with average pore size between 150 and 200 μm. The contact angle of less than 75° and high swelling ratio of more than 700% showed the excellent hydrophilic performance of these scaffolds. Degradation of the PLGA/CS composite scaffolds could be modified and more CS content contributed a higher resistance to biodegradation. The mechanical properties of the scaffolds could be controlled by varying the PLGA/CS molar ratio and solid content. The scaffolds exhibited good elastic behavior in wet state. In vitro culture of rabbit adipose-derived stem cells (ASCs) indicated that the selected PLGA/CS porous scaffolds supported cell attachment and growth. In summary, the PLGA/CS porous scaffolds show excellent properties, such as an interconnected porous structure, mechanical strength, hydrophilicity, biodegradability and biocompatibility. The successful repair of articular cartilage defects showed the potentiality of using PLGA/CS scaffolds in cartilage 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.

Novel injectable porous poly(γ-benzyl-L-glutamate) microspheres for cartilage tissue engineering: preparation and evaluation

In search of an injectable cellular delivery vehicle for tissue regeneration, porous microspheres fabricated from the synthetic polypeptide of poly(γ-benzyl-l-glutamate) (PBLG) were developed. The structural and morphological characteristics of the microspheres could be adjusted by changing the amounts of the gelatin porogen. PBLG microspheres fabricated from 6.5% gelatin content exhibited an average pore diameter of 50.9 ± 10.3 μm and a porosity of 86.58 ± 2.37%. Degradation in vitro of the microspheres could be well controlled by adjusting the molecular weight of PBLG, and the degradability in vivo showed a satisfactory degradation time range from 8 to 12 weeks. Articular chondrocytes, which were seeded within the PBLG porous microspheres, exhibited progressive proliferation and deposition of the cartilaginous extracellular matrix. After cultivation for 2 days in vitro, the PBLG porous microspheres loaded with chondrocytes were injected subcutaneously into nude mice. At 4, 8 and 12 weeks post-injection, neo-generated tissue was harvested for histological observations, which showed a typical cartilage structure and cartilaginous matrix accumulation. A gradual increase of GAG and COL II content in neo-generated tissue was detected by biochemical analysis. These results indicate that the fabricated porous microspheres showing controllable degradation properties, good biocompatibility and cytocompatibility are potentially useful as an injectable vehicle for cartilage 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.

A Poly(acrylic acid)-block-Poly(L-glutamic acid) Diblock Copolymer with Improved Cell Adhesion for Surface Modification

A novel PAA-b-PLGA diblock copolymer is synthesized and characterized that has excellent cell adhesion and biocompatibility. Fluorescent DiO labeling is used to monitor the attachment and growth of hASCs on the film surface, and cell proliferation over time is studied. Results show that PLLA modified by a CS/PAA-b-PLGA multilayer film can promote the attachment of human hASCs and provide an advantageous environment for their proliferation. The multilayer film presents excellent biocompatibility and cell adhesive properties, which will provide a new choice for improving the cell attachment in surface modification for tissue engineering. Hydroxyl, carboxyl and amine groups in the CS/PAA-b-PLGA multilayer film may be combined with drugs and growth factors for therapy and differentiation.

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.

Layer-by-Layer Assembled Multilayer Films of Methoxypoly(ethylene glycol)-block-poly(α,L-glutamic acid) and Chitosan with Reduced Cell Adhesion

A methoxypoly(ethylene glycol)-block-poly(α,L-glutamic acid) (mPEGGA) diblock copolymer is synthesized. Using QCM measurements, it is shown that (CS/mPEGGA)(n) film construction takes place over two build-up stages (exponential-to-linear). UV-vis spectra reveal the regular increase of the multilayer film growth at different molecular weights of mPEGGA. Contact angle and surface morphology investigation prove that the hydrophilicity of CS/mPEGGA multilayer film-modified substrate becomes better and the surface becomes rough. Significantly reduced cell adhesion is observed on the CS/mPEGGA multilayer film coated surface.