In situ sequestration of endogenous PDGF-BB with an ECM-mimetic sponge for accelerated wound healing.

Recapitulating the typical features of extracellular matrix (ECM) in engineered biomaterials is crucial for preparing a suitable niche to activate endogenous tissue repair. Here, we report the design and evaluation of an ECM-mimetic scaffold that successfully accelerated wound healing through enriching endogenous platelet-derived growth factor-BB (PDGF-BB). Specifically, we prepared a electrospun hydrogel sponge (EGS) comprising a PDGF-BB-binding polysaccharide (EUP3) and gelatin. The two polymers in concert exerted a 'retention-and-release' function: upon the application of EGS in vivo, EUP3 started to bind and sequester endogenous PDGF-BB at the wound site; gradually, gelatin was degraded to free the PDGF-BB/EUP3 complex that acted on the cells in situ. Our serial in vitro and in vivo tests validated the efficacy of EGS in retaining PDGF-BB, releasing PDGF-BB/EUP3 in response to collagenase, and promoting various PDGF-BB-mediated regenerative activities. Particularly, EGS accelerated the repair of a full-thickness skin wound in mice and induced optimal neo-tissue formation, without the addition of any exogenous GFs, cells or genes. Collectively, our results suggest that, by mimicking the distinctive GF-affinitive feature of ECM, EGS as an engineered biomaterial can effectively harness the endogenous regenerative power of the native tissue. Our investigation may inspire the design of new, effective and safer approaches for tissue regeneration.

Three-Dimensional Nanofiber Hybrid Scaffold Directs and Enhances Axonal Regeneration after Spinal Cord Injury.

Spinal cord injuries (SCIs) are followed by a complex series of events that contribute to the failure of regeneration. To date, there is no robust treatment that can restore the injury-induced loss of function. Since damaged spinal axons do not spontaneously regenerate in their native inhibitory microenvironment, a combined application of biomaterials and neurotrophic factors that induce nerve regeneration emerges as an attractive treatment for SCIs. In this study, we report the novel use of a three-dimensional (3D) hybrid scaffold to provide contact guidance for regrowth of axons in vivo. The scaffold comprises 3D aligned sparsely distributed poly(ε-caprolactone-co-ethyl ethylene phosphate) nanofibers that are supported and dispersed within a collagen hydrogel. Neurotrophin-3 was incorporated into the scaffold as an additional biochemical signal. To evaluate the efficacy of the scaffold in supporting nerve regeneration after SCIs, the construct was implanted into an incision injury, which was created at level C5 in the rat spinal cord. After 3 months of implantation, scaffolds with NT-3 incorporation showed the highest average neurite length (391.9 ± 12.9 µm, p ≤ 0.001) as compared to all the other experimental groups. In addition, these regenerated axons formed along the direction of the aligned nanofibers, regardless of their orientation. Moreover, the presence of the hybrid scaffolds did not affect tissue scarring and inflammatory reaction. Taken together, these findings demonstrate that our scaffold design can serve as a potential platform to support axonal regeneration following SCIs.

Effects of nucleus pulposus cell-derived acellular matrix on the differentiation of mesenchymal stem cells.

Recent attempts to treat disc degeneration with mesenchymal stem cells (MSCs) showed encouraging results. Differentiating MSCs towards nucleus pulposus cell (NPC)-like lineages represents a speculative mechanism. Niche factors including hypoxia, growth factors and cell-cell interactions have been suggested but the matrix niche factor has not been studied. Our collagen microencapsulation provides a 3D model to study matrix niche as it enables the encapsulated cells to remodel the template matrix. We previously demonstrated the chondro-inductive role of of chondrocytes-derived matrix in MSCs and showed that NPCs maintained their phenotype and remodeled the template matrix of collagen microspheres into a glycosaminoglycan (GAG)-rich one. Here we aim to study the effects of NPC-derived matrix on MSC differentiation towards NPC-like lineages by firstly producing an NPC-derived matrix in collagen microspheres, secondly optimizing a decellularization protocol to discard NPCs yet retaining the matrix, thirdly repopulating the acellular NPC-derived matrix with MSCs and fourthly evaluating their phenotype. Finally, we injected these microspheres in a pilot rabbit disc degeneration model. Results showed that NPCs survived, maintained their phenotypic markers and produced GAGs. A decellularization protocol with maximal removal of the NPCs, minimal loss in major matrix components and partial retention of NPC-specific markers was identified. The resulting acellular matrix supported MSC survival and matrix production, and up-regulated the gene expression of NPC markers including type II collagen and glypican 3. Finally, injection of MSC in these microspheres in rabbit degenerative disc better maintained hydration level with more pronounced staining of GAGs and type II collagen than controls.

Simultaneous regeneration of articular cartilage and subchondral bone in vivo using MSCs induced by a spatially controlled gene delivery system in bilayered integrated scaffolds.

Engineering complex tissues is important but difficult to achieve in tissue regeneration. Osteochondral tissue engineering for the repair of osteochondral defect, involving simultaneous regeneration of bone and cartilage, has attracted considerable attention and also serves as an optimal model system for developing effective strategies aimed at regenerating complex tissues. In the present study, we formulated a bilayered gene-activated osteochondral scaffold consisting of plasmid TGF-ß1-activated chitosan-gelatin scaffold for chondrogenic layer and plasmid BMP-2-activated hydroxyapatite/chitosan-gelatin scaffold for osteogenic layer. Mesenchymal stem cells seeded in each layer of the bilayered gene- activated osteochondral scaffold showed significant cell proliferation, high expression of TGF-ß1 protein and BMP-2 protein respectively. The results showed that spatially controlled and localized gene delivery system in the bilayered integrated scaffolds could induce the mesenchymal stem cells in different layers to differentiate into chondrocytes and osteoblasts in vitro, respectively, and simultaneously support the articular cartilage and subchondral bone regeneration in the rabbit knee ostochondral defect model. This study gives the evidence that multi-tissue regeneration through the combination of biomimetic and multi-phasic scaffold design, spatially controlled and localized gene delivery system and multi-lineage differentiation of a single stem cell population represents a promising strategy for facilitating the development of complex tissue or organ systems.

Antitumor activity and toxicological properties of doxorubicin conjugated to [alpha],[beta]-poly[(2-hydroxyethyl)-L-aspartamide] administered intraperitoneally in mice.

A polymer-drug conjugate was developed by conjugating doxorubicin (DOX) to [alpha],[beta]-poly[(2-hydroxyethyl)-L-aspartamide] (PHEA) with a succinic spacer. The suitability of PHEA-DOX in intraperitoneal chemotherapy was investigated both in vitro and in vivo. The results showed that the release rate of DOX from PHEA-DOX in S180 ascites was faster than that in mouse serum or in buffer solutions. An in-vivo antitumor study revealed that PHEA-DOX was more effective than DOX against solid S180 tumor after intraperitoneal injection at the same dose of 10 or 15 mg (DOX eq.)/kg, respectively. At a high dose of 28 mg (DOX eq.)/kg, which was lethal for free DOX to mice, PHEA-DOX could inhibit 61.5% of solid S180 tumor growth and markedly prolonged the survival time of ascetic S180-bearing mice. The toxicological effects of PHEA-DOX injected intraperitoneally in normal mice were assessed by using LD50, body weight increment, electrocardiography, blood biochemical indices, and myocardium histology, giving evidence that PHEA-DOX displayed considerably reduced systemic and cardiotoxicity compared with free DOX. All results suggest that PHEA-DOX has great potential for intraperitoneal chemotherapy because of its high therapeutic effects and few adverse side effects.

A physiologically active polysaccharide hydrogel promotes wound healing.

When the skin is injured, the subcutaneous tissues and organs are threatened by pathogens and excessive water loss. Wound dressings are, therefore, needed to protect the wound site from infection and improve the wound closure. Natural polysaccharides have been applied for various biomaterials including wound dressings, which show their advantages in biocompatibility, low toxicity, and pharmaceutical biomedical activity. In this study, a natural polysaccharide Bletilla striata polysaccharide (BSP) hydrogel is prepared by an oxidation and crosslinking methods. This BSP hydrogel represents preferable swelling ability and appropriate water vapor transmission rate. Using a full-thickness trauma mouse model, the hydrogel is applied on the in vivo cutaneous wound healing. Compared with the control groups, the BSP hydrogel achieves the much better healing results. The quantification of the infiltrating inflammatory cells and the level of tumor necrosis factor alpha (TNF-alpha) in the BSP group are attenuated, whereas the secretion of the epidermal growth factor (EGF) is highly elevated. On the 11th day after surgery, the wound area in the BSP hydrogel group is only 1/5-1/3 of those in the control groups. This new BSP hydrogel is proved to control the inflammatory responses and accelerate the wound closure and has potential application in wound healing. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Improved cartilage regeneration utilizing mesenchymal stem cells in TGF-beta1 gene-activated scaffolds.

Recently, bone marrow-derived mesenchymal stem cells (MSCs) have been paid more attention for cartilage regeneration. This study evaluated the potential of using MSCs seeded in plasmid transforming growth factor beta1 (pTGF-beta1)-activated three-dimensional chitosan/gelatin scaffolds for improving cartilage repair in vivo. Significant cell proliferation and transforming growth factor beta1 protein expression were observed in vitro in pTGFbeta1-activated scaffolds. Transforming growth factor beta1-activated scaffolds showed high collagen type II and aggrecan expression and low collagen type I expression during in vitro cultivation. MSC-based pTGF-beta1-activated scaffolds also exhibited cartilage histology with high secretion of collagen type II in vitro under the stimulation of pTGF-beta1. In rabbits with full-thickness cartilage defects, the implantation of MSC-based pTGF-beta1-activated scaffolds not only significantly promoted chondrogenic differentiation of MSCs and hyalin-like cartilage matrix synthesis, but also remarkably improved the overall repair of rabbit cartilage defects and exhibited favorable tissue integrity at 10 weeks postsurgery. These results suggest that MSC-based localized pTGF-beta1-activated scaffolds have potential applications for in vivo cartilage repair.

Targeting delivery oligonucleotide into macrophages by cationic polysaccharide from Bletilla striata successfully inhibited the expression of TNF-alpha.

Competent vehicles based on natural biopolymers are highly demanded in the practice of gene-assisted cell therapy. In this study, a novel gene carrier was developed based on a bioactive glucomannan that was a polysaccharide isolated from an herb Bletilla striata (BSP) and modified with N, N'-carbonyldiimidazole (CDI)/ethylenediamine in order to acquire nucleic acid binding affinity. Particle size observation and electrophoretic mobility tests indicated that the cationized BSP (cBSP) could efficiently combine DNA to form nano-scaled compact and stable complexes and promote the transfection of oligodeoxynucleotide (ODN). Specifically, cBSP exhibited significantly high affinity to macrophages, as evidenced by transfection examination on multiple cell types and competitive test with mannose/glucomannan. In addition, the efficacy of the delivered ODN by cBSP was evaluated by the quantification of gene expression and a dramatic enhancement in suppressing target gene expression was observed. All the findings suggested the possible existence of interaction between cBSP ligand and receptor on macrophage surface. In this way, the ubiquitous mannose receptors and beta-glucan receptors on macrophage could recognize the mannose and beta-glucose residues in BSP framework, thus further mediated the oriented ODN delivery. We expect cBSP to be capable of conveying antisense nucleotides (e.g., oligodeoxynucleotide and small interference RNA) for the practical anti-inflammatory therapy.

Antisense oligonucleotide targeting TNF-alpha can suppress Co-Cr-Mo particle-induced osteolysis.

The most common cause of implant failure in joint replacement is aseptic loosening due to particle-induced osteolysis. TNF-alpha has been shown to be one of the key factors in the process of osteoclastogenesis. Anti-TNF agents are useful in the treatment of joint inflammation related to osteolysis. This study investigated the effect of a single subcutaneous dose of an antisense oligonucleotide (ASO) on particle-induced osteolysis. We utilized the murine calvaria osteolysis model in C57BL/J6 mice. Bone resorption was measured by the toluidine blue staining. Osteoclasts were detected by tartrate resistant acid phosphatase (TRAP) staining assay and were quantified by a TRAP quantification kit. Results show that bone resorption is 0.347 +/- 0.09 mm(2) in mice with particle implantation, and decreased to 0.123 +/- 0.05 mm(2) and 0.052 +/- 0.02 mm(2) after ASO treatment with low and high doses, respectively. The number of osteoclasts in animal calvaria treated with ASO is reduced compared with that of untreated animals, and the quantification results indicate that about 90% of osteoclastogenesis is suppressed by the ASO. In addition, the osteoclastogenesis can be reestablished by the addition of TNF-alpha. In conclusion, we demonstrate that the antisense oligonucleotide targeting to TNF-alpha can suppress osteolysis induced by metal particles in a murine calvaria model. This new finding may be of value in the search for novel therapeutic methods for implant loosening.

Bletilla striata polysaccharide stimulates inducible nitric oxide synthase and proinflammatory cytokine expression in macrophages.

Bletilla striata, a traditional Chinese medicine, has been used for the treatment of alimentary canal mucosal damage, ulcers, bleeding, bruises and burns. B. striata polysaccharide (BSP) isolated from B. striata was found to enhance vascular endothelial cell (EC) proliferation and vascular endothelial growth factor (VEGF) expression. However, the wound healing mechanism of BSP is not well understood. In this study, the results show that treatment with BSP induces coordinate changes in inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1beta) mRNA levels and enhances the expression of these cytokines, but has no effect on interferon gamma (IFN-gamma) level. In this study, we partially elucidate the wound healing mechanism of BSP.

Galactosylated low molecular weight chitosan as a carrier delivering oligonucleotides to Kupffer cells instead of hepatocytes in vivo.

The in vivo cellular localization of oligodeoxynucleotides (ODNs) delivered by galactosylated low molecular weight chitosan (gal-LMWC) was investigated. The gal-LMWCs preference for Kupffer cells was confirmed by in vivo and in vitro experiments. Furthermore, asialoglycoprotein receptor (ASGPr) was studied as a possible surface lectin which may involved in the endocytosis of the gal-LMWC/ODN complexes. Results showed that the gal-LMWC/ODN complex accumulated in liver when injected intravenously (i.v.). Further studies revealed that 50.6% of the complex was taken up by Kupffer cells in liver, 33.2% was taken up by endothelial cells, and only 16.2% of the complex was taken up by parenchymal cells. In vitro results also confirmed the affinity of gal-LMWC to murine Kupffer cells. Inhibition of the transfection by lactose and N-acetyl galactosamine (GalNAc) suggested that the particles might enter macrophages via ASGPr and the inhibition by LMWC implied that there might be other lectins involved in the endocytosis. In summary, our studies revealed that gal-LMWC/ODN complex is inclined to enter into Kupffer cells rather than into liver parenchymal cells in vivo. Galactosylation may not be a proper means for targeting chitosan/DNA nanoparticles to hepatocytes but it does have the potential to be a Kupffer cells targeting strategy especially for delivering drugs for antiinflammation.

Encapsulation of platinum anticancer drugs by apoferritin.

Apoferritin derived from the native protein ferritin was employed to encapsulate anticancer drugs cisplatin and carboplatin.

An efficient vector for gene delivery: alpha,beta-poly (3-dimethylaminopropyl-D,L-aspartamide).

PSI, as the potential peptide-like intermediate, is subject to simple chemical modification in order to obtain good non-viral carriers for gene delivery. This paper describes the facile synthesis and preliminary evaluation of alpha,beta-poly (3-dimethylaminopropyl-D,L-aspartamide) (PDAI) as a vector. Reaction of PSI with 3-dimethylamino-1-propylamine afforded PDAI in N,N-dimethylformamide (DMF) solution. Such biophysical properties of PDAI/DNA complexes as the particle size and the zeta potential were determined by dynamic light scattering assay. The complexes prepared at weight ratios ranging from 2 to 3 have an average size of around 200 nm and a zeta potential of around 10.0 mV. Gel electrophoresis assays confirmed that PDAI could compact DNA to form the complexes and protect DNA from enzymatic degradation by DNase I at the weight ratio above 2.0. Furthermore, PDAI was found to transfect HepG2 cells at a much higher efficiency than commercially available polyethylenimine (PEI) (W(w)=75,000 Da). MTT cytotoxicity assay demonstrated that PDAI also showed much less toxicity than did PEI, suggesting that PDAI is a new class of transfection reagent to be used as a safe vector.

A polysaccharide isolated from the medicinal herb Bletilla striata induces endothelial cells proliferation and vascular endothelial growth factor expression in vitro.

A polysaccharide from the traditional Chinese medicinal herb, Bletilla striata (Thunb.) Reichb. f., was isolated, purified and characterized. It induced the proliferation of human umbilical vascular endothelial cells and the expression of vascular endothelial growth factor up to 156% and 147% of control after 72 h, respectively.