Kai Chiang Yang

The use of biotinylated-EGF-modified gelatin nanoparticle carrier to enhance cisplatin accumulation in cancerous lungs via inhalation

To develop a polymer-anticancer drug conjugate, we employed gelatin nanoparticles (GPs) as carriers of cisplatin (CDDP) with anticipated improved therapeutic effect and reduced side effects. The anticancer activities of CDDP-incorporated in GPs (GP-Pt) with biotinylated-EGF (bEGF) modification (GP-Pt-bEGF) were studied. GP-Pt-bEGF with EGFR affinity produced much higher Pt concentrations in A549 cells (high EGFR expression) than in HFL1 cells (low EGFR expression). An in vitro anticancer study showed that GP-Pt-bEGF was more potent than free CDDP or GP-Pt because of its rapid effect on the cell cycle as well as a lower IC(50) (1.2microg/ml) that inhibits A549 cell growth. PI staining showed that cells treated with GP-Pt-bEGF for only 4h had the highest sub-G1 population. The CDDP formulations - free CDDP, GP-Pt, and GP-Pt-bEGF - were given by intratumorous injections to SCID mice in a subcutaneous model. This treatment showed that GP-Pt-bEGF had stronger anti-tumor activity and was less toxic than free CDDP in vivo. Mice treated with GP-Pt-bEGF showed slight body weight loss, whereas free CDDP treatment at the same dose caused a body weight loss of 20-30%. Furthermore, these formulations were given to mice with lung cancer via aerosol delivery. This treatment showed that inhaled GP-Pt-bEGF could target EGFR-overexpressing cells to achieve high cisplatin dosage in cancerous lungs. To summarize, gelatin nanoparticles loaded with CDDP and decorated with EGF tumor-specific ligand were successfully developed. Their in vitro and in vivo targeting ability and anticancer effect were confirmed. The aerosol delivery of the nanodrug carrier was demonstrated. Simple aerosol delivery of targeted drug carriers may prove useful for the clinical treatment of lung cancer patients.

A highly organized three-dimensional alginate scaffold for cartilage tissue engineering prepared by microfluidic technology.

Osteoarthritis is a degenerative disease and frequently involves the knee, hip and phalangeal joints. Current treatments used in small cartilage defects including multiple drilling, abrasion arthroplasty, mosaicplasty, and autogenous chondrocyte transplantation, however, there are problems needed to be solved. The standard treatment for severe osteoarthritis is total joint arthroplasty. The disadvantages of this surgery are the possibility of implant loosening. Therefore, tissue engineering for cartilage regeneration has become a promising topic. We have developed a new method to produce a highly organized single polymer (alginate) scaffold using microfluidic device. Scanning electron microscope and confocal fluoroscope examinations showed that the scaffold has a regular interconnected porous structure in the scale of 250 μm and high porosity. The scaffold is effective in chondrocyte culture; the cell viability test (WST-1 assay), cell toxicity (lactate dehydrogenase assay), cell survival rate, extracellular matrix production (glycosaminoglycans contents), cell proliferation (DNA quantification), and gene expression (real-time PCR) all revealed good results for chondrocyte culture. The chondrocytes can maintain normal phenotypes, highly express aggrecan and type II collagen, and secrete a great deal of extracellular matrix when seeded in the alginate scaffold. This study demonstrated that a highly organized alginate scaffold can be prepared with an economical microfluidic device, and this scaffold is effective in cartilage tissue engineering.

Chitosan/gelatin hydrogel as immunoisolative matrix for injectable bioartificial pancreas

Abstract:  Background:  The feasibility of using chitosan/gelatin hydrogel as immunoisolative matrix to provide an additional protection to the microencapsulated islet cells was demonstrated in this study. We hope that the use of hydrogel can extend the functional longevity of microencapsulated islet cells during xenotransplantation.

Cartilage regeneration in SCID mice using a highly organized three-dimensional alginate scaffold

Tissue engineering for cartilage regeneration provides an alternative to surgery for degenerative osteoarthritis. Recently, a highly organized three-dimensional (3D) alginate scaffold was prepared using a microfluidic device; this scaffold is effective for chondrocyte culture in vitro. The performance of this scaffold was further demonstrated; an alginate scaffold seeded with porcine chondrocytes was implanted in the dorsal subcutaneous site of SCID mice. The recipients were sacrificed at 2, 4, and 6 weeks after transplantation. The grafted implants retrieved from the subcutaneous site were analyzed with histologic examinations. Real-time PCR was used to identify the gene expression patterns of the chondrocytes. The hematoxylin and eosin staining showed that the chondrocytes survived normally in SCID mice; cartilage-like structures were formed after 4 weeks implantation. Immunohistochemical staining revealed cells secreted type II collagen, produced glycosaminoglycans (proved by alcian blue stain), and maintained the expression of S-100. On the other hand, the cells were negative for type I and type X collagen staining. PCR showed that the mRNA expressions of aggrecan and type II collagen were up-regulated at weeks two and four, while type I and type X collagen were down-regulated during the study period. In summary, this highly organized 3D alginate scaffold provided a suitable environment and maintained functional phenotypes for chondrocytes in this animal study.

Bone marrow combined with dental bud cells promotes tooth regeneration in miniature pig model

Growth factors and morphogens secreted by bone marrow mesenchymal stem cells (BMSCs) of bone marrow fluid may promote tooth regeneration. Accordingly, a tissue engineering approach was utilized to develop an economical strategy for obtaining the growth factors and morphogens from BMSCs. Unerupted second molar tooth buds harvested from miniature pigs were cultured in vitro to obtain dental bud cells (DBCs). Bone marrow fluid, which contains BMSCs, was collected from the porcine mandible before operation. DBCs suspended in bone marrow fluid were seeded into a gelatin/chondoitin-6-sulfate/hyaluronan tri-copolymer scaffold (GCHT scaffold). The DBCs/bone marrow fluid/GCHT scaffold was autografted into the original alveolar sockets of the pigs. Radiographic and histological examinations were applied to identify the structure of regenerated tooth at 40 weeks postimplantation. The present results showed that one pig developed a complete tooth with crown, root, pulp, enamel, dentin, odontoblast, cementum, blood vessel, and periodontal ligament in indiscriminate shape. Three animals had an unerupted tooth that expressed dentin matrix protein-1, vascular endothelial growth factor, and osteopontin; and two other pigs also had dental-like structure with dentin tubules. This study reveals that DBCs adding bone marrow fluid and a suitable scaffold can promote the tooth regeneration in autogenic cell transplantation.

The cytoprotection of chitosan based hydrogels in xenogeneic islet transplantation: An in vivo study in streptozotocin-induced diabetic mouse.

Immune rejection and scarcity of donor tissues are the restrictions of islets transplantation. In this study, the cytoprotection of chitosan hydrogels in xenogeneic islet transplantation was demonstrated. Wistar rat islets encapsulated in chitosan hydrogels were performed glucose challenge test and live/dead cell staining in vitro. Islets/chitosan hydrogels were transplanted into the renal subcapsular space of diabetic C57BL/6 mice. Non-fasting blood glucose level (NFBG), body weight, intraperitoneal glucose tolerance test (IPGTT), and glucose disappearance rate were determined perioperatively. The serum insulin level was analyzed, and the kidney transplanted with islets/chitosan hydrogels were retrieved for histological examination after sacrifice. The present results showed that islets encapsulated in chitosan hydrogels secreted insulin in response to the glucose stimulation as naked islets with higher cell survival. The NFBG of diabetic mice transplanted with islets/chitosan hydrogels decreased from 487+/-46 to 148+/-32 at one day postoperation and maintained in the range of 201+/-36 mg/dl for four weeks with an increase in body weight. IPGTT showed the glucose disappearance rate of mice transplanted with islets/chitosan hydrogels was significant faster than that of mice transplanted with naked islets; the serum insulin level increased from 0.29+/-0.06 to 1.69+/-0.65 microg/dl postoperatively. Histological examination revealed that the islets successfully engrafted at renal subcapsular space with positive insulin staining. The immunostain was negative for neither the T-cell lineages nor the monocyte/macrophages. This study indicates that the chitosan hydrogels deliver and protect encapsulated islets successfully in xenotransplantation.

Calcium phosphate cement delivering zoledronate decreases bone turnover rate and restores bone architecture in ovariectomized rats

Patients sustaining bony fractures frequently require the application of bone graft substitutes to fill the bone defects. In the meantime, anti-osteoporosis drugs may be added in bone fillers to treat osteoporosis, especially in postmenopausal women and the elderly. The effects of zoledronate-impregnated calcium phosphate cement (ZLN/CPC) on ovariectomized (OVX) rats were evaluated. OVX rats were implanted with ZLN/CPC, containing 0.025 mg ZLN in the greater omentum. Afterward the clinical sign of toxicity was recorded for eight weeks. The rats were sacrificed and blood samples were collected for hematology and serum bone turnover markers analyses. The four limbs of the rats were harvested and micro-computer tomography (micro-CT) scanning and bone ash analyses were performed. No clinical toxicity was observed in the treated rats. Compared to the OVX rats, levels of bone resorption markers (fragments of C-telopeptides of type I collagen) and bone formation markers (alkaline phosphatase and osteocalcin) decreased significantly in the treated rats. Osteopontin, which mediates the anchoring of osteoclasts to the mineral matrix of bones, also decreased significantly. Micro-CT scanning and histologic examinations of the distal femoral metaphyses showed that the cancellous bone architectures were restored, with a concomitant decrease in bone porosity. The bone mineral content in the bone ashes also increased significantly. This study indicates that ZLN-impregnated CPC reduces bone turnover rate and restores bone architecture in OVX rats. CPC may be an appropriate carrier to deliver drugs to treat osteoporosis, and this approach may also reduce rates of post-dosing symptoms for intravenous ZLN delivery.

Hyaluronan up-regulates IL-10 expression in fibroblast-like synoviocytes from patients with tibia plateau fracture.

Progression to osteoarthritis (OA) is a frequent sequela of severe articular fracture, particularly when weight‐bearing joints are involved. Prevention from post‐traumatic OA remains a challenge. Hyaluronan (HA) therapy is reported to represent a safe and effective treatment for patients with OA and rheumatoid arthritis. However, the capacity of HA to prevent the occurrence of osteoarthritic changes in fractured joints has not been demonstrated. The present study was undertaken to examine the effects of HA on expression of six OA‐related proteins in fibroblast‐like synoviocytes (FLS) from 10 patients with tibia plateau fracture. OA‐related factors were quantified using a sandwich enzyme‐linked immunosorbent assay. Regardless of induction of the FLS with interleukin (IL)‐1β, HA was found to down‐regulate expression of catabolic factors (IL‐1β, matrix metalloproteinase‐3, and tumor necrosis factor‐α) and to up‐regulate production of anti‐catabolic factors (tissue inhibitors of metalloproteinase‐1 and metalloproteinase‐2). HA also enhanced expression of IL‐10, an anti‐inflammatory cytokine, in FLS. Our results indicated that HA can promote the expression of both antiinflammatory and structure‐protective factors in FLS of patients with tibia plateau fracture.

Fabrication of large perfusable macroporous cell-laden hydrogel scaffolds using microbial transglutaminase

In this study, we developed a method to fabricate large, perfusable, macroporous, cell-laden hydrogels. This method is suitable for efficient cell seeding, and can maintain sufficient oxygen delivery and mass transfer. We first loaded three types of testing cells (including NIH 3T3, ADSC and Huh7) into gelatin hydrogel filaments, then cross-linked the cell-laden gelatin hydrogel filaments using microbial transglutaminase (mTGase). In situ cross-linking by mTGase was found to be non-cytotoxic and prevented the scattering of the cells after delivery. The gelatin hydrogel constructs kept the carried cells viable; also, the porosity and permeability were adequate for a perfusion system. Cell proliferation was better under perfusion culture than under static culture. When human umbilical vein endothelial cells were seeded into the constructs, we demonstrated that they stably formed an even coverage on the surface of the hydrogel filaments, serving as a preliminary microvasculature network. We concluded that this method provides a viable solution for cell seeding, oxygen delivery, and mass transfer in large three-dimensional (3-D) tissue engineering. Furthermore, it has the potential for being a workhorse in studies involving 3-D cell cultures and tissue engineering.

Electrofusion of Mesenchymal Stem Cells and Islet Cells for Diabetes Therapy: A Rat Model

Islet transplantation is a minimally invasive treatment for severe diabetes. However, it often requires multiple donors to accomplish insulin-independence and the long-term results are not yet satisfying. Therefore, novel ways to overcome these problems have been explored. Isolated islets are fragile and susceptible to pro-apoptotic factors and poorly proliferative. In contrast, mesenchymal stem cells (MSCs) are highly proliferative, anti-apoptotic and pluripotent to differentiate toward various cell types, promote angiogenesis and modulate inflammation, thereby studied as an enhancer of islet function and engraftment. Electrofusion is an efficient method of cell fusion and nuclear reprogramming occurs in hybrid cells between different cell types. Therefore, we hypothesized that electrofusion between MSC and islet cells may yield robust islet cells for diabetes therapy. We establish a method of electrofusion between dispersed islet cells and MSCs in rats. The fusion cells maintained glucose-responsive insulin release for 20 days in vitro. Renal subcapsular transplantation of fusion cells prepared from suboptimal islet mass (1,000 islets) that did not correct hyperglycemia even if co-transplanted with MSCs, caused slow but consistent lowering of blood glucose with significant weight gain within the observation period in streptozotocin-induced diabetic rats. In the fusion cells between rat islet cells and mouse MSCs, RT-PCR showed new expression of both rat MSC-related genes and mouse β-cell-related genes, indicating bidirectional reprogramming of both β-cell and MSCs nuclei. Moreover, decreased caspase3 expression and new expression of Ki-67 in the islet cell nuclei suggested alleviated apoptosis and gain of proliferative capability, respectively. These results show that electrofusion between MSCs and islet cells yield special cells with β-cell function and robustness of MSCs and seems feasible for novel therapeutic strategy for diabetes mellitus.