Chang Chin Wu

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.

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.

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.

Strontium-impregnated bioabsorbable composite for osteoporotic fracture fixation

Osteoporosis impairs the bone-healing process as well as bone fracture fixation. The intervention of osteoporosis is considered to be one part of bone fracture treatment. Thus, orthopedic fixators impregnated with antiosteoporosis regimens will improve fracture fixation in osteoporotic bone. In this study, the strontium (Sr) and calcium phosphate ceramic (CPC) were mixed first and then mixed with poly(ε-caprolactone) (PCL) to fabricate a bioactive and bioabsorbable bone fixators. The prepared Sr-CPC/PCL screws were implanted into the distal femur of ovariectomized rabbits. The results showed that Sr-CPC/PCL composite had the appropriate mechanical properties, good biocompatibility, and radio-opacity. The Sr addition created a porous structure and accelerated the degradation of bone screws, but the degradation products did not acidify the surrounding environment. For osteoporotic animals, favorable osteointegration around the Sr-CPC/PCL screws was found, and the total porosity of trabecular bone was decreased under the inspections of micro-computerized tomography. Compared with PCL or CPC/PCL screw, animals which received Sr-CPC/PCL were found to have better results in terms of trabecular number, thickness, and separation. This study reveals that the Sr-impregnated bone fixator improves osseointegration in osteoporotic animals. Sr-CPC/PCL composite is a good candidate material for osteofixation in osteoporotic patients.

A biomimetic honeycomb‐like scaffold prepared by flow‐focusing technology for cartilage regeneration

A tissue engineering chondrocytes/scaffold construct provides a promise to cartilage regeneration. The architecture of a scaffold such as interconnections, porosities, and pore sizes influences the fates of seeding cells including gene expression, survival, migration, proliferation, and differentiation thus may determine the success of this approach. Scaffolds of highly ordered and uniform structures are desirable to control cellular behaviors. In this study, a newly designed microfluidic device based on flow‐focusing geometry was developed to fabricate gelatin scaffolds of ordered pores. In comparison with random foam scaffolds made by the conventional freeze‐dried method, honeycomb‐like scaffolds exhibit higher swelling ratio, porosity, and comparable compressive strength. In addition, chondrocytes grown in the honeycomb‐like scaffolds had good cell viability, survival rate, glycosaminoglycans production, and a better proliferation than ones in freeze‐dried scaffolds. Real‐time PCR analysis showed that the mRNA expressions of aggrecan and collagen type II were up‐regulated when chondrocytes cultured in honeycomb‐like scaffolds rather than cells cultured as monolayer fashion. Oppositely, chondrocytes expressed collagen type II as monolayer culture when seeded in freeze‐dried scaffolds. Histologic examinations revealed that cells produced proteoglycan and distributed uniformly in honeycomb‐like scaffolds. Immunostaining showed protein expression of S‐100 and collagen type II but negative for collagen type I and X, which represents the chondrocytes maintained normal phenotype. In conclusion, a highly ordered and honeycomb‐like scaffold shows superior performance in cartilage tissue engineering. Biotechnol. Bioeng. 2014;111: 2338–2348.

In Vitro Studies of Composite Bone Filler Based on Poly(Propylene Fumarate) and Biphasic α‐Tricalcium Phosphate/Hydroxyapatite Ceramic Powder

While many different filler materials have been applied in vertebral augmentation procedures, none is perfect in all biomechanical and biological characteristics. To minimize possible shortages, we synthesized a new biodegradable, injectable, and premixed composite made from poly(propylene fumarate) (PPF) and biphasic α-tricalcium phosphate (α-TCP)/hydroxyapatite (HAP) ceramics powder and evaluated the material properties of the compound in vitro. We mixed the PPF cross-linked by N-vinyl pyrrolidinone and biphasic α-TCP/HAP powder in different ratios with benzoyl peroxide as an initiator. The setting time and temperature were recorded, although they could be manipulated by modulating the concentrations of hydroquinone and N,N-dimethyl-p-toluidine. Degradation, cytocompatibility, mechanical properties, and radiopacity were analyzed after the composites were cured by a cylindrical shape. We also compared the study materials with poly(methyl methacrylate) (PMMA) and PPF with pure HAP particles. Results showed that lower temperature during curing process (38-44°C), sufficient initial mechanical compressive fracture strength (61.1±3.7MPa), and gradual degradation were observed in the newly developed bone filler. Radiopacity in Hounsfield units was similar to PMMA as determined by computed tomography scan. Both pH value variation and cytotoxicity were within biological tolerable limits based on the biocompatibility tests. Mixtures with 70% α-TCP/HAP powder were superior to other groups. This study indicated that a composite of PPF and biphasic α-TCP/HAP powder is a promising, premixed, injectable biodegradable filler and that a mixture containing 70% α-TCP/HAP exhibits the best properties.

Comparison of Bioartificial Pancreas Performance in the Bone Marrow Cavity and Intramuscular Space

BACKGROUND AND AIMS Bone marrow with a widely distributed and well-vascularized microenvironment that is capable of sustaining grafts is a potential site for islet transplantation. The femur bone marrow cavity offers sufficient space that may also receive the implantation of bioartificial pancreas (BAP). METHODS Mouse insulinoma cells encapsulating in agarose gel were further enclosed in a calcium phosphate cement chamber to create a BAP. BAPs implanted into the femur bone marrow cavity of diabetics were compared with those implanted in the intramuscular space. Blood glucose level and C-peptide were determined perioperatively. RESULTS The blood glucose level of the diabetics receiving BAPs in the intramuscular space decreased from 413 +/- 24 to 285 +/- 47 mg/dL at 1 day post-surgery. However, the blood glucose level returned to 398 +/- 35 mg/dL with undetectable serum C-peptide at 2 weeks postoperatively that reveals implant failure. The blood glucose level of diabetics receiving BAPs into the femur bone marrow cavity decreased from 422 +/- 32 to 247 +/- 52 mg/dL and maintained in the range of 288 +/- 47 mg/dL during the experimental period with an increase in C-peptide level from 6.1 +/- 2.8 to 104.7 +/- 16.4 pmol/L. CONCLUSIONS This preliminary study indicates that the effectiveness of BAPs transplanted into the femur bone marrow cavity is superior to that implanted in the intramuscular space, which reveals the bone marrow may be a potential receptor site for the BAP transplantation.

Investigating the suspension culture on aggregation and function of mouse pancreatic β-cells†

The integrity and hierarchical structure of islet influence β-cells physiology dramatically. A culture substrate which can maintain or improve β-cells aggregation shall benefit cell therapy for diabetics. In this study, nontreated, type IV collagen, Lipidure, and ultralow attachment dishes were used to culture a murine β-cell line, MIN-6. The formation and biological performances of pseudoislets were investigated. Results showed that β-cells formed loose and irregular aggregates on nontreated dishes. Oppositely, pseudoislets formed on other three substrates. Most pseudoislets on Lipidure and type IV collagen dishes had a diameter between 100-150 μm with high survival rate, while large pseudoislets (>250 μm) with seriously central necrosis were found on ultralow attachment dishes. Western blot analysis revealed that pseudoislets had relatively higher connexin 36 protein productions relative to single cells. The glucose-stimulated insulin secretion test showed pseudoislets on type IV collagen have high stimulation index. Monolayers from TCPS dishes and pseudoislets from type IV collagen or Lipidure dishes were further transplanted into diabetic mice. Animals received both single cells and pseudoislets had decreasing blood glucose level and regained body weight. Histologic examination revealed that all implants successfully engrafted with positive insulin staining. Interestingly, the area under curve for the intraperitoneal glucose tolerance test showed pseudoislets had superior glucose disappearance rate. This study reveals that isolated islets or insulin-producing cells can be cultured on type IV collagen or Lipidure dishes to improve/maintain integrity prior to transplantation.