Development of Cryogels as Adipose-Derived Stem Cells Carriers for Adipose and Bone Tissue Engineering Applications

• Main Authors: Kun Hung Chang, 張琨鴻
• Other Authors: J. P. Chen
• Format: Others
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/82116431064310036368

博士 === 長庚大學 === 化工與材料工程學系 === 101 === Adipose tissue engineering. Macroporous elastic scaffolds containing gelatin (4% or 10%) and 0.25% hyaluronic acid (HA) were fabricated by cryogelation for application in adipose tissue engineering. These cryogels have interconnected pores (~ 200 m), high porosity (> 90%) and a high degree of crosslinking (> 99%). The higher gelatin concentration reduced the pore size, porosity, and swelling ratio of the cryogel but improved its swelling kinetics. Compressive mechanical testing of cryogel samples demonstrated non–linear stress–strain behavior and hysteresis loops during loading–unloading cycles, but total recovery from large strains. The presence of more gelatin increased the elastic modulus, toughness and storage modulus and yielded a cryogel that was highly elastic, with a loss tangent equal to 0.03. Porcine adipose–derived stem cells (ADSCs) were seeded in the cryogel scaffolds to assess their proliferation and differentiation. In vitro studies demonstrated a good proliferation rate and the adipogenic differentiation of the ADSCs in the cryogel scaffolds, as shown by their morphological change from a fibroblast–like shape to a spherical shape, decreased actin cytoskeleton content, growth arrest, secretion of the adipogenesis marker protein leptin, Oil Red O staining for triglycerides and expression of early (LPL and PPAR) and late (aP2 and leptin) adipogenic marker genes. In vivo studies of ADSCs/cryogel constructs implanted in nude mice and pigs demonstrated adipose tissue and new capillary formation, the expression of PPARleptin and CD31 in immunostained explants, and the continued expression of adipocyte-specific genes. Both the in vitro and in vivo studies indicated that the gelatin/HA cryogel provided a structural and chemical environment that enabled cell attachment and proliferation and supported the biological functions and adipogenesis of the ADSCs. Bone tissue engineering. Macroporous elastic scaffolds containing gelatin (4%), chitosan (2%), nano-hydroxyapatite (nHAP) (2%), and surface immobilized with bone morphogenetic protein 2 (BMP-2) were fabricated by cryogelation for application in bone tissue engineering. The cryogels are with interconnected pores (~100 m), high porosity (>80%) and degree of crosslinking (>95%) with nHAP dispersed throughout the frame structure. nHAP could substantially improve the mechanical properties of the cryogel. In vitro studies show that rabbits ADSCs show good proliferation rate and the osteogenic differentiation in the cryogel scaffolds. Up-regulation of osteogenic genes (collagen type I, osteopontin and chordin) expression and bone marker protein (osteocalcin and alkaline phosphatase) secretion were observed in the presence of BMP-2 due to its osteoinduction ability. In vivo animal experiments were carried out with osteo-induced ADSCs/cryogels constructs implanted in nude mice subcutaneously and in critical size rabbit calvarial defects. Masson trichrome stain, immunohistochemical stainings of bone marker proteins (osteocalcin and collagen type Ι), and computed tomography (CT) analysis indicate ectopic new bone formation and show robust osseous regeneration. Both the in vitro and in vivo studies indicated that the gelatin/chitosan/n-HAP/BMP-2 cryogel provided a structural and chemical environment that enabled cell attachment and proliferation and supported the biological functions and osteogenesis of the ADSCs for applications in bone tissue engineering.