Han-Tsung Liao

Preparation and characterization of gelatin/hyaluronic acid cryogels for adipose tissue engineering: in vitro and in vivo studies.

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 μm), high porosity (>90%) and a high degree of cross-linking (>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 PPARγ) 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 PPARγ, 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.

Incorporation of biphasic calcium phosphate microparticles in injectable thermoresponsive hydrogel modulates bone cell proliferation and differentiation.

To provide osteoblast cells with a three-dimensional environment closer to bone matrix, an engineered construct mimicking bone components have been designed and evaluated. A biocompatible injectable thermo-responsive hydrogel, hyaluronic acid-g-chitosan-g-poly (N-isopropylacrylamide) (HA-CPN), was used as a biomimetic organic gel matrix while embedded biphasic calcium phosphate (BCP) ceramic micoparticles were used to replace mineralized matrix. The physicochemical properties of HA-CPN/BCP hydrogel composite were characterized by X-ray diffraction, thermogravimeric analysis, differential scanning calorimetric, and Fourier transform infrared spectroscopy. Human fetal osteoblast cells were cultured in vitro in HA-CPN and HA-CPN/BCP scaffold to study cell viability, proliferation, gene expression, and maintenance of their osteoblastic differential potentials. Overall, HA-CPN/BCP can serve as a better injectable carrier for bone cells than HA-CPN with enhanced proliferation rate and alkaline phosphatase activity from biochemical assays, improved calcium deposition and mineralization of extracellular matrix by histological examinations, and elevated gene expression of all essential osteoblastic genes from quantitative real-time PCR, to provide a cell/scaffold construct with higher mechanical strength and elasticity. From in vivo nude mice subcutaneous implantation experiments, the injected cell mass can form ectopic bone tissue in HA-CPN/BCP hydrogel composite.

Osteogenesis of adipose-derived stem cells on polycaprolactone-β-tricalcium phosphate scaffold fabricated via selective laser sintering and surface coating with collagen type I.

The current study aimed to fabricate three‐dimensional (3D) polycaprolactone (PCL), polycaprolactone and β‐tricalcium phosphate (PCL–TCP) scaffolds via a selective laser‐sintering technique (SLS). Collagen type I was further coated onto PCL–TCP scaffolds to form PCL–TCP–COL scaffolds. The physical characters of these three scaffolds were analysed. The osteogenic potential of porcine adipose‐derived stem cells (pASCs) was compared among these three scaffolds in order to find an optimal scaffold for bone tissue engineering. The experimental results showed no significant differences in pore size and porosity among the three scaffolds; the porosity was ca. 75–77% and the pore size was ca. 300–500 µm in all three. The compressive modulus was increased from 6.77 ± 0.19 to 13.66 ± 0.19 MPa by adding 30% β‐TCP into a 70% PCL scaffold. No significant increase of mechanical strength was found by surface‐coating with collagen type I. Hydrophilicity and swelling ratios showed statistical elevation (p < 0.05) after collagen type I was coated onto the PCL–TCP scaffolds. The in vitro study demonstrated that pASCs had the best osteogenic differentiation on PCL–TCP–COL group scaffolds, due to the highest ALP activity, osteocalcin mRNA expression and mineralization. A nude mice experiment showed better woven bone and vascular tissue formation in the PCL–TCP–COL group than in the PCL group. In conclusion, the study demonstrated the ability to fabricate 3D, porous PCL–TCP composite scaffolds (PCL:TCP = 70:30 by weight) via an in‐house‐built SLS technique. In addition, the osteogenic ability of pASCs was found to be enhanced by coating COL onto the PCL–TCP scaffolds, both in vitro and in vivo. Copyright

Osteogenic Differentiation and Ectopic Bone Formation of Canine Bone Marrow-Derived Mesenchymal Stem Cells in Injectable Thermo-Responsive Polymer Hydrogel

This study describes an injectable, thermo-responsive hyaluronic acid-g-chitosan-g-poly(N-isopropylacrylamide) (HA-CPN) copolymer for bone tissue engineering. The wettability, temperature-dependent change of water content, and volume of HA-CPN hydrogel were measured, together with its biocompatibility in vitro and in vivo. The dried hydrogel morphology shows a three-dimensional, porous structure with interconnected pores. Canine bone marrow-derived mesenchymal stem cells (cBMSCs) were encapsulated in HA-CPN hydrogel and osteoinduction was assessed by comparing samples with different osteogenic differentiation induction times but with the same total cell culture time. Cell proliferation and time-dependent osteogenic differentiation, evident from secretion of extracellular matrix and formation of mineral deposits, were observed. The cells showed better proliferation in HA-CPN hydrogel than on tissue culture polystyrene after osteo-induced for 21 days and higher alkaline phosphatase activity regardless of osteo-induction times. Mineralization extent of cBMSCs in HA-CPN followed by Alizarin red stains showed positive stained nodules after osteo-induced longer than 7 days. The cells/hydrogel construct also showed increased mechanical strength and elasticity after osteogenic differentiation, and the increase could be correlated with osteo-induction time. In vivo studies confirmed the biocompatibility and bioresorption of the HA-CPN hydrogel and ectopic bone formation when the hydrogel was used as a cell carrier for osteo-induced cBMSCs and implanted in nude mice subcutaneously. Taken together, the results indicate the feasibility and efficacy of HA-CPN hydrogel as an injectable bone tissue engineering scaffold with cBMSCs.

Combination of guided osteogenesis with autologous platelet-rich fibrin glue and mesenchymal stem cell for mandibular reconstruction.

BACKGROUND This study examined whether a combination of autologous platelet-rich fibrin glue (PRFG) with mesenchymal stem cells (MSCs) and MEDPOR as guided tissue regeneration (GTR) could act as an osteogenic substitute and whether this treatment yields faster new bone formation than MEDPOR alone or PRFG plus MSC. MATERIAL MSCs were harvested and isolated from the bone marrow of dog ilium. Full-thickness bony defects (1.5×1.5 cm) were created in the bilateral mandible angles of the dog. Treatments for bone defect in each group were as follows: group I (n=4), MEDPOR sheet as GTR and autologous PRFG/MSCs admixtures; group II (n=4), autologous PRFG/MSCs admixtures; group III (n=4), MEDPOR sheet as GTR; and group IV (n=4), control (empty defect). The percentage of new bone regeneration in computerized tomography at 2 months and 4 months was calculated by Analyze version 7.0 software. The mandibles were harvested from all specimens at 4 months, and the grafted sites were evaluated by gross, histologic, and X-ray examination. RESULTS By radiographic analysis at 16 weeks posttransplantation, it was shown that an average of 72.8%±8.02% new bone formation in group I, 53.34%±6.87% in group II, 26.58%±6.41% in group III, and 15.14%±2.37% in group IV. Histologic examination revealed that the defect was repaired by typical bone tissue in groups I and II, whereas only minimal bone formation with fibrous connection was observed in the groups III and IV group. Besides, muscle incarceration was found in groups II and IV without MEDPOR as GTR. CONCLUSION Autologous PRFG plus osteoinduced MSCs have good potential for bone regeneration. In combination with MEDPOR as GTR, bone regeneration is enhanced by preventing soft tissue ingrowth hindering bone regeneration.

Fabrication of tissue engineered PCL scaffold by selective laser-sintered machine for osteogeneisis of adipose-derived stem cells

Polycaprolactone (PCL) is a bioresorbable polymer with potential applications for bone and cartilage repair. In this work, three-dimensional (3D) and porous PCL scaffolds were designed and fabricated via selective laser sintering (SLS). The aim of this study was to evaluate the osteogenic potential of porcine adipose-derived stem cells (pASCs) in a laser-sintered PCL (lsPCL) scaffold. The character of the lsPCL scaffold was evaluated. The pore size and the microstructure were observed by SEM. The pASCs were harvested and isolated from pig inguinal area. Then, the lsPCL scaffold was seeded with ASCs and cultured in osteogenic medium for 0 and 14 days. Cell proliferation was measured by MTS. Alkaline phosphatase activity (ALP) was detected using biochemical methods. SEM was used to observe the interaction between scaffold and cell. An energy dispersive spectrum (EDS) was used to analyze the mineralization in each group. Porosity was around 83%; pore size was around 300–400 µm. Both MTS and ALP showed significant increase after subcultivation in osteogenic medium for 14 days. SEM detailed that the pASCs cell can attach well to the lsPCL scaffold. The energy dispersive spectrum (EDS) also demonstrated calcium deposits around pASCs after osteo-induction for 14 days. In contrast, no mineralization was found around ASCs after osteo-induction of 0 days. In conclusion, the laser-sintered PCL is a suitable scaffold for the proliferation of ASCs. The ASCs were also well differentiated into osteoblasts in the 3D, porous, laser-sintered PCL scaffold.

Investigation of synergistic effects of inductive and conductive factors in gelatin-based cryogels for bone tissue engineering

Macroporous and biocompatible scaffolds for bone tissue engineering were prepared from 4% gelatin (G) and 4% gelatin/2% nanohydroxyapatite (nHAP), (GN), by cryogelation. The cryogels have interconnected pores with pore size around 100 μm and a high degree of cross-linking. The incorporation of nHAP slightly reduced the porosity, degree of crosslinking, swelling kinetics and equilibrium water uptake, but enhanced the toughness of the cryogel scaffolds. The osteo-regeneration potential of GN cryogels was further enhanced by binding with bone morphogenetic protein (BMP-2) to produce the gelatin/nHAP/BMP-2 (GNB) scaffold. The efficacy of BMP-2 incorporation was tested through in vitro release studies and a sustained release profile could be observed from the cumulative BMP-2 release curve. To elucidate the effect of cryogel composition on cell proliferation and differentiation, rabbit adipose-derived stem cells (ADSCs) were seeded in cryogel scaffolds. In vitro studies demonstrated a reduced proliferation rate and enhanced osteogenic differentiation of ADSCs in GNB cryogel scaffolds from the combined effect of nHAP and BMP-2, judging from the elevated alkaline phosphatase activity and the degree of mineralization. Confocal microscopy confirmed high viability and good cytoskeletal spreading of ADSCs on cryogels while osteocalcin (OCN) protein quantification affirmed the dominance of GNB in the osteogenic differentiation of ADSCs compared to G and GN cryogels. The maximum osteogenesis capability of GNB was also confirmed through the up-regulation of specific bone maker genes of early marker protein collagen I (COL I) and late marker protein osteopontin (OPN). From an in vivo animal model, computed tomography analysis confirmed the superior bone regeneration capability of ADSCs in GNB cryogels by implanting ADSCs/GNB cryogel constructs in rabbit calvarial critical size defects. Histological and immunohistochemical analysis demonstrated new bone formation and continued expression of COL I and OCN bone-specific proteins at the defect site. Taken together, the results demonstrate that G cryogels modified with osteo-conductive nHAP and osteo-inductive BMP-2 could provide cues to synergistically promote the osteogenesis of ADSCs in vitro and in vivo.

Bone Tissue Engineering with Adipose-Derived Stem Cells in Bioactive Composites of Laser-Sintered Porous Polycaprolactone Scaffolds and Platelet-Rich Plasma

Three-dimensional porous polycaprolactone (PCL) scaffolds with consistent inter-pore channels, 83% porosity and 300–400 μm pore size were fabricated via selective laser sintering. The PCL scaffold was combined with platelet-rich plasma (PRP) to form a bioactive composite and studied for potential application in bone tissue engineering using porcine adipose-derived stem cells (PASCs). The PCL/PRP/PASCs construct showed enhanced cell seeding efficiency and synergistically increased the differentiation capability of PASCs in osteogenic medium toward the osteoblast lineage, judging from elevated alkaline phosphatase activity and up-regulated osteogenic genes expression. For in vivo study, a 3 cm × 3 cm mandible defect was created in pigs and reconstructed by implanting acellular PCL scaffolds or PCL/PRP/PASCs constructs. Both groups showed new bone formation, however, the new bone volume was 5.1 times higher for PCL/PRP/PASCs 6 months post-operation. The bone density was less and loose in the acellular PCL group and the Young’s modulus was only 29% of normal bone. In contrast, continued and compact bone formation was found in PCL/PRP/PASCs and the Young’s modulus was 81% that of normal bone. Masson’s trichrome stain, immunohistochemical analysis of osteocalcin and collagen type I also confirmed new bone formation.

Microsphere-Based Hierarchically Juxtapositioned Biphasic Scaffolds Prepared from Poly(Lactic-co-Glycolic Acid) and Nanohydroxyapatite for Osteochondral Tissue Engineering

This study aims to prepare biphasic osteochondral scaffolds based on seamless joining of sintered polymer and polymer/ceramic microspheres for co-culture of chondrocytes and bone marrow stem cells (BMSCs). Poly(lactide-co-glycolide) (PLGA) microspheres and 10% nanohydroxyapatite (nHAP)-incorporated PLGA (PGA/nHAP) microspheres were prepared through the oil-in-water precipitation method. Virgin (V) and composite (C) scaffolds were prepared from 250–500 µm PLGA and PLGA/nHAP microspheres, respectively, while osteochondral (OC) scaffolds were fabricated through the combination of V and C scaffolds. Physico-chemical properties of scaffolds were characterized through microscopic-spectroscopic evaluations. The effect of nHAP in scaffolds was investigated through thermogravimetric analysis and mechanical testing, while surface hydrophobicity was tested through contact angle measurements. Rabbit chondrocytes and BMSCs were used for cell culture, and cell morphology and proliferation were determined from SEM and DNA assays. Alizarin red and Alcian blue stains were used to identify the in vitro bone and cartilage tissue-specific regeneration, while cetylpyridinium chloride was used to quantitatively estimate calcium in mineralized bone. For co-culture in OC scaffolds, BMSCs were first seeded in the bone part of the scaffold and cultured in osteogenic medium, followed by seeding chondrocytes in the cartilage part, and cultured in chondrocyte medium. High cell viability was confirmed from the Live/Dead assays. Actin cytoskeleton organization obtained by DAPI-phalloidin staining revealed proper organization of chondrocytes and BMSCs in OC scaffolds. Immunofluorescent staining of bone (type I collagen and osteocalcin (OCN)) and cartilage marker proteins (type II collagen (COL II)) confirmed cellular behavior of osteoblasts and chondrocytes in vitro. Using an ectopic osteochondral defect model by subcutaneous implantation of co-cultured OC scaffolds in nude mice confirmed cell proliferation and tissue development from gross view and SEM observation. IF staining of OCN and COL II in the bone and cartilage parts of OC scaffolds and tissue-specific histological analysis exhibited a time-dependent tissue re-modeling and confirmed the potential application of the biphasic scaffold in osteochondral tissue engineering.

CULTIVATION OF CHONDROCYTES AND MENISCUS CELLS IN THERMO-RESPONSIVE HYDROGELS CONTAINING CHITOSAN AND HYALURONIC ACID UNDER MECHANICAL TENSILE STIMULATION

Temperature-responsive hydrogel scaffold containing chitosan and hyaluronic acid was used to entrap primary chondrocytes and meniscus cells. The effect of dynamic tensile strain on the cells/hydrogel constructs was evaluated by measuring cell proliferation, biosynthetic activity, and viability. The results demonstrated that mechanical deformation applied at 15% tensile strain, 0.5 Hz, and 10 min per day for 43 days resulted in substantial increases in glycosaminoglycan (36% for chondrocytes and 31% for meniscus cells) and collagen productions (37% for chondrocytes and 52% for meniscus cells) over static controls while not significantly affecting cell proliferation and viability.