Osteogenic differentiation of bone marrow stromal cells on poly(ε-caprolactone) nanofiber scaffolds

Nanofiber poly(ε-caprolactone) (PCL) scaffolds were fabricated by electrospinning, and their ability to enhance the osteoblastic behavior of marrow stromal cells (MSCs) in osteogenic media was investigated. MSCs were isolated from Wistar rats and cultured on nanofiber scaffolds to assess short-term cytocompatibility and long-term phenotypic behavior. Smooth PCL substrates were used as control surfaces. The short-term cytocompatibility results indicated that nanofiber scaffolds supported greater cell adhesion and viability compared with control surfaces. In osteogenic conditions, MSCs cultured on nanofiber scaffolds also displayed increased levels of alkaline phosphatase activity for 3 weeks of culture. Calcium phosphate mineralization was substantially accelerated on nanofiber scaffolds compared to control surfaces as indicated through von Kossa and calcium staining, scanning electron microscopy and energy-dispersive X-ray spectroscopy. Increased levels of intra- and extracellular levels of osteocalcin and osteopontin were observed on nanofiber scaffolds using immunofluorescence techniques after 3 weeks of culture. These results demonstrate the enhanced tissue regeneration property of nanofiber scaffolds, which may be of potential use for engineering osteogenic scaffolds for orthopedic applications.     

β(1)-Adrenoceptor stimulation enhances the differentiation of mouse induced pluripotent stem cells into neural progenitor cells

The cyclic AMP/protein kinase A signaling pathway is thought to be involved in neural differentiation of mesenchymal stem cells. In the present study, we examined the involvement of β-adrenoceptor signaling on the differentiation of mouse induced pluripotent stem (iPS) cells into neural progenitor cells. Mouse iPS cells were cultured on ultra-low-attachment dishes to induce embryoid body (EB) formation. All-trans retinoic acid (ATRA, 1μM) and/or the β-adrenoceptor agonist l-isoproterenol (0.3 or 1μM) were added to the EB cultures for 4 days, then EBs were plated on gelatin-coated plates and cultured for 7 or 14 days. Subtype-specific antibody staining revealed that mouse iPS cells express β(1)-adrenoceptors predominantly. Although treatment with l-isoproterenol alone did not affect the expression of Nestin (a specific marker for neural progenitor cells), l-isoproterenol significantly enhanced ATRA-induced Nestin expression. Pretreatment of EBs with either atenolol (a selective β(1)-adrenoceptor antagonist) or H89 (a protein kinase A inhibitor) significantly inhibited the l-isoproterenol-enhancement of ATRA-induced Nestin expression. In addition, the l-isoproterenol treatment significantly enhanced ATRA-induced expression of NeuN (a neuron-specific nuclear protein). These findings suggest that β(1)-adrenoceptor stimulation enhances ATRA-induced neural differentiation of mouse iPS cells.     

Preparation of anionic poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) copolymeric nanoparticles as basic protein antigen carrier

It is an useful method for polymeric nanoparticles to load protein by electrostatic method to improve the immunogenicity of protein antigen. In this article, anionic poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCEC) nanoparticles were prepared by modified emulsion solvent evaporation method, and human basic fibroblast growth factor (bFGF), as a model protein, was absorbed onto its surface due to electrostatic interaction. The prepared anionic PCEC nanoparticles, with mean diameter of 136.9 nm, had zeta potential of ? 33.14 mV. The surface charge and particle size of bFGF/nanoparticles complex increased with increase of bFGF/nanoparticles mass ratio. The encapsulated bFGF could be released slowly from bFGF/nanoparticle complexes. The animal experiment indicated that the humoral immunity induced by bFGF/PCEC complex was improved greatly than that created by naked bFGF. Otherwise, the cytotoxicity of anionic PCEC nanoparticles was also evaluated by 293 cell viability. The prepared anionic PCEC nanoparticles might have great potential application as basic protein vaccine delivery system.     

Superparamagnetic iron oxide labeling influences in vitro differentiation of induced pluripotent stem cells北大核心

BACKGROUND: Superparamagnetic iron oxide (SPIO) labeling technology is a classic noninvasive tracing method, which has been widely used in the stem cell transplantation. Induced pluripotent stem cells (iPSCs) are currently one of the most promising seed cells for cell transplantation. Whether SPIO labeling can also be used to noninvasively trace induced pluripotent stem cells is rarely reported, and concern has been raised about whether SPIO markedly impacts the differentiation of iPSCs. OBJECTIVE: To investigate the effects of SPIO labeling on the differentiation of iPSCs in vitro. METHODS: Rat fibroblasts were isolated and cultured. Efficient recombinant vector and plasmids that were packaged by virus and contained target genes (Oct4, Sox2, Klf4 and c-Myc) were transfected into 293T cells for virus packaging and production. The packaging lentiviral vectors that contained target genes infected rat fibroblasts to obtain iPSCs. SPIO-labeled (experimental) or unlabeled (control) iPSCs were subjected to neural induction and differentiation. Prussian blue staining and transmission electron microscope observation were performed for SPIO-labeled iPSCs. Immunohistochemical method was used to detect neuron-specific enolase expression after induced differentiation. Flow cytometry was used to detect the proportion of neurons and glial cells differentiated from iPSCs. RESULTS AND CONCLUSION: There were dense iron particles in the cytoplasm of SPIO-labeled iPSCs shown by Prussian staining and under transmission electron microscope. Differentiated iPSCs were positive for neuron-specific enolase. In addition, the proportion of neurons and glial cells showed no difference between the experimental and control groups. To conclude, SPIO labeling has no obvious effect on the capacity of iPSCs differentiating into neurons. Reasonable application of this new cell labeling technique will promote the development of seed cells in regenerative medicine. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

In vivo biocompatibility and osteogenesis of electrospun poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone)/nano-hydroxyapatite composite scaffold

A flexible and fibrous composite scaffold composed of poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL, PCEC) and 30?wt.% nano-hydroxyapatite (n-HA) was fabricated through electrospinning. In the present study, we investigated its in?vitro and in?vivo performance by means of hydrolytic degradation, muscle pouch implantation, as well as repair the calvarial defects in New Zealand white rabbits. The results demonstrated that the degradable scaffold held good biocompatibility. Qualitative analysis of bone regeneration process was performed by radiological examination and histological analysis. The results indicated that new bone formed originally from the margin of host bone, and then grew toward the center of defects. Moreover, the quantitative determination of newly formed bone was performed using statistical analysis of histological sections at predetermined time points. At 20th week, the defects of treatment group were covered with the new solid cortical bone. In comparison, the control group was filled with a large amount of cancelous bone and bone marrow. It suggested that the composite scaffold had better activity of guided bone regeneration than that of self-healing. So the electrospun PCEC/n-HA fibrous scaffold had the great potential application in bone tissue engineering.     

Thermosensitive β-cyclodextrin modified poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) micelles prolong the anti-inflammatory effect of indomethacin following local injection

A novel biodegradable and injectable in situ gel-forming controlled drug delivery system based on thermosensitive β-cyclodextrin-modified poly(ε-caprolactone)–poly(ethylene glycol)–poly(ε-caprolactone) co-polymer (PCEC–β-CD) was studied in this work. The drug encapsulating capacity has been improved by introducing β-CD bound to the PCEC co-polymer. The prepared PCEC–β-CD co-polymers self-assembled in water to form micelles, and underwent a temperature-dependent gel–sol transition, which was in the form of a flowing injectable solution at low temperatures but became a non-flowing gel at around physiological body temperature. Furthermore, a small hydrophobic drug molecule indomethacin (IND) was successfully encapsulated in PCEC–β-CD micelles by dialysis at a high encapsulation efficiency and drug loading capacity. The IND-loaded micelles (IND-M) exhibited controlled release in vitro. Additionally, a pharmacodynamic study in vivo based on both the carrageenan-induced acute and complete Freund’s adjuvant-induced adjuvant arthritis models indicated that sustained therapeutic efficacy could be achieved through subcutaneous injection of IND-loaded micelles. A significant improvement in the anti-inflammatory effect of IND in rats occurred on encapsulation in PCEC–β-CD micelles.     

Electrospun tubular scaffolds: On the effectiveness of blending poly(ε-caprolactone) with poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Tissue engineering can effectively contribute to the development of novel vascular prostheses aimed to overcome the well-known drawbacks of small-diameter grafts. To date, poly(ε-caprolactone) (PCL), a bioresorbable synthetic poly(α-hydroxyester), is considered one of the most promising materials for vascular tissue engineering. In this work, the potential advantage of intimate blending soft PCL and hard poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a polymer of microbial origin, has been evaluated. Nonwoven mats and small-diameter tubular scaffolds of PCL, PHBV, and PCL/PHBV were fabricated by means of electrospinning technique. Mechanical properties and suture retention strength were investigated according to the international standard for cardiovascular implants. Biological tests demonstrated that both PCL-based scaffolds supported survival and growth of rat cerebral endothelial cells in a short time. The fiber alignment of the electrospun tubular scaffolds contributed to a more rapid and homogeneous cell colonization of the luminal surface. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.     

Effect of topology of poly(L-lactide-co-ε-caprolactone) scaffolds on the response of cultured human umbilical cord Wharton’s jelly-derived mesenchymal stem cells and neuroblastoma cell lines

In this study, for the first time, a biodegradable poly(L-lactide-co-ε-caprolactone), PLC 67:33 copolymer was developed for use as temporary scaffolds in reconstructive nerve surgery. The effect of the surface topology and pore architecture were studied on the biocompatibility for supporting the growth of human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) and human neuroblastoma cells (hNBCs) as cell models. Porous PLC membranes were prepared by electrospinning and phase immersion precipitation with particulate leaching and nonporous PLC membranes were prepared by solvent casting. From the results, the porous PLC membranes can support hWJ-MSCs and hNBCs cells better than the nonporous PLC membrane, and the interconnected pore scaffold prepared by electrospinning exhibited a more significant supporting attachment of the cells than the open pore and nonporous membranes. We can consider that these electrospun PLC membranes with 3-D interconnecting fiber networks and a high porosity warrant a potential use as nerve guides in reconstructive nerve surgery.     

Electrospun gelatin/poly(L-lactide-co-ε-caprolactone) nanofibers for mechanically functional tissue-engineering scaffolds

Recently, much attention has been given to the fabrication of tissue-engineering scaffolds with nano-scaled structure to stimulate cell adhesion and proliferation in a microenvironment similar to the natural extracellular matrix milieu. In the present study, blends of gelatin and poly(L-lactide-co-ε-caprolactone) (PLCL) (blending ratio: 0, 30, 70 and 100 wt% gelatin to PLCL) were electrospun to prepare nano-structured non-woven fibers for the development of mechanically functional engineered skin grafts. The resulting nanofibers demonstrated the uniform and smooth fibers with mean diameters ranging from approx. 50 to 500 nm with interconnected pores, regardless of the composition. The contact angle decreased with increasing amount of gelatin in the blend and the water content of the nanofibers increased concurrently. PLCL nanofibers retained significant levels of recovery following application of uniaxial stress; GP-3 with 70% PLCL blend returned to the original length within less than 10% of deformation following 200% of uniaxial elongation. The overall tensile strength was inversely affected by increase in the gelatin content and degradation rates of the nanofibers were accelerated as the gelatin concentration increased. When seeded with human primary dermal fibroblasts and keratinocytes on the nanofibers, both initial cell adhesion and proliferation rate increased as a function of the gelatin content in the blend. Additionally, the total cell number was significantly greater on the nanofiber scaffolds than on polymer-coated glasses, indicating that nanofibrous structure facilitates cell proliferation. Taken together, gelatin/PLCL blend nanofiber scaffolds may serve as a promising artificial extracellular matrix for regeneration of mechanically functional skin tissue.     

Whole-cell SELEX aptamer-functionalised poly(ethyleneglycol)-poly(ε-caprolactone) nanoparticles for enhanced targeted glioblastoma therapy

Though there has been substantial advancement in the knowledge about tumour development and treatment in the past 40 years, the prognosis of brain glioblastoma is still very grim due to the difficulty of targeting drugs to glioblastoma cells. An active targeting delivery system helps increase intracellular drug delivery, which is promising for the treatment of glioblastoma. For an active targeting delivery system, targeting ligands are crucial for efficient intracellular drug delivery. Current methods include systematic evolution of ligands by exponential enrichment (SELEX), which has been utilised for selecting specific ligands with better targeting effects. The GMT8 aptamer was a short DNA sequence selected by SELEX that could specifically bind with U87 cells. In this study, nanoparticles functionalised with GMT8 aptamers (ApNP) were utilised for glioblastoma therapy. In vitro cell uptake and U87 tumour spheroid uptake demonstrated that nanoparticles functionalised with GMT8 aptamer significantly enhanced intracellular drug delivery and tumour spheroid penetration. Assays for cell apoptosis and growth inhibition of tumour spheroids identified docetaxel-loaded ApNP to significantly induce cell apoptosis and inhibit tumour spheroid growth. In vivo imaging of glioblastoma-bearing mice demonstrated that ApNP could target glioblastoma and accumulate at the tumour site, which was further verified by fluorescence imaging of brain slices. Pharmacodynamic results indicated that docetaxel-loaded ApNP significantly prolonged the median survival time of glioblastoma-bearing mice compared to NP, DTX and control. In conclusion, GMT8 aptamer-functionalised nanoparticles enhanced tumour penetration and targeted glioblastoma therapy, which is promising for the prognosis of brain glioblastoma.     

Lamellar stack formation and degradative behaviors of hydrolytically degraded poly(ε-caprolactone) and poly(glycolide-ε-caprolactone) blended fibers

Electrospun fibrous mats have gained popularity in bioengineering over the past decade, but few papers detail their degradative mechanisms. To address this, blends of hydrophobic poly(ε-caprolactone) (PCL) and hydrophilic PGA-PCL-PGA triblock copolymer were electrospun into aligned fibrous mats to assess the copolymers' mechanical and degradative properties. Increased hydrophilic triblock content led to enhanced morphological uniformity of fiber, tightening of fiber diameters, increased storage and Young's modulus, and decreased elongation. The corresponding decrease in hydrophobic PCL content led to faster hydrolytic degradation rate, as reflected by enhanced decrease in mass, molecular weight, and modulus loss at 25, 37, and 45°C. The activation energy for hydrolytic degradation for 15:85 PCL: triblock copolymer was approximately half that of 85:15 PCL: triblock copolymer. Detailed examination of fiber morphology and crystallinity revealed initial surface erosion followed by the evolution of crystalline lamellar stacks and bulk degradation at 37°C. Because of the high surface to volume and short diffusion length scale of the small diameter fibers, surface and bulk degradation may both contribute to the hydrolytic degradative behavior of these electrospun fibrous mats. Electrospun mats' distinct architecture that embodies high specific surface to volume and interfiber porous ultrastructures that lead to their unique degradative behaviors hold much potential for significant impact in the field of tissue engineering and controlled drug delivery.     

Porous alginate/poly(ε-caprolactone) scaffolds: preparation, characterization and in vitro biological activity

In bone tissue engineering, scaffolds with controlled porosity are required to allow cell ingrowth, nutrient diffusion and sufficient formation of vascular networks. The physical properties of synthetic scaffolds are known to be dependent on the biomaterial type and its processing technique. In this study, we demonstrate that the separation phase technique is a useful method to process poly(ε-caprolactone) (PCL) into a desired shape and size. Moreover, using poly(ethylene glycol), sucrose, fructose and Ca2+ alginate as porogen agents, we obtained PCL scaffolds with three-dimensional porous structures characterized by different pore size and geometry. Scanning electron microscopy and porosity analysis indicated that PCL scaffolds prepared with Ca2+ alginate threads resemble the porosity and the homogeneous pore size distribution of native bone. In parallel, MicroCT analysis confirmed the presence of interconnected void spaces suitable to guarantee a biological environment for cellular growth, as demonstrated by a biocompatibility test with MC3T3-E1 murine preosteoblastic cells. In particular, scaffolds prepared with Ca2+ alginate threads increased adhesion and proliferation of MC3T3-E1 cells under basal culture conditions, and upon stimulation with a specific differentiation culture medium they enhanced the early and later differentiated cell functions, including alkaline phosphatase activity and mineralized extracellular matrix production. These results suggest that PCL scaffolds, obtained by separation phase technique and prepared with alginate threads, could be considered as candidates for bone tissue engineering applications, possessing the required physical and biological properties.     

Phase transfer and characterization of poly(ε-caprolactone) and poly(L-lactide) microspheres

A method suitable for transfer of poly(ε-caprolactone) and poly(L-lactide) microspheres (synthesized by pseudoanionic dispersion polymerization of ε-caprolactone and L-lactide in heptane1,4-dioxane mixed solvent) from heptane to water was developed. This method consists of treating the microspheres with KOH-ethanol in the presence of surfactants (nonionic Triton X-405, anionic sodium dodecyl sulfate (SDS), and zwitterionic ammonium sulfobetaine-2 (ASB)). Partial hydrolysis of polyesters results in the formation of hydroxyl and carboxyl groups in the surface layer of microspheres and enhances their stability in water-based media. Minimal concentrations of surfactants, needed to obtain stable suspensions of particles, were equal to 3 × 10^-2, and 6 × 10^-2, and 3 × 10^-2 mol l^-1 for Triton X-405, SDS, and ASB, respectively. In the case of poly(ε-caprolactone) microspheres, suspensions in water were stable for all three surfactants for pH values ranging from 3 to 11. Suspensions of poly(L-lactide) were stable in the same range of pH values only for ASB. Surface charge density determined by electrophoretic mobility varied for poly(ε-caprolactone) microspheres from 2.6 × 10^-7 to 8.9 × 10^-7 mol m^-2, for particles stabilized with Triton X-405 and ASB, respectively. In the case of poly(L-lactide) microspheres, surface charge density varied from 3.9 × 10-7 (stabilizer: Triton X-405) to 7.4 × 10^-7 mol m^-2 (stabilizer: ASB). Carboxyl groups located in the surface layer of poly(L-lactide) microspheres were used for covalent immobilization of 6-aminoquinoline, a fluorophore with an amino group. Maximum surface concentration of immobilized 6-aminoquinoline was equal to 1.9 × 10^-6 mol m^-2. Poly(ε-caprolactone) microspheres transferred into water were loaded with ethyl salicylate. Loading up to 38% (w/w) was obtained.     

Small diameter vascular graft with fibroblast cells and electrospun poly (L-lactide-co-ε-caprolactone) scaffolds: Cell Matrix Engineering

Abstract

Electrospun scaffolds have been widely used in tissue engineering due to their similar structure to native extracellular matrices (ECM). However, one of the obstacles limiting the application of electrospun scaffolds for tissue engineering is the nano-sized pores, which inhibit cell infiltration into the scaffolds. To overcome this limitation, we approached to make layers which are consisted of cells onto the electrospun sheet and then tubular structure was constructed by rolling. We called this as ‘Cell Matrix Engineering’ because the electrospun sheets were combined with the cells to form one matrix. They maintained 3-D tubular structures well and their diameters were 4.1 mm (±0.1 mm). We compared the mechanical and biological properties of various vascular grafts with the electrospun PLCL sheets of different thickness. In these experiments, the vascular graft made with thin sheets showed a better cell proliferation and attachment than the grafts made with thick sheets because the thin layer allowed for more efficient mass transfer and better permeability than the thick layer. Culturing under physiological pulsatile flow condition was demonstrated in this work. These dynamic conditions provided the improved mass transport and aerobic cell metabolism. Therefore, the Cell Matrix Engineered vascular graft holds a great promise for clinical applications by overcoming the limitations associated with conventional scaffolds.     

The effect of hydrophilic chain length and iRGD on drug delivery from poly(ε-caprolactone)-poly(N-vinylpyrrolidone) nanoparticles

Poly(ε-caprolactone)-b-Poly(N-vinylpyrrolidone) (PCL-b-PVP) copolymers with different PVP block length were synthesized by xanthate-mediated reverse addition fragment transfer polymerization (RAFT) and the xanthate chain transfer agent on chain end was readily translated to hydroxy or aldehyde for conjugating various functional moieties, such as fluorescent dye, biotin hydrazine and tumor homing peptide iRGD. Thus, PCL-PVP nanoparticles were prepared by these functionalized PCL-b-PVP copolymers. Furthermore, paclitaxel-loaded PCL-PVP nanoparticles with satisfactory drug loading content (15%) and encapsulation efficiency (>90%) were obtained and used in?vitro and in?vivo antitumor examination. It was demonstrated that the length of PVP block had a significant influence on cytotoxicity, anti-BSA adsorption, circulation time, stealth behavior, biodistribution and antitumor activity for the nanoparticles. iRGD on PCL-PVP nanoparticle surface facilitated the nanoparticles to accumulate in tumor site and enhanced their penetration in tumor tissues, both of which improved the efficacy of paclitaxel-loaded nanoparticles in impeding tumor growth and prolonging the life time of H22 tumor-bearing mice.     

Mechanical properties and cytocompatibility of poly(ε-caprolactone)-infiltrated biphasic calcium phosphate scaffolds with bimodal pore distribution

Biphasic calcium phosphate scaffolds have attracted interest because they have good osteoconductivity and a resorption rate close to that of new bone ingrowth, but their brittleness limits their potential applications. In this study, we show how the infiltration of biphasic calcium phosphate scaffolds with poly(ε-caprolactone) improves their mechanical properties. It was found that the polymer effectively contributes to energy to failure enhancement in bending, compressive and tensile tests. The main toughening mechanism in these composites is crack bridging by polymer fibrils. The presence of fibrils at two different size scales – as found in scaffolds with a bimodal pore distribution – results in a more effective toughening effect as compared to scaffolds with a monomodal pore size distribution, especially in the early stage of mechanical deformation. An optimized infiltration process allowed the preservation of micropore interconnection after infiltration, which is beneficial for cells adhesion. In addition, it is shown that biphasic calcium phosphates infiltrated with poly(ε-caprolactone) are cytocompatible with human bone marrow stromal cells, which makes them good candidates for bone substitution.     

Synthesis and characterization of phosphoryl-choline-capped poly(ε-caprolactone)-poly(ethylene oxide) di-block co-polymers and its surface modification on polyurethanes

By sequential ring-opening polymerization of ethylene oxide and ε-caprolactone, poly(ethylene oxide) (PEO)-poly(ε-caprolactone) (PCL) di-block co-polymers with a phosphoryl choline (PC)-terminated group were synthesized. Using FT-IR, NMR, DSC and SEC, the products were characterized and the results proved the successful synthesis of functionalized di-block co-polymer. After blending the products with polyurethane (PU) and casting the result as film, the PEO segments migrated to the surface of the blend and the PCL segments acted as an anchor to fix the co-polymer on PU matrix, while the PEO segments provided PU the hydrophibility to prevent the fibrinogen adsorption on PU. This specific di-block co-polymer and the method of processing are hoped to be applied in the biomedical field to improve the biocompatibility of polymer materials.     

A combined compression molding,heating, and leaching process for fabrication of micro-porous poly(ε-caprolactone) scaffolds

Abstract

Biomaterial scaffolds have been increasingly used for tissue engineering applications as well as three dimensional (3D) cell culture models. Herein, we report a simple procedure combining compression molding, heating, and leaching methods for the fabrication of 3D micro-porous poly(ε-caprolactone) (PCL) biomaterial scaffolds. In this procedure, PCL micro particles are mixed with NaCl of defined sizes and compression molded, followed by heating and subsequent leaching of NaCl particles. This technique eliminates the gas foaming method, which is commonly used in the fabrication of PCL scaffolds. Process and scaffold parameters (i.e., heating time, NaCl concentration, and NaCl particle size) were varied and analyzed to determine their impact on the overall scaffold structural and mechanical properties. An increase in NaCl particle size led to an increase in pore area but did not significantly impact the mechanical properties of the scaffolds. Additionally, NaCl concentration did not show a significant effect on pore area, but considerably impacted the mechanical properties, water absorption capacity and porosity of the scaffolds. Variations in the heating time did not have an effect in the pore area, porosity, water absorption capacity or mechanical properties of the scaffolds. We also demonstrated the ability of these scaffolds to support the proliferation of breast cancer cells. Overall, these results elucidated structure-property relationships in the fabricated micro-porous PCL scaffolds. Further, this procedure could be potentially scaled up for the fabrication of micro-porous PCL scaffolds.     

In situ chondrogenic differentiation of human adipose tissue-derived stem cells in a TGF-β1 loaded fibrin–poly(lactide-caprolactone) nanoparticulate complex

When conducting cartilage tissue engineering with stem cells, it is well known that chemical and physical stimulations are very important for the induction and maintenance of chondrogenesis. In this study, we induced chondrogenic differentiation of human adipose tissue-derived stem cells (hASCs) in situ by effective stimulation via the continuous controlled release of TGF-β1 from a heparin-functionalized nanoparticle–fibrin–poly(lactide-co-caprolactone) (PLCL) complex. PLCL scaffolds were fabricated with 85% porosity and 300–500 μm pore size by a gel-pressing method. Heparin-functionalized nanoparticles were prepared by a solvent-diffusion method, composed of poly(lactide-co-glycolide) (PLGA), Pluronic F-127, and heparin, and then TGF-β1 was loaded to the nanoparticles. A mixture of hASCs, fibrin gels and TGF-β1 loaded nanoparticles was then seeded onto PLCL scaffolds and cultured in vitro, after which they were subcutaneously implanted into nude mice for up to five weeks. The results of in vitro and in vivo studies revealed that chondrogenic differentiation of the hASCs on the complex was induced and sustained by continuous stimulation by TGF-β1 from the heparin-functionalized nanoparticles. In addition, there was no significant difference between the predifferentiation condition prior to incubation in chondrogenic medium and the proliferation condition, which suggests that in situ chondrogenic differentiation of hASCs was induced by the TGF-β1 loaded nanoparticles. Consequently, the hybridization of fibrin and PLCL scaffolds for three-dimensional spatial organization of cells and the effective delivery of TGF-β1 using heparin-functionalized nanoparticles can induce hASCs to differentiate to a chondrogenic lineage and maintain their phenotypes.     

Controlled release of transforming growth factor-β3 from cartilage-extra-cellular-matrix-derived scaffolds to promote chondrogenesis of human-joint-tissue-derived stem cells

The objective of this study was to develop a scaffold derived from cartilaginous extracellular matrix (ECM) that could be used as a growth factor delivery system to promote chondrogenesis of stem cells. Dehydrothermal crosslinked scaffolds were fabricated using a slurry of homogenized porcine articular cartilage, which was then seeded with human infrapatellar-fat-pad-derived stem cells (FPSCs). It was found that these ECM-derived scaffolds promoted superior chondrogenesis of FPSCs when the constructs were additionally stimulated with transforming growth factor (TGF)-β3. Cell-mediated contraction of the scaffold was observed, which could be limited by the additional use of 1-ethyl-3-3dimethyl aminopropyl carbodiimide (EDAC) crosslinking without suppressing cartilage-specific matrix accumulation within the construct. To further validate the utility of the ECM-derived scaffold, we next compared its chondro-permissive properties to a biomimetic collagen–hyaluronic acid (HA) scaffold optimized for cartilage tissue engineering (TE) applications. The cartilage-ECM-derived scaffold supported at least comparable chondrogenesis to the collagen–HA scaffold, underwent less contraction and retained a greater proportion of synthesized sulfated glycosaminoglycans. Having developed a promising scaffold for TE, with superior chondrogenesis observed in the presence of exogenously supplied TGF-β3, the final phase of the study explored whether this scaffold could be used as a TGF-β3 delivery system to promote chondrogenesis of FPSCs. It was found that the majority of TGF-β3 that was loaded onto the scaffold was released in a controlled manner over the first 10 days of culture, with comparable long-term chondrogenesis observed in these TGF-β3-loaded constructs compared to scaffolds where the TGF-β3 was continuously added to the media. The results of this study support the use of cartilage-ECM-derived scaffolds as a growth factor delivery system for use in articular cartilage regeneration.