Fabrication of chitosan/collagen/hydroxyapatite scaffolds with encapsulated Cissus quadrangularis extract

Here, we demonstrated the fabrication of a composite scaffold (chitosan [CS], collagen [Col], and hydroxyapatite [HA]) with the incorporation of encapsulated Cissus quadrangularis (CQ) extract for tissue engineering applications. First, the crude extract of CQ loaded nanoparticles were synthesized via double emulsion technique using polycaprolactone (PCL) and polyvinyl alcohol (PVA) as oil and aqueous phases, respectively. Both PCL (20, 40, and 80 mg/mL) and PVA (0.5%, 1%, and 3% w/v) concentrations were varied to determine the optimum concentrations for CQ‐loaded nanoparticle preparation. The CQ‐loaded PCL nanoparticles (CQ‐PCL NPs), prepared with 20 mg/mL PCL and 0.5% (w/v) PVA, exhibited the smallest size of 334.22 ± 43.21 nm with 95.54 ± 1.49% encapsulation efficiency. Then, the CQ‐PCL NPs were incorporated into the CS/Col/HA scaffolds. These scaffolds were also studied for their ultrastructure, pore sizes, chemical composition, compressive modulus, water swelling, weight loss, and biocompatibility. The results showed that the addition of CQ‐PCL NPs into the scaffolds did not dramatically alter the ultrastructure and properties of the scaffolds, compared to CS/Col/HA scaffolds alone. However, incorporation of CQ‐PCL NPs in the scaffolds improved the release profile of CQ by preventing the initial burst release and prolonging the release rate of CQ. In addition, the CQ‐PCL NPs‐loaded CS/Col/HA scaffolds supported the attachment and proliferation of MC3T3‐E1 osteoblast cells.     

Thermoplastic elastomers based on poly(l‐Lysine)‐Poly(ε‐Caprolactone) multi‐block copolymers

Novel thermoplastic elastomers based on multi‐block copolymers of poly(l ‐lysine) (PLL), poly(N‐ε‐carbobenzyloxyl‐l ‐lysine) (PZLL), poly(ε‐caprolactone) (PCL), and poly(ethylene glycol) (PEG) were synthesized by combination of ring‐opening polymerization (ROP) and chain extension via l ‐lysine diisocyanate (LDI). SEC and ¹H NMR were used to characterize the multi‐block copolymers, with number‐average molecular weights between 38,900 and 73,400 g/mol. Multi‐block copolymers were proved to be good thermoplastic elastomers with Young's modulus between 5 and 60 MPa and tensile strain up to 1300%. The PLL‐containing multi‐block copolymers were electrospun into non‐woven mats that exhibited high surface hydrophilicity and wettability. The polypeptide–polyester materials were biocompatible, bio‐based and environment‐friendly for promising wide applications. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 3012–3018     

Development of Electrospun Three‐arm Star Poly(ε‐caprolactone) Meshes for Tissue Engineering Applications

We have developed three‐dimensional electrospun microfibrous meshes of a novel star branched three‐arm poly(ε‐caprolactone) (*PCL) as potential scaffolds for tissue engineering applications. The processing conditions required to obtain uniform fibers were optimized by studying their influence on fiber morphology and size. Polymer molecular weight and solution feed rate influenced both the mesh microstructure and the tensile properties of the developed mats. Electrospun samples were also tested for their mechanical properties in wet conditions, showing higher yield strength and strain in comparison to that observed in dry conditions. Cell culture experiments employing MC3T3‐E1 osteoblast like cells showed good cell viability adhesion and collagen production on the *PCL scaffolds.

     

Electrospun poly(ε‐caprolactone)‐based composites using synthesized β‐tricalcium phosphate

Non‐woven hybrid membranes based on poly(ε‐caprolactone) (PCL) and as‐synthesized β‐tricalcium phosphate (β‐TCP) were obtained by the electrospinning technique. A wide range of composition was investigated, the filler content spanning between 2 and 60 wt%. The synthesis of the β‐TCP powder was accomplished by titration of calcium hydroxide with phosphoric acid followed by calcination of the resulting precipitate at 1100°C. The as‐dried calcium phosphate was characterized by Inductive Coupled Plasma (AES‐ICP), thermal analysis (TG‐DTA), Fourier Transform Infrared Spectroscopy (FT‐IR), Scanning Electron Microscopy (SEM), and high temperature X‐ray diffraction analysis (HT‐XRD). The specific surface area (SSA) was evaluated by N₂ adsorption. Microstructure of PCL/TCP membranes was investigated by SEM, energy dispersion spectroscopy (EDS), XRD analysis, and SSA measurements. The average fiber diameter ranged between 1 and 2 µm, the porosity was 80–90%, and the SSA 16 m²/g. Mechanical properties were determined by uniaxial tensile test. A remarkable enhancement of the tensile modulus was observed for composites containing up to 4 wt% β‐TCP. The ultimate tensile strength ranged between 2 and 3 MPa for samples loaded up to 8 wt%. For most of the samples, the elongation at break was in the range 100–150%. Copyright © 2010 John Wiley & Sons, Ltd.     

Mechanical,thermal and morphological properties of poly(ε‐caprolactone)/zein blends

Blends of poly(ε‐caprolactone) (PCL) with zein (PCL/zein) in different proportions (100/0, 75/25, 50/50, 25/75 and 0/100 wt% containing 5 wt% glycerol) were compared based on their mechanical properties (tensile strength, elongation at break, and Young's modulus), and on their thermal properties, the latter determined by thermogravimetric analysis (TGA) and dynamic mechanical thermal analysis (DMTA). The morphology of the materials was studied by scanning electron microscopy (SEM). Blends of PCL/zein showed reduced tensile strength and elongation at break, but increased Young's modulus compared to the pure polymers, in agreement with the DMTA and SEM results. These findings indicated that PCL and zein were incompatible. TGA showed that the thermal stability was enhanced by the addition of zein to PCL, whereas SEM showed a poor interfacial interaction between the polymers. Copyright © 2004 John Wiley & Sons, Ltd.     

Fabrication of 3D electrospun structures from poly(lactide‐co‐glycolide acid)–nano‐hydroxyapatite composites

The fabrication of three‐dimensional (3D) electrospun composite scaffolds was presented in this study. Layers of electrospun meshes made from composites of poly(lactide‐co‐glycolide acid) (PLGA) and hydroxyapatite (HA) were stacked and sintered using pressurized gas. Three HA concentrations of 5, 10, and 20 wt % were tested, and the addition of the HA nanoparticles decreased the tensile mechanical properties of the meshes with 20 wt % HA. However, after the gas absorption process, the fibers within the mesh sintered, which improved the mechanical properties more than twofold. The fabrication of 3D, porous, electrospun scaffolds was also demonstrated. The resulting 3D scaffolds had open porosity of up to 70% and modulus of ～20 MPa. This technique improves on the current electrospinning technology by overcoming the challenges of depositing a thick, 3D structure. © 2011 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2011     

Incorporation of nanohydroxyapatite and vitamin D3 into electrospun PCL/Gelatin scaffolds: The influence on the physical and chemical properties and cell behavior for bone tissue engineering

Scaffold, an essential element of tissue engineering, should provide proper physical and chemical properties and evolve suitable cell behavior for tissue regeneration. Polycaprolactone/Gelatin (PCL/Gel)‐based nanocomposite scaffolds containing hydroxyapatite nanoparticles (nHA) and vitamin D3 (Vit D3) were fabricated using the electrospinning method. Structural and mechanical properties of the scaffold were determined by scanning electron microscopy (SEM) and tensile measurement. In this study, smooth and bead‐free morphology with a uniform fiber diameter and optimal porosity level with appropriate pore size was observed for PCL/Gel/nHA nanocomposite scaffold. The results indicated that adding nHA to PCL/Gel caused an increase of the mechanical properties of scaffold. In addition, chemical interactions between PCL, gelatin, and nHA molecules were shown with XRD and FT‐IR in the composite scaffolds. MG‐63 cell line has been cultured on the fabricated composite scaffolds; the results of viability and adhesion of cells on the scaffolds have been confirmed using MTT and SEM analysis methods. Here in this study, the culture of the osteoblast cells on the scaffolds showed that the addition of Vit D3 to PCL/Gel/nHA scaffold caused further attachment and proliferation of the cells. Moreover, DAPI staining results showed that the presence and viability of the cells were greater in PCL/Gel/nHA/Vit D3 scaffold than in PCL/Gel/nHA and PCL/Gel scaffolds. The results also approved increasing cell proliferation and alkaline phosphatase (ALP) activity for MG‐63 cells cultured on PCL/Gel/nHA/Vit D3 scaffold. The results indicated superior properties of hydroxyapatite nanoparticles and vitamin D3 incorporated in PCL/Gel scaffold for use in bone tissue engineering.     

Cell(MC3T3‐E1)‐Printed Poly(ϵ‐caprolactone)/Alginate Hybrid Scaffolds for Tissue Regeneration

A new cell‐printed scaffold consisting of poly(ϵ‐caprolactone) (PCL) and cell‐embedded alginate struts is designed. The PCL and alginate struts are stacked in an interdigitated pattern in successive layers to acquire a three‐dimensional (3D) shape. The hybrid scaffold exhibits a two‐phase structure consisting of cell (MC3T3‐E1)‐laden alginate struts able to support biological activity and PCL struts able to provide controllable mechanical support of the cell‐laden alginate struts. The hybrid scaffolds exhibit an impressive increase in tensile modulus and maximum strength compared to pure alginate scaffolds. Laden cells are homogeneously distributed throughout the alginate struts and the entire scaffold, resulting in cell viability of approximately 84%.     

Magnesium-containing silk fibroin/polycaprolactone electrospun nanofibrous scaffolds for accelerating bone regeneration

Bone tissue engineering has become one of the most effective methods for treating bone defects. In this study, an electrospun tissue engineering membrane containing magnesium was successfully fabricated by incorporating magnesium oxide (MgO) nanoparticles into silk fibroin and polycaprolactone (SF/PCL)-blend scaffolds. The release kinetics of Mg²⁺ and the effects of magnesium on scaffold morphology, and cellular behavior were investigated. The obtained Mg-functionalized nanofibrous scaffolds displayed controlled release of Mg²⁺, satisfactory biocompatibility and osteogenic capability. The in vivo implantation of magnesium-containing electrospun nanofibrous membrane in a rat calvarial defect resulted in the significant enhancement of bone regeneration twelve weeks post-surgery. This work represents a valuable strategy for fabricating functional magnesium-containing electrospun scaffolds that show potential in craniofacial and orthopedic applications.     

Polycaprolactone Electrospun Fiber Mats Prepared Using Benign Solvents: Blending with Copper(II)‐Chitosan Increases the Secretion of Vascular Endothelial Growth Factor in a Bone Marrow Stromal Cell Line

Inducing the formation of new blood vessels (angiogenesis) is an essential requirement for successful tissue engineering. Approaches have been proposed to enhance angiogenesis using growth factors and other biomolecules; however, this approaches present drawbacks in terms of high cost and patient safety. Copper is known to effectively regulate angiogenesis and can offer a more cost‐effective alternative than the direct use of growth factors. With this study, a strategy to incorporate copper in electrospun fibrous scaffolds with pro‐angiogenic properties is presented. Polycaprolactone (PCL) and copper(II)‐chitosan are electrospun using benign solvents. The morphological and physicochemical properties of the fiber mats are investigated through scanning electron microscopy (SEM), static contact angle measurements, energy dispersive X‐ray, and Fourier‐transform infrared spectroscopies. Scaffold stability in phosphate buffered saline at 37 °C is monitored over 1 week. A bone marrow stromal cell line (ST‐2) is cultured for 7 days and its behavior is evaluated using SEM, fluorescence microscopy and a tetrazolium salt‐based colorimetric assay. Results confirm that PCL/copper(II)‐chitosan is suitable for electrospinning. The fiber mats are biocompatible and favor cell colonization and infiltration. Most notably, the angiogenic potential of PCL/copper(II)‐chitosan blends is confirmed by a three‐fold increase in VEGF secretion by ST‐2 cells in the presence of copper(II)‐chitosan.     

Preparation and characterization of novel bone scaffolds based on electrospun polycaprolactone fibers filled with nanoparticles

Novel bone-scaffolding materials were successfully fabricated by electrospinning from polycaprolactone (PCL) solutions containing nanoparticles of calcium carbonate (CaCO(3)) or hydroxyapatite (HA). The diameters of the as-spun fibers were found to increase with the addition and increasing amounts of the nanoparticles. The observed increase in the diameters of the as-spun fibers with the addition and increasing amounts of the nanoparticulate fillers was responsible for the observed increase in the tensile strength of the obtained fiber mats. An increase in the concentration of the base PCL solution caused the average diameter of the as-spun PCL/HA composite fibers to increase. Increasing applied electrical potential also resulted in an increase in the diameters of the obtained PCL/HA composite fibers. Lastly, indirect cytotoxicity evaluation of the electrospun mats of PCL, PCL/CaCO(3), and PCL/HA fibers based on human osteoblasts (SaOS2) and mouse fibroblasts (L929) revealed that these as-spun mats posed no threat to the cells, a result that implied their potential for utilization as bone-scaffolding materials.     

Synthesis and Characterization of Biodegradable Poly(ϵ-caprolactone)-b-Poly(L-lactide) and Study on Their Electrospun Scaffolds

A series of multi-block copolymers, poly(L-lactide)-b-poly (?-caprolactone) (PLLA-b-PCL) were synthesized. The first step of the synthesis consisted of the transesterification between the PLLA and 1,4-Butanediol, followed by the copolymerization of PLLA-diols and PCL, using isophorone diisocyanate (IPDI) as a coupling agent. The synthesized polymers were characterized by Fourier transform infrared (FTIR) spectra, differential scanning calorimetry (DSC) and wide angle X-ray diffraction (WAXD). PLLA/PCL block copolymers were electrospun into ultrafine fibers. The morphology of the electrospun fibrous scaffolds were investigated by Scanning Electron Microscopy (SEM). Results showed that the morphology and diameter of the fibers were affected by the electrospinning solution concentrationan and different weight ratio of PLLA/PCL. These electrospun PLLA-b-PCL fibrous membranes exhibited good flexibility and deformability. In comparison with the electrospun PLLA membrane, the electrospun fibrous membranes of PLLA-b-PCL demonstrated an enhanced elongation with still high tensile strength and Young's modulus to be beneficial for tissue engineering scaffolds.     

Biodegradable highly porous interconnected poly(ε-caprolactone)/poly(L-lactide-co-ε-caprolactone) scaffolds by supercritical foaming for small-diameter vascular tissue engineering

Biodegradable ?4 mm tubular porous poly(ε-caprolactone)/poly(L-lactide-co-ε-caprolactone) (PCL/PLCL) scaffolds are fabricated successfully via one-step microcellular supercritical carbon dioxide foaming process. The effect of blending ratio on the rheology, pore structures, mechanical property, wettability, and biocompatibility of PCL/PLCL blends tubular scaffold are reported. Rheological results show that PCL matrix and PLCL dispersed phase has good compatibility. The melt strength of PCL can be enhanced obviously by adding PLCL. With an increase of PLCL content from 10 to 30 wt%, the pore size increases from 7.6 to 24.9 μm due to the homogeneous nucleation effect. The maximum open-cell content can reach 77% for PCL/PLCL foamed sample. Cyclical tensile and compliance tests show that few content of dispersed PLCL (10–20 wt%) improves the flexibility and recoverability. Cell viability results demonstrate that human umbilical vein endothelial cells (HUVECs) cultured on all PCL/PLCL porous scaffolds exhibit a typical spindle-like cell morphology. Moreover, HUVECs have a higher density and spreading areas on surface of 10% PLCL scaffold. The results gathered in this paper may open a new perspective for the fabrication of small-diameter vascular tissue engineering scaffold.     

Polycaprolactone-polymethyl methacrylate electrospun blends for biomedical applications

Electrospinning is one of most versatile process to fabricate porous scaffolds in biomedical field. Synthetic polymers such as polycaprolactone (PCL) and polymethyl methacrylate (PMMA) provide excellent properties for biomedical applications due to their biocompatibility and tunable mechanical properties. PCL-PMMA electrospun blends combine compressive/tensile properties of individual polymers as well as biocompatibility/biodegradability. Together with porosity of scaffold, drug/nutrient supply is required in tissue regeneration and healing. High pressure CO₂ has been investigated to plasticize many biopolymers and impregnate drugs in scaffolds. This study explores several compositions of PCL-PMMA electrospun scaffolds for morphological and mechanical properties. These scaffolds are impregnated with hydrophilic (Rhodamine B) and hydrophobic (Fluorescein) dyes using high pressure CO₂ and air plasma treatment. Furthermore, release profiles of dyes have been studied from thin films and porous scaffolds to understand several controlling factors for controlled release applications. Results show dye-polymer interactions, CO₂ impregnation and stress relaxation of electrospun fibers are key factors in release profile from electrospun fibers. This study is a step forward in developing PCL-PMMA based electrospun scaffolds for drug delivery and tissue engineering.     

Deformation behavior of electrospun poly(L‐lactide‐co‐ε‐caprolactone) nonwoven membranes under uniaxial tensile loading

Biodegradable poly(L ‐lactide‐co‐ε‐caprolactone) copolymers with different L ‐lactide (LLA)/ε‐caprolactone (CL) ratios of 75/25 and 50/50 were electrospun into fine fibers. The deformation behavior of the electrospun membranes with randomly oriented structures was evaluated under uniaxial tensile loading. The electrospun membrane with a higher LLA content showed a significantly higher tensile modulus but a similar maximum stress and a lower ultimate strain in comparison with the membrane with a lower LLA content. The beaded fibers that formed in the membranes caused lower tensile properties. X‐ray diffraction and differential scanning calorimetry results suggested that the electrospun fine fibers developed highly oriented structures in CL‐unit sequences during the electrospinning process even though the concentration was only 25 wt %. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 3205–3212, 2005     

Scaffolds for Sustained Release of Ambroxol Hydrochloride,a Pharmacological Chaperone That Increases the Activity of Misfolded β‐Glucocerebrosidase

Ambroxol is a pharmacological chaperone (PC) for Gaucher disease that increases lysosomal activity of misfolded β‐glucocerebrosidase (GCase) while displaying a safe toxicological profile. In this work, different poly(ε‐caprolactone) (PCL)‐based systems are developed to regulate the sustained release of small polar drugs in physiological environments. For this purpose, ambroxol is selected as test case since the encapsulation and release of PCs using polymeric scaffolds have not been explored yet. More specifically, ambroxol is successfully loaded in electrospun PCL microfibers, which are subsequently coated with additional PCL layers using dip‐coating or spin‐coating. The time needed to achieve 80% release of loaded ambroxol increases from ≈15 min for uncoated fibrous scaffolds to 3 days and 1 week for dip‐coated and spin‐coated systems, respectively. Furthermore, it is proven that the released drug maintains its bioactivity, protecting GCase against induced thermal denaturation.     

In vitro biocompatibility of electrospun hexanoyl chitosan fibrous scaffolds towards human keratinocytes and fibroblasts

With the ability to form a submicron-sized fibrous structure with interconnected pores mimicking the extracellular matrix (ECM) for tissue formation, electrospinning was used to fabricate ultra-fine fiber mats of hexanoyl chitosan (H-chitosan) for potential use as skin tissue scaffolds. In the present communication, the in vitro biocompatibility of the electrospun fiber mats was evaluated. Indirect cytotoxicity evaluation of the fiber mats with mouse fibroblasts (L929) revealed that the materials were non-toxic and did not release substances harmful to living cells. The potential for use of the fiber mats as skin tissue scaffolds was further assessed in terms of the attachment and the proliferation of human keratinocytes (HaCaT) and human foreskin fibroblasts (HFF) that were seeded or cultured on the scaffolds at different times. The results showed that the electrospun fibrous scaffolds could support the attachment and the proliferation of both types of cells, especially for HaCaT. In addition, the cells cultured on the fibrous scaffolds exhibited normal cell shapes and integrated well with surrounding fibers. The obtained results confirmed the potential for use of the electrospun H-chitosan fiber mats as scaffolds for skin tissue engineering.     

Preparation of Small‐Diameter Tissue‐Engineered Vascular Grafts Electrospun from Heparin End‐Capped PCL and Evaluation in a Rabbit Carotid Artery Replacement Model

Aiming to construct small diameter (ID <6 mm) off‐the‐shelf tissue‐engineered vascular grafts, the end‐group heparinizd poly(ε‐caprolactone) (PCL) is synthesized by a three‐step process and then electrospun into an inner layer of double‐layer vascular scaffolds (DLVSs) showing a hierarchical double distribution of nano‐ and microfibers. Afterward, PCL without the end‐group heparinization is electrospun into an outer layer. A steady release of grafted heparin and the existence of a glycocalyx structure give the grafts anticoagulation activity and the conjugation of heparin also improves hydrophilicity and accelerates degradation of the scaffolds. The DLVSs are evaluated in six rabbits via a carotid artery interpositional model for a period of three months. All the grafts are patent until explantation, and meanwhile smooth endothelialization and fine revascularization are observed in the grafts. The composition of the outer layer of scaffolds exhibits a significant effect on the aneurysm dilation after implantation. Only one aneurysm dilation is detected at two months and no calcification is formed in the follow‐up term. How to prevent aneurysms remains a challenging topic.     

Viscoelastic properties of poly(ε‐caprolactone) – hydroxyapatite micro‐ and nano‐composites

Various composites have been proposed in the literature for the fabrication of bioscaffolds for bone tissue engineering. These materials include poly(ε‐caprolactone) (PCL) with hydroxyapatite (HA). Since the biomaterial acts as the medium that transfers mechanical signals from the body to the cells, the fundamental properties of the biomaterials should be characterized. Furthermore, in order to control the processing of these materials into scaffolds, the characterization of the fundamental properties is also necessary. In this study, the physical, thermal, mechanical, and viscoelastic properties of the PCL‐HA micro‐ and nano‐composites were characterized. Although the addition of filler particles increased the compressive modulus by up to 450%, the thermal and viscoelastic properties were unaffected. Furthermore, although the presence of water plasticized the polymer, the viscoelastic behavior was only minimally affected. Testing the composites under various conditions showed that the addition of HA can strengthen PCL without changing its viscoelastic response. The results found in this study can be used to further understand and approximate the time‐dependent behavior of scaffolds for bone tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis and properties of photocurable biodegradable multiblock copolymers based on poly(ε‐caprolactone) and poly(L‐lactide) segments

Photocurable biodegradable multiblock copolymers were synthesized from poly(ε‐caprolactone) (PCL) diol and poly(L ‐lactide) (PLLA) diol with 4,4′‐(adipoyldioxy)dicinnamic acid (CAC) dichloride as a chain extender derived from adipoyl chloride and 4‐hydroxycinnamic acid, and they were characterized with Fourier transform infrared and ¹H NMR spectroscopy, gel permeation chromatography, wide‐angle X‐ray diffraction, differential scanning calorimetry, and tensile tests. The copolymers were irradiated with a 400‐W high‐pressure mercury lamp from 30 min to 3 h to form a network structure in the absence of photoinitiators. The gel concentration increased with time, and a concentration of approximately 90% was obtained in 90–180 min for all the films. The photocuring hardly affected the crystallinity and melting temperature of the PCL segments but reduced the crystallinity of the PLLA segments. The mechanical properties, such as the tensile strength, modulus, and elongation, were significantly affected by the copolymer compositions and gel concentrations. Shape‐memory properties were determined with cyclic thermomechanical experiments. The CAC/PCL and CAC/PCL/PLLA (75/25) films photocured for 30–120 min showed good shape‐memory properties with strain fixity rates and recovery rates of approximately 100%. The formation of the network structure and the crystallization and melting of the PCL segments played very important roles for the typical shape‐memory properties. Finally, the degradation characteristics of these copolymers were investigated in a phosphate buffer solution at 37 °C with proteinase‐k and Pseudomonas cepacia lipase. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 2426–2439, 2005