Shih-Hsien Chen

Composite chitosan/silk fibroin nanofibers for modulation of osteogenic differentiation and proliferation of human mesenchymal stem cells

Nanofibrous membrane scaffolds of chitosan (CS), silk fibroin (SF) and CS/SF blend were prepared by electrospinning and studied for growth and osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). The morphology and physico-chemical properties of all membrane scaffolds were compared. The influence of CS and SF on cell proliferation was assessed by the MTS assay, whereas osteogenic differentiation was determined from the Alizarin Red staining, alkaline phosphatase activity and expression of osteogenic marker genes. The osteogenic differentiation and proliferation of hMSCs were enhanced by CS and SF nanofibers, respectively. Blending CS with SF retained the osteogenesis nature of CS without negatively influencing the cell proliferative effect of SF. By taking advantage of the differentiation/proliferation cues from individual components, the electrospun CS/SF composite nanofibrous membrane scaffold is suitable for bone tissue engineering.

Preparation and characterization of biomimetic silk fibroin/chitosan composite nanofibers by electrospinning for osteoblasts culture.

In this study, we have successfully fabricated electrospun bead-free silk fibroin [SF]/chitosan [CS] composite nanofibers [NFs] covering the whole range of CS content (0%, 25%, 50%, 75%, and 100%). SF/CS spinning solutions were prepared in a mixed solvent system of trifluoroacetic acid [TFA] and dichloromethane. The morphology of the NFs was observed by scanning electron microscope, and the average fiber diameter ranges from 215 to 478 nm. Confocal laser scanning microscopy confirms the uniform distribution of SF and CS within the composite NFs. To increase biocompatibility and preserve nanostructure when seeded with cells in culture medium, NFs were treated with an ethanol/ammonia aqueous solution to remove residual TFA and to change SF protein conformation. After the chemical treatment, SF/CS NFs could maintain the original structure for up to 54 days in culture medium. Properties of pristine and chemically treated SF/CS NFs were investigated by Fourier transform infrared spectroscopy [FT-IR], X-ray diffraction [XRD], and thermogravimetry/differential scanning calorimetry [TG/DSC]. Shift of absorption peaks in FT-IR spectra confirms the conformation change of SF from random coil to β-sheet by the action of ethanol, which is also consistent with the SF crystalline diffraction patterns measured by XRD. From TG/DSC analysis, the decomposition temperature peaks due to salt formation from TFA and protonated amines disappeared after chemical treatment, indicating complete removal of TFA by binding with ammonium ions during the treatment. This was also confirmed with the disappearance of F1s peak in X-ray photoelectron spectroscopy spectra and disappearance of TFA salt peaks in FT-IR spectra. The composite NFs could support the growth and osteogenic differentiation of human fetal osteoblastic [hFOB] cells, but each component in the composite NF shows distinct effect on cell behavior. SF promotes hFOB proliferation while CS enhances hFOB differentiation. The composite SF/CS NFs will be suitable for bone tissue engineering applications by choosing a suitable blend composition.PACS: 87.85.jf; 87.85.Rs; 68.37.Hk.

Dual functional core–sheath electrospun hyaluronic acid/polycaprolactone nanofibrous membranes embedded with silver nanoparticles for prevention of peritendinous adhesion

Peritendinous adhesions, one of the common complications after tendon injury and subsequent surgery, could be minimized by directly placing a physical barrier between the injured site and the surrounding tissue. We used silver (Ag) nanoparticles embedded in electrospun hyaluronic acid (HA)/polycaprolactone (PCL) nanofibrous membranes (NFMs) (HA/PCL+Ag NFMs) to prevent peritendinous adhesions and bacterial infection after tendon surgery. HA was used for effective lubrication, and Ag provided antibacterial activity. A dual functional anti-adhesion barrier with core-sheath nanofibrous architecture was made from an HA core solution and a photo-reduced silver nitrate/PCL sheath solution. Polycaprolactone NFMs (PCL NFMs), hyaluronic acid/polycaprolactone core-sheath NFMs (HA/PCL NFMs) and HA/PCL+Ag NFMs with comparable fiber diameters and pore sizes were prepared and analyzed. The microporous structure of NFMs is expected to effectively block the penetration of adhesion-forming fibroblasts during tendon healing. The release of Ag from HA/PCL+Ag NFMs plateaued after 4 days, which confirmed the short-term anti-bacterial effect, and this result was verified with agar diffusion tests. In contrast, the release of HA was extended up to 21 days to simulate the lubrication effect offered by HA in the synovial fluid of the tendon sheath. In vitro cell culture experiments revealed that HA/PCL+Ag NFMs exhibited the highest inhibition of fibroblast attachment and proliferation without significant cytotoxicity due to the synergistic effect of Ag and HA. In vivo studies with a rabbit flexor tendon model further confirmed the efficacy of HA/PCL+Ag NFMs in reducing peritendinous adhesion as determined by gross observation, histology, joint range-of-motion, tendon gliding and biomechanical tests.

Prevention of peritendinous adhesions with electrospun chitosan-grafted polycaprolactone nanofibrous membranes.

As one of the common complications after tendon injury and subsequent surgery, peritendinous adhesions could be minimized by directly placing a physical barrier between the injured site and the surrounding tissue. With the aim of solving the shortcomings of current biodegradable anti-adhesion barrier membranes, we propose the use of an electrospun chitosan-grafted polycaprolactone (PCL-g-CS) nanofibrous membrane (NFM) to prevent peritendinous adhesions. After introducing carboxyl groups on the surface by oxygen plasma treatment, the polycaprolactone (PCL) NFM was covalently grafted with chitosan (CS) molecules, with carbodiimide as the coupling agent. Compared with PCL NFM, PCL-g-CS NFM showed a similar fiber diameter, permeation coefficient for bovine serum albumin, ultimate tensile strain, reduced pore diameter, lower water contact angle, increased water sorption and tensile strength. With its submicrometer pore diameter (0.6-0.9μm), both NFMs could allow the diffusion of nutrients and waste while blocking fibroblast penetration to prevent adhesion formation after tendon surgery. Cell culture experiments verified that PCL-g-CS NFM can reduce fibroblast attachment while maintaining the biocompatibility of PCL NFM, implicating a synergistic anti-adhesion effect to raise the anti-adhesion efficacy. In vivo studies with a rabbit flexor digitorum profundus tendon surgery model confirmed that PCL-g-CS NFM effectively reduced peritendinous adhesion from gross observation, histology, joint flexion angle, gliding excursion and biomechanical evaluation. An injured tendon wrapped with PCL-g-CS NFM showed the same tensile strength as the naturally healed tendon, indicating that the anti-adhesion NFM will not compromise tendon healing.

Preparation and characterization of antiadhesion barrier film from hyaluronic acid-grafted electrospun poly(caprolactone) nanofibrous membranes for prevention of flexor tendon postoperative peritendinous adhesion.

Peritendinous adhesion is one of the common complications encountered after tendon injury and subsequent surgery, and it can be minimized by introducing a physical barrier between the injured site and the surrounding tissue. An electrospun hyaluronic acid-grafted poly(caprolactone) (PCL-g-HA) nanofibrous membrane (NFM) is proposed as an alternative to current antiadhesion barrier films. HA is covalently grafted to surface-aminolyzed PCL nanofibers, using carbodiimide as the coupling agent. Pristine PCL and PCL-g-HA NFMs were characterized by scanning electron microscopy, thermogravimetric analysis, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, and mechanical testing. In vitro cell culture with fibroblasts showed that PCL-g-HA NFMs reduced cellular adhesion on the membrane surface while maintaining cell proliferation. Animal experiments using a rabbit flexor digitorum profundus tendon model confirmed the efficacy of PCL-g-HA in reducing peritendinous adhesion, based on gross observation, histology, joint flexion-angle measurements, gliding tests, and biomechanical evaluation.

Prevention of peritendinous adhesions with electrospun polyethylene glycol/polycaprolactone nanofibrous membranes

Postoperative adhesion formation is the major complication that could occur after acute tendon surgery. The application of an anti-adhesive membrane at the post-surgical site is deemed as a potential way to solve this problem by preventing adhesive fibrotic tissue development. In this study, we fabricated electrospun composite poly(ethylene glycol) (PEG)/poly(caprolactone) (PCL) nanofibrous membrane (NFM) to prevent peritendinous adhesions, which could act as a barrier between the tendon and surrounding tissues, without interrupting mass transfer and normal tendon gliding. PCL/PEG NFMs of 0% PEG (PCL), 25% PEG (25PECL), 50% PEG (50PECL) and 75% PEG (75PECL) were prepared and characterized for physico-chemical properties. The PCL NFM shows the lowest protein permeability while 25PECL NFM exhibited the largest fiber diameter, smallest pore size and the largest ultimate stress and strain. The 75PECL NFM had the lowest water contact angle and the highest Youngs modulus. In vitro cell adhesion and migration experiments with fibroblasts indicate that all NFMs could prevent cell penetration, with 75PECL NFM having the least cell attachment. In vivo application of 75PECL NFM on the repaired site of rabbit flexor tendon rupture model demonstrated improved efficacy compared with the PCL NFM and a commercial anti-adhesion barrier (Seprafilm™), from gross observation, histological analysis and functional assays. We concluded that 75PECL NFM could function as an effective anti-adhesion membrane after tendon surgery in a clinical setting.

Response of Dermal Fibroblasts to Biochemical and Physical Cues in Aligned Polycaprolactone/Silk Fibroin Nanofiber Scaffolds for Application in Tendon Tissue Engineering

Silk fibroin (SF) and fiber alignment were introduced into polycaprolactone (PCL)-based electrospun nanofibers as chemical and physical cues for tendon tissue engineering applications. The physicochemical properties of random PCL (RP) nanofibers, random PCL/SF (RPSF) nanofibers and aligned PCL/SF (APSF) nanofibers were characterized for fiber orientation and SF blending effects. An in vitro cell culture with rabbit dermal fibroblasts (RDFBs) on nanofibers indicated that SF promotes cell proliferation to a higher extent than fiber alignment. Cells aligned in the direction of fiber axes could be confirmed through scanning electron microscopy (SEM) observation and cytoskeleton staining. The quantitative real-time polymerase chain reaction (qRT-PCR) experiments indicated up-regulated gene expression of tendon marker proteins (type I collagen (Col I), fibronectin and biglycan) on APSF nanofibers and tendon reconstruction was confirmed from Col III gene expression. Animal experiments with Achilles tendon defect repairs in rabbits were carried out with RPSF and APSF scaffolds. The beneficial effects of fiber alignment were verified from histological and immunohistochemical staining, where cell migration and extracellular matrix protein deposition tend to stretch in a parallel direction along the axial direction of APSF nanofibers with enhanced Col I and tenascin C production. Biomechanical testing indicated the tensile stiffness and maximum load of cell-seeded APSF scaffolds were 60.2 and 81.3% of normal tendon values, respectively, which are significantly higher than cell-seeded RPSF or acellular APSF and RPSF scaffolds. These results suggest that APSF nanofiber scaffolds combined with RDFBs have the potential to repair the gap defects of Achilles tendons in vivo and to effectively restore the function and structure of tendons.

Injectable thermosensitive hydrogel containing hyaluronic acid and chitosan as a barrier for prevention of postoperative peritoneal adhesion

Peritoneal adhesion is one of the common complications after abdominal surgery. Injectable thermosensitive hydrogel could serve as an ideal barrier to prevent this postoperative tissue adhesion. In this study, poly(N-isopropylacrylamide) (PNIPAm) was grafted to chitosan (CS) and the polymer was further conjugated with hyaluronic acid (HA) to form thermosensitive HA-CS-PNIPAm hydrogel. Aqueous solutions of PNIPAm and HA-CS-PNIPAm at 10%(w/v) are both free-flowing and injectable at room temperature and exhibit sol-gel phase transition around 31°C; however, HA-CS-PNIPAm shows less volume shrinkage after gelation and higher complex modulus than PNIPAm. Cell culture studies indicate both injectable hydrogel show barrier effects to reduce fibroblasts penetration while induce little cytotoxicity in vitro. From a sidewall defect-bowel abrasion model in rats, significant reduction of postoperative peritoneal adhesion was found for peritoneal defects treated with HA-CS-PNIPAm compared with those treated with PNIPAm and untreated controls from gross and histological evaluation. Furthermore, HA-CS-PNIPAm did not interfere with normal peritoneal tissue healing and did not elicit acute toxicity from blood analysis and tissue biopsy examination. By taking advantage of the easy handling and placement properties of HA-CS-PNIPAm during application, this copolymer hydrogel would be a potentially ideal injectable anti-adhesion barrier after abdominal surgeries.

Thermo-responsive in-situ forming hydrogels as barriers to prevent post-operative peritendinous adhesion

In this study, we aimed to assess whether thermo-responsive in-situ forming hydrogels based on poly(N-isopropylacrylamide) (PNIPAM) could prevent post-operative peritendinous adhesion. The clinical advantages of the thermo-responsive hydrogels are acting as barrier material to block penetration of fibroblasts, providing mobility and flexibility during application and enabling injection through a small opening to fill spaces of any shape after surgery. The thermo-responsiveness of hydrogels was determined to ensure their clinic uses. By grafting hydrophilic biopolymers chitosan (CS) and hyaluronic acid (HA) to PNIPAM, the copolymer hydrogels show enhanced water retention and lubrication, while reduced volume shrinkage during phase transition. In cell culture experiments, the thermo-responsive hydrogel has good biocompatibility and reduces fibroblast penetration. In animal experiments, the effectiveness of preventing post-operative peritendinous adhesion was studied in a rabbit deep flexor tendon model. From gross examination, histology, bending angles of joints, tendon gliding excursion and pull-out force, HA-CS-PNIPAM (HACPN) was confirmed to be the best barrier material to prevent post-operative peritendinous adhesion compared to PNIPAM and CS-PNIPAM (CPN) hydrogels and a commercial barrier film Seprafilm®. There was no significant difference in the breaking strength of HACPN-treated tendons and spontaneously healed ones, indicating HACPN hydrogel application did not interfere with normal tendon healing. We conclude that HACPN hydrogel can provide the best functional outcomes to significantly prevent post-operative tendon adhesion in vivo. STATEMENT OF SIGNIFICANCE We prepared thermo-responsive in-situ forming hydrogels based on poly(N-isopropylacrylamide) (PNIPAM) to prevent post-operative peritendinous adhesion. The injectable barrier hydrogel could have better anti-adhesive properties than current commercial products by acting as barrier material to block penetration of fibroblasts, providing mobility and flexibility during application and enabling injection through a small opening to fill spaces of any shape after surgery. The effectiveness of preventing post-operative peritendinous adhesion was studied in a rabbit deep flexor tendon model. From gross examination, histology, bending angles of joints, tendon gliding excursion and pull-out force, HA-CS-PNIPAM (HACPN) was confirmed to be the best barrier material to prevent post-operative peritendinous adhesion compared to PNIPAM and CS-PNIPAM (CPN) hydrogels and a commercial barrier film Seprafilm®.

Thermosensitive Injectable Hydrogel for Simultaneous Intraperitoneal Delivery of Doxorubicin and Prevention of Peritoneal Adhesion

To improve intraperitoneal chemotherapy and to prevent postsurgical peritoneal adhesion, we aimed to develop a drug delivery strategy for controlled release of a chemotherapeutic drug from the intraperitoneally injected thermosensitive poly(N-isopropylacrylamide)-based hydrogel (HACPN), which is also endowed with peritoneal anti-adhesion properties. Anticancer drug doxorubicin (DOX) was loaded into the hydrogel (HACPN-DOX) to investigate the chemotherapeutic and adhesion barrier effects in vivo. A burst release followed by sustained release of DOX from HACPN-DOX was found due to gradual degradation of the hydrogel. Cell culture studies demonstrated the cytotoxicity of released DOX toward CT-26 mouse colon carcinoma cells in vitro. Using peritoneal carcinomatosis animal model in BALB/c mice with intraperitoneally injected CT-26 cells, animals treated with HACPN-DOX revealed the best antitumor efficacy judging from tumor weight and volume, survival rate, and bioluminescence signal intensity when compared with treatment with free DOX at the same drug dosage. HACPN (or HACPN-DOX) also significantly reduced the risk of postoperative peritoneal adhesion, which was generated by sidewall defect-cecum abrasion in tumor-bearing BALB/c mice, from gross and histology analyses. This study could create a paradigm to combine controlled drug release with barrier function in a single drug-loaded injectable hydrogel to enhance the intraperitoneal chemotherapeutic efficacy while simultaneously preventing postsurgical adhesion.