Photo-Crosslinked Hyaluronic Acid/Carboxymethyl Cellulose Composite Hydrogel as a Dural Substitute to Prevent Post-Surgical Adhesion.

A dural substitute is frequently used to repair dura mater during neurosurgical procedures. Although autologous or commercially available dural substitutes matched most of the requirements; difficulties during dural repair, including insufficient space for suturing, insufficient mechanical strength, easy tear and cerebrospinal fluid leakage, represent major challenges. To meet this need, a photo-crosslinked hydrogel was developed as a dural substitute/anti-adhesion barrier in this study, which can show sol-to-gel phase transition in situ upon short-time exposure to visible light. For this purpose, hyaluronic acid (HA) and carboxymethyl cellulose (CMC), materials used in abdominal surgery for anti-adhesion purposes, were reacted separately with glycidyl methacrylate to form hyaluronic acid methacrylate (HAMA) and carboxymethyl cellulose methacrylate (CMCMA). The HA/CMC (HC) hydrogels with different HA compositions could be prepared by photo-crosslinking HAMA and CMCMA with a 400 nm light source using lithium phenyl-2,4,6-trimethylbenzoylphosphinate as a photo-initiator. From studies of physico-chemical and biological properties of HC composite hydrogels, they are bio-compatible, bio-degradable and mechanically robust, to be suitable as a dural substitute. By drastically reducing attachment and penetration of adhesion-forming fibroblasts in vitro, the HC hydrogel can also act as an anti-adhesion barrier to prevent adhesion formation after dural repair. From in vivo study in rabbits, the HC hydrogel can repair dural defects as well as protect the dura from post-operative adhesion, endorsing the possible application of this hydrogel as a novel dural substitute.

Polycaprolactone/Chitosan Composite Nanofiber Membrane as a Preferred Scaffold for the Culture of Mesothelial Cells and the Repair of Damaged Mesothelium.

Mesothelial cells are specific epithelial cells lining the serosal cavity and internal organs. Nonetheless, few studies have explored the possibility to culture mesothelial cells in a nanostructure scaffold for tissue engineering applications. Therefore, this study aims to fabricate nanofibers from a polycaprolactone (PCL) and PCL/chitosan (CS) blend by electrospinning, and to elucidate the effect of CS on the cellular response of mesothelial cells. The results demonstrate that a PCL and PCL/CS nanofiber membrane scaffold could be prepared with a comparable fiber diameter (~300 nm) and porosity for cell culture. Blending CS with PCL influenced the mechanical properties of the scaffold due to interference of PCL crystallinity in the nanofibers. However, CS substantially improves scaffold hydrophilicity and results in a ~6-times-higher cell attachment rate in PCL/CS. The mesothelial cells maintain high viability in both nanofiber membranes, but PCL/CS provides better maintenance of cobblestone-like mesothelial morphology. From gene expression analysis and immunofluorescence staining, the incorporation of CS also results in the upregulated expression of mesothelial marker genes and the enhanced production of key mesothelial maker proteins, endorsing PCL/CS to better maintain the mesothelial phenotype. The PCL/CS scaffold was therefore chosen for the in vivo studies, which involved transplanting a cell/scaffold construct containing allograft mesothelial cells for mesothelium reconstruction in rats. In the absence of mesothelial cells, the mesothelium wound covered with PCL/CS showed an inflammatory response. In contrast, a mesothelium layer similar to native mesothelium tissue could be obtained by implanting the cell/scaffold construct, based on hematoxylin and eosin (H&E) and immunohistochemical staining.

Preparation and Characterization of Surface Heat Sintered Nanohydroxyapatite and Nanowhitlockite Embedded Poly (Lactic-co-glycolic Acid) Microsphere Bone Graft Scaffolds: In Vitro and in Vivo Studies.

In the context of using bone graft materials to restore and improve the function of damaged bone tissues, macroporous biodegradable composite bone graft scaffolds have osteoinductive properties that allow them to provide a suitable environment for bone regeneration. Hydroxyapatite (HAP) and whitlockite (WLKT) are the two major components of hard tissues such as bone and teeth. Because of their biocompatibility and osteoinductivity, we synthesized HAP (nHAP) and WLKT nanoparticles (nWLKT) by using the chemical precipitation method. The nanoparticles were separately incorporated within poly (lactic-co-glycolic acid) (PLGA) microspheres. Following this, the composite microspheres were converted to macroporous bone grafts with sufficient mechanical strength in pin or screw shape through surface sintering. We characterized physico-chemical and mechanical properties of the nanoparticles and composites. The biocompatibility of the grafts was further tested through in vitro cell adhesion and proliferation studies using rabbit bone marrow stem cells. The ability to promote osteogenic differentiation was tested through alkaline phosphate activity and immunofluorescence staining of bone marker proteins. For in vivo study, the bone pins were implanted in tibia bone defects in rabbits to compare the bone regeneration ability though H&E, Masson's trichrome and immunohistochemical staining. The results revealed similar physico-chemical characteristics and cellular response of PLGA/nHAP and PLGA/nWLKT scaffolds but the latter is associated with higher osteogenic potential towards BMSCs, pointing out the possibility to use this ceramic nanoparticle to prepare a sintered composite microsphere scaffold for potential bone grafts and tissue engineered implants.

Docosahexaenoic Acid-Loaded Polylactic Acid Core-Shell Nanofiber Membranes for Regenerative Medicine after Spinal Cord Injury: In Vitro and In Vivo Study.

Spinal cord injury (SCI) is associated with disability and a drastic decrease in quality of life for affected individuals. Previous studies support the idea that docosahexaenoic acid (DHA)-based pharmacological approach is a promising therapeutic strategy for the management of acute SCI. We postulated that a nanostructured material for controlled delivery of DHA at the lesion site may be well suited for this purpose. Toward this end, we prepare drug-loaded fibrous mats made of core-shell nanofibers by electrospinning, which contained a polylactic acid (PLA) shell for encapsulation of DHA within the core, for delivery of DHA in situ. In vitro study confirmed sustained DHA release from PLA/DHA core-shell nanofiber membrane (CSNM) for up to 36 days, which could significantly increase neurite outgrowth from primary cortical neurons in 3 days. This is supported by the upregulation of brain-derived neurotropic factor (BDNF) and neurotrophin-3 (NT-3) neural marker genes from qRT-PCR analysis. Most importantly, the sustained release of DHA could significantly increase the neurite outgrowth length from cortical neuron cells in 7 days when co-cultured with PLA/DHA CSNM, compared with cells cultured with 3 µM DHA. From in vivo study with a SCI model created in rats, implantation of PLA/DHA CSNM could significantly improve neurological functions revealed by behavior assessment in comparison with the control (no treatment) and the PLA CSNM groups. According to histological analysis, PLA/DHA CSNM also effectively reduced neuron loss and increased serotonergic nerve sprouting. Taken together, the PLA/DHA CSNM may provide a nanostructured drug delivery system for DHA and contribute to neuroprotection and promoting neuroplasticity change following SCI.

Preparation of Gelatin and Gelatin/Hyaluronic Acid Cryogel Scaffolds for the 3D Culture of Mesothelial Cells and Mesothelium Tissue Regeneration.

Mesothelial cells are specific epithelial cells that are lined in the serosal cavity and internal organs. Nonetheless, few studies have explored the possibility to culture mesothelial cells in a three-dimensional (3D) scaffold for tissue engineering applications. Towards this end, we fabricated macroporous scaffolds from gelatin and gelatin/hyaluronic acid (HA) by cryogelation, and elucidated the influence of HA on cryogel properties and the cellular phenotype of mesothelial cells cultured within the 3D scaffolds. The incorporation of HA was found not to significantly change the pore size, porosity, water uptake kinetics, and swelling ratios of the cryogel scaffolds, but led to a faster scaffold degradation in the collagenase solution. Adding 5% HA in the composite cryogels also decreased the ultimate compressive stress (strain) and toughness of the scaffold, but enhanced the elastic modulus. From the in vitro cell culture, rat mesothelial cells showed quantitative cell viability in gelatin (G) and gelatin/HA (GH) cryogels. Nonetheless, mesothelial cells cultured in GH cryogels showed a change in the cell morphology and cytoskeleton arrangement, reduced cell proliferation rate, and downregulation of the mesothelium specific maker gene expression. The production of key mesothelium proteins E-cadherin and calretinin were also reduced in the GH cryogels. Choosing the best G cryogels for in vivo studies, the cell/cryogel construct was used for the transplantation of allograft mesothelial cells for mesothelium reconstruction in rats. A mesothelium layer similar to the native mesothelium tissue could be obtained 21 days post-implantation, based on hematoxylin and eosin (H&E) and immunohistochemical staining.

Effect of Cyclic Dynamic Compressive Loading on Chondrocytes and Adipose-Derived Stem Cells Co-Cultured in Highly Elastic Cryogel Scaffolds.

In this study, we first used gelatin/chondroitin-6-sulfate/hyaluronan/chitosan highly elastic cryogels, which showed total recovery from large strains during repeated compression cycles, as 3D scaffolds to study the effects of cyclic dynamic compressive loading on chondrocyte gene expression and extracellular matrix (ECM) production. Dynamic culture of porcine chondrocytes was studied at 1 Hz, 10% to 40% strain and 1 to 9 h/day stimulation duration, in a mechanical-driven multi-chamber bioreactor for 14 days. From the experimental results, we could identify the optimum dynamic culture condition (20% and 3 h/day) to enhance the chondrocytic phenotype of chondrocytes from the expression of marker (Col I, Col II, Col X, TNF-α, TGF-ß1 and IGF-1) genes by quantitative real-time polymerase chain reactions (qRT-PCR) and production of ECM (GAGs and Col II) by biochemical analysis and immunofluorescence staining. With up-regulated growth factor (TGF-ß1 and IGF-1) genes, co-culture of chondrocytes with porcine adipose-derived stem cells (ASCs) was employed to facilitate chondrogenic differentiation of ASCs during dynamic culture in cryogel scaffolds. By replacing half of the chondrocytes with ASCs during co-culture, we could obtain similar production of ECM (GAGs and Col II) and expression of Col II, but reduced expression of Col I, Col X and TNF-α. Subcutaneous implantation of cells/scaffold constructs in nude mice after mono-culture (chondrocytes or ASCs) or co-culture (chondrocytes + ASCs) and subject to static or dynamic culture condition in vitro for 14 days was tested for tissue-engineering applications. The constructs were retrieved 8 weeks post-implantation for histological analysis by Alcian blue, Safranin O and Col II immunohistochemical staining. The most abundant ectopic cartilage tissue was found for the chondrocytes and chondrocytes + ASCs groups using dynamic culture, which showed similar neo-cartilage formation capability with half of the chondrocytes replaced by ASCs for co-culture. This combined co-culture/dynamic culture strategy is expected to cut down the amount of donor chondrocytes needed for cartilage-tissue engineering.

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.

Dual Function of Glucosamine in Gelatin/Hyaluronic Acid Cryogel to Modulate Scaffold Mechanical Properties and to Maintain Chondrogenic Phenotype for Cartilage Tissue Engineering.

Glucosamine (GlcN) fulfills many of the requirements as an ideal component in scaffolds used in cartilage tissue engineering. The incorporation of GlcN in a gelatin/hyaluronic acid (GH) cryogel scaffold could provide biological cues in maintaining the phenotype of chondrocytes. Nonetheless, substituting gelatin with GlcN may also decrease the crosslinking density and modulate the mechanical properties of the cryogel scaffold, which may be beneficial as physical cues for chondrocytes in the scaffold. Thus, we prepared cryogel scaffolds containing 9% GlcN (GH-GlcN9) and 16% GlcN (GH-GlcN16) by carbodiimide-mediated crosslinking reactions at -16 °C. The crosslinking density and the mechanical properties of the cryogel matrix could be tuned by adjusting the content of GlcN used during cryogel preparation. In general, incorporation of GlcN did not influence scaffold pore size and ultimate compressive strain but increased porosity. The GH-GlcN16 cryogel showed the highest swelling ratio and degradation rate in hyaluronidase and collagenase solutions. On the contrary, the Young's modulus, storage modulus, ultimate compressive stress, energy dissipation level, and rate of stress relaxation decreased by increasing the GlcN content in the cryogel. The release of GlcN from the scaffolds in the culture medium of chondrocytes could be sustained for 21 days for GH-GlcN16 in contrast to only 7 days for GH-GlcN9. In vitro cell culture experiments using rabbit articular chondrocytes revealed that GlcN incorporation affected cell proliferation, morphology, and maintenance of chondrogenic phenotype. Overall, GH-GlcN16 showed the best performance in maintaining chondrogenic phenotype with reduced cell proliferation rate but enhanced glycosaminoglycans (GAGs) and type II collagen (COL II) secretion. Quantitative real-time polymerase chain reaction also showed time-dependent up-regulation of cartilage-specific marker genes (COL II, aggrecan and Sox9) for GH-GlcN16. Implantation of chondrocytes/GH-GlcN16 constructs into full-thickness articular cartilage defects of rabbits could regenerate neocartilage with positive staining for GAGs and COL II. The GH-GlcN16 cryogel will be suitable as a scaffold for the treatment of articular cartilage defects.

Injectable gelatin/glucosamine cryogel microbeads as scaffolds for chondrocyte delivery in cartilage tissue engineering.

In this study, we fabricate squeezable cryogel microbeads as injectable scaffolds for minimum invasive delivery of chondrocytes for cartilage tissue engineering applications. The microbeads with different glucosamine concentrations were prepared by combining the water-in-oil emulsion and cryogelation through crosslinking of gelatin with glutaraldehyde in the presence of glucosamine. The physicochemical characterization results show the successful preparation of cryogel microbeads with uniform shape and size, high porosity, large pore size, high water uptake capacity, and good injectability. In vitro analysis indicates proliferation, migration, and differentiated phenotype of rabbit chondrocytes in the cryogel scaffolds. The seeded chondrocytes in the cryogel scaffold can be delivered by injecting through an 18G needle to fully retain the cell viability. Furthermore, the incorporation of glucosamine in the cryogel promoted the differentiated phenotype of chondrocytes in a dose-dependent manner, from cartilage-specific gene expression and protein production. The in vivo study by injecting the cryogel microbeads into the subcutaneous pockets of nude mice indicates good retention ability as well as good biocompatibility and suitable biodegradability of the cryogel scaffold. Furthermore, the injected chondrocyte/cryogel microbead constructs can form ectopic functional neocartilage tissues following subcutaneous implantation in 21 days, as evidenced by histological and immunohistochemical analysis.

Development of high resilience spiral wound suture-embedded gelatin/PCL/heparin nanofiber membrane scaffolds for tendon tissue engineering.

This study develops a spiral wound scaffold based on gelatin/PCL/heparin (GPH) nanofiber membranes for tendon tissue engineering. By embedding sutures in dual layers of aligned GPH nanofiber membranes, prepared from mixed electrospinning of gelatin and PCL/heparin solutions, we fabricate a high resilience scaffold intended for the high loading environment experienced by tendons. The basic fibroblast growth factor (bFGF) was anchored to GPH scaffold through bioaffinity between heparin and bFGF, aim to provide biological cues for maintenance of tenogenic phenotype. In addition, the aligned nanofiber morphology is expected to provide physical cues toward seeded tenocytes. With sustained release of bFGF, GPH-bFGF can enhance proliferation, up-regulate tenogenic gene expression, and increase synthesis of tendon-specific proteins by tenocytes in vitro. Furthermore, by properly maintaining tendon phenotypes, GPH-bFGF/tenocytes constructs showed improved mechanical properties over GPH-bFGF. From in vivo study using GPH-bFGF/tenocytes constructs to repair rabbit Achilles tendon defects, neotendon tissue formation was confirmed from histological staining and biomechanical analysis. These findings collectively demonstrate that the newly designed GPH-bFGF scaffold could provide a niche for inducing tendon tissue regeneration by effectively restoring the tendon tissue structure and function.

Co-Delivery of Docosahexaenoic Acid and Brain-Derived Neurotropic Factor from Electrospun Aligned Core-Shell Fibrous Membranes in Treatment of Spinal Cord Injury.

To restore lost functions while repairing the neuronal structure after spinal cord injury (SCI), pharmacological interventions with multiple therapeutic agents will be a more effective modality given the complex pathophysiology of acute SCI. Toward this end, we prepared electrospun membranes containing aligned core-shell fibers with a polylactic acid (PLA) shell, and docosahexaenoic acid (DHA) or a brain-derived neurotropic factor (BDNF) in the core. The controlled release of both pro-regenerative agents is expected to provide combinatory treatment efficacy for effective neurogenesis, while aligned fiber topography is expected to guide directional neurite extension. The in vitro release study indicates that both DHA and BDNF could be released continuously from the electrospun membrane for up to 50 days, while aligned microfibers guide the neurite extension of primary cortical neurons along the fiber axis. Furthermore, the PLA/DHA/BDNF core-shell fibrous membrane (CSFM) provides a significantly higher neurite outgrowth length from the neuron cells than the PLA/DHA CSFM. This is supported by the upregulation of genes associated with neuroprotection and neuroplasticity from RT-PCR analysis. From an in vivo study by implanting a drug-loaded CSFM into the injury site of a rat suffering from SCI with a cervical hemisection, the co-delivery of DHA and BDNF from a PLA/DHA/BDNF CSFM could significantly improve neurological function recovery from behavioral assessment, as well as provide neuroprotection and promote neuroplasticity changes in recovered neuronal tissue from histological analysis.

A bioactive multi-functional heparin-grafted aligned poly(lactide-co-glycolide)/curcumin nanofiber membrane to accelerate diabetic wound healing.

Curcumin is reported to possess excellent efficacy to treat wounds that exhibit impaired healing. Heparin shows high affinity for many growth factors that are key biological mediators during the wound healing process. In this study, we aimed to prepare wound dressing membranes, for sustained release of an exogenous factor curcumin as well as sequestering endogenous growth factors at the wound site, to promote wound healing in diabetic rats. Toward this end, we prepared aligned curcumin-loaded poly(lactide-co-glycolide) (PLGA) nanofiber membranes (PC NFMs), followed by high density surface grafting of heparin to fabricate PLGA/curcumin (PCH) NFMs. Both PC and PCH NFMs show high tensile strength, low cytotoxicity and suitable water vapor transmission rate for application as wound dressings. Nonetheless, the PCH NFM shows higher curcumin release rate than PC due to enhanced hydrophilicity, which leads to higher cell migration rate and induced oxidative stress protection of HS68 fibroblast cells in vitro. In vivo study indicated the PCH exhibits the fastest wound closure rate among all membranes with accelerated re-epithelization rate, higher angiogenesis rate and more collagen deposition at the wound site. The accelerated and better skin tissue regeneration could be suggested to correlate with the multi-functionality of nanofibers, where grafted heparin attracting and stabilizing the growth factors important for wound healing in situ, together with relieving the high oxidative stress and the inflammatory cascade from released curcumin during diabetic wound healing.

Rational design of gelatin/nanohydroxyapatite cryogel scaffolds for bone regeneration by introducing chemical and physical cues to enhance osteogenesis of bone marrow mesenchymal stem cells.

Identification of key components in the chemical and physical milieu for directing osteogenesis is a requirement in the investigation of tissue engineering scaffolds for advancement of bone regeneration. In this study, we engineered different gelatin-based cryogels and studied the effect of nanohydroxyapatite (nHAP) and crosslinking agents on scaffold properties and its osteogenic response towards bone marrow stem cells (BMSCs). The cryogels examined are 5% gelatin and 5% gelatin/2.5% nHAP, crosslinked either with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) or glutaraldehyde (GA). We confirmed that nHAP or the crosslinking agent has no effects on scaffold pore size and porosity. Nonetheless, incorporation of nHAP increased mechanical strength, swelling ratio and degree of crosslinking, but decreased degradation rate. Cryogels crosslinked with EDC showed faster degradation and promoted osteogenic differentiation of BMSCs while those prepared from GA crosslinking promoted proliferation of BMSCs. Furthermore, osteogenic differentiation was always enhanced in the presence of nHAP irrespective of the culture medium (normal or osteogenic) used but osteogenic medium always provide a higher extent of osteogenic differentiation. Employing gelatin/nHAP cryogel crosslinked by EDC in a bioreactor for dynamic culture of BMSCs, cyclic compressive mechanical simulation was found to be beneficial for both cell proliferation and osteogenic differentiation. However, the optimum conditions for osteogenic differentiation and cell proliferation were found at 30% and 60% strain, respectively. We thus demonstrated that osteogenic differentiation of BMSCs could be tuned by taking advantages of chemical cues generated from scaffold chemistry or physical cues generated from dynamic cell culture in vitro. Furthermore, by combining the best cryogel preparation and in vitro cell culture condition for osteogenesis, we successfully employed in vitro cultured cryogel/BMSCs constructs for repair of rabbit critical-sized cranial bone defects.

Gelatin/Nanohyroxyapatite Cryogel Embedded Poly(lactic-co-glycolic Acid)/Nanohydroxyapatite Microsphere Hybrid Scaffolds for Simultaneous Bone Regeneration and Load-Bearing.

It is desirable to combine load-bearing and bone regeneration capabilities in a single bone tissue engineering scaffold. For this purpose, we developed a high strength hybrid scaffold using a sintered poly(lactic-co-glycolic acid) (PLGA)/nanohydroxyapatite (nHAP) microsphere cavity fitted with gelatin/nHAP cryogel disks in the center. Osteo-conductive/osteo-inductive nHAP was incorporated in 250⁻500 µm PLGA microspheres at 40% (w/w) as the base matrix for the high strength cavity-shaped microsphere scaffold, while 20% (w/w) nHAP was incorporated into gelatin cryogels as an embedded core for bone regeneration purposes. The physico-chemical properties of the microsphere, cryogel, and hybrid scaffolds were characterized in detail. The ultimate stress and Young's modulus of the hybrid scaffold showed 25- and 21-fold increases from the cryogel scaffold. In vitro studies using rabbit bone marrow-derived stem cells (rBMSCs) in cryogel and hybrid scaffolds through DNA content, alkaline phosphatase activity, and mineral deposition by SEM/EDS, showed the prominence of both scaffolds in cell proliferation and osteogenic differentiation of rBMSCs in a normal medium. Calcium contents analysis, immunofluorescent staining of collagen I (COL I), and osteocalcin (OCN) and relative mRNA expression of COL I, OCN and osteopontin (OPN) confirmed in vitro differentiation of rBMSCs in the hybrid scaffold toward the bone lineage. From compression testing, the cell/hybrid scaffold construct showed a 1.93 times increase of Young's modulus from day 14 to day 28, due to mineral deposition. The relative mRNA expression of osteogenic marker genes COL I, OCN, and OPN showed 5.5, 18.7, and 7.2 folds increase from day 14 to day 28, respectively, confirming bone regeneration. From animal studies, the rBMSCs-seeded hybrid constructs could repair mid-diaphyseal tibia defects in rabbits, as evaluated by micro-computed tomography (µ-CT) and histological analyses. The hybrid scaffold will be useful for bone regeneration in load-bearing areas.

Multi-functional electrospun antibacterial core-shell nanofibrous membranes for prolonged prevention of post-surgical tendon adhesion and inflammation.

The possibility of endowing an electrospun anti-adhesive barrier membrane with multi-functionality, such as lubrication, prevention of fibroblast attachment and anti-infection and anti-inflammation properties, is highly desirable for the management of post-surgical tendon adhesion. To this end, we fabricated core-shell nanofibrous membranes (CSNMs) with embedded silver nanoparticles (Ag NPs) in the poly(ethylene glycol) (PEG)/poly(caprolactone) (PCL) shell and hyaluronic acid (HA)/ibuprofen in the core. HA imparted a lubrication effect for smooth tendon gliding and reduced fibroblast attachment, while Ag NPs and ibuprofen functioned as anti-infection and anti-inflammation agents, respectively. CSNMs with a PEG/PCL/Ag shell (PPA) and HA core containing 0% (H/PPA), 10% (HI10/PPA), 30% (HI30/PPA) and 50% (HI50/PPA) ibuprofen were fabricated through co-axial electrospinning and assessed through microscopic, spectroscopic, thermal, mechanical and drug release analyses. Considering nutrient passage through the barrier, the microporous CSNMs exerted the same barrier effect but drastically increased the mass transfer coefficients of bovine serum albumin compared with the commercial anti-adhesive membrane SurgiWrap®. Cell attachment/focal adhesion formation of fibroblasts revealed effective reduction of initial cell attachment on the CSNM surface with minimum cytotoxicity (except HI50/PPA). The anti-bacterial effect against both Gram-negative and Gram-positive bacteria was verified to be due to the Ag NPs in the membranes. In vivo studies using H/PPA and HI30/PPA CSNMs and SurgiWrap® in a rabbit flexor tendon rupture model demonstrated the improved efficacy of HI30/PPA CSNMs in reducing inflammation and tendon adhesion formation based on gross observation, histological analysis and functional assays. We conclude that HI30/PPA CSNMs can act as a multifunctional barrier membrane to prevent peritendinous adhesion after tendon surgery. STATEMENT OF SIGNIFICANCE: A multi-functional anti-adhesion barrier membrane that could reduce fibroblasts attachment and penetration while simultaneously prevent post-surgical infection and inflammation is urgently needed. To this end, we prepared electrospun core-shell hyaluronic acid + ibuprofen/polyethylene glycol + polycaprolactone + Ag nanoparticles nanofibrous membranes by co-axial electrospinning as an ideal anti-adhesive membrane. The core-shell structure could meet the need of a desirable anti-adhesion barrier through release of ibuprofen and Ag nanoparticles to reduce infection and inflammation while hyaluronic acid can reduce fibroblasts adhesion. The superior performance of this multi-functional core-shell nanofibrous membrane in preventing peritendinous adhesion and post-surgical inflammation was demonstrated in a rabbit flexor tendon rupture model.

Magnetic Graphene Oxide for Dual Targeted Delivery of Doxorubicin and Photothermal Therapy.

To develop a pH-sensitive dual targeting magnetic nanocarrier for chemo-phototherapy in cancer treatment, we prepared magnetic graphene oxide (MGO) by depositing Fe3O4 magnetic nanoparticles on graphene oxide (GO) through chemical co-precipitation. MGO was modified with polyethylene glycol (PEG) and cetuximab (CET, an epidermal growth factor receptor (EGFR) monoclonal antibody) to obtain MGO-PEG-CET. Since EGFR was highly expressed on the tumor cell surface, MGO-PEG-CET was used for dual targeted delivery an anticancer drug doxorubicin (DOX). The physico-chemical properties of MGO-PEG-CET were fully characterized by dynamic light scattering, transmission electron microscopy, X-ray diffraction, Fourier transform Infrared spectroscopy, thermogravimetric analysis, and superconducting quantum interference device. Drug loading experiments revealed that DOX adsorption followed the Langmuir isotherm with a maximal drug loading capacity of 6.35 mg/mg, while DOX release was pH-dependent with more DOX released at pH 5.5 than pH 7.4. Using quantum-dots labeled nanocarriers and confocal microscopy, intracellular uptakes of MGO-PEG-CET by high EGFR-expressing CT-26 murine colorectal cells was confirmed to be more efficient than MGO. This cellular uptake could be inhibited by pre-incubation with CET, which confirmed the receptor-mediated endocytosis of MGO-PEG-CET. Magnetic targeted killing of CT-26 was demonstrated in vitro through magnetic guidance of MGO-PEG-CET/DOX, while the photothermal effect could be confirmed in vivo and in vitro after exposure of MGO-PEG-CET to near-infrared (NIR) laser light. In addition, the biocompatibility tests indicated MGO-PEG-CET showed no cytotoxicity toward fibroblasts and elicited minimum hemolysis. In vitro cytotoxicity tests showed the half maximal inhibitory concentration (IC50) value of MGO-PEG-CET/DOX toward CT-26 cells was 1.48 µg/mL, which was lower than that of MGO-PEG/DOX (2.64 µg/mL). The IC50 value could be further reduced to 1.17 µg/mL after combining with photothermal therapy by NIR laser light exposure. Using subcutaneously implanted CT-26 cells in BALB/c mice, in vivo anti-tumor studies indicated the relative tumor volumes at day 14 were 12.1 for control (normal saline), 10.1 for DOX, 9.5 for MGO-PEG-CET/DOX, 5.8 for MGO-PEG-CET/DOX + magnet, and 0.42 for MGO-PEG-CET/DOX + magnet + laser. Therefore, the dual targeting MGO-PEG-CET/DOX could be suggested as an effective drug delivery system for anticancer therapy, which showed a 29-fold increase in therapeutic efficacy compared with control by combining chemotherapy with photothermal therapy.

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.

Self-Targeting, Immune Transparent Plasma Protein Coated Nanocomplex for Noninvasive Photothermal Anticancer Therapy.

Cancer cells exhibit specific physiological differences compared to normal cells. Most surface membranes of cancer cells are characterized by high expression of given protein receptors, such as albumin, transferrin, and growth factors that are also present in the plasma of patients themselves, but are lacking on the surface of normal cells. These distinct features between cancer and normal cells can serve as a niche for developing specific treatment strategies. Near-infrared (NIR)-light-triggered therapy platforms are an interesting novel avenue for use in clinical nanomedicine. As a photothermal agent, conducting polymer nanoparticles, such as polypyrrole (PPy), of great NIR light photothermal effects and good biocompatibility, show promising applications in cancer treatments through the hyperthermia mechanism. Autologous plasma proteins coated PPy nanoparticles for hyperthermia therapy as a novel core technology platform to treat cancers through secreted protein acid and rich in cysteine targeting are developed here. This approach can provide unique features of specific targeting toward cancer cell surface markers and immune transparency to avoid recognition and attack by defense cells and achieve prolonged circulation half-life. This technology platform unveils new clinical options for treatment of cancer patients, supporting the emergence of innovative clinical products.

Dual crosslinked hyaluronic acid nanofibrous membranes for prolonged prevention of post-surgical peritoneal adhesion.

Peritoneal adhesions are a common complication following pelvic and abdominal surgery, which could be reduced using anti-adhesion barriers. However, the shortcomings of existing hyaluronic acid (HA)-based anti-adhesion film, such as the rapid degradation rate and poor operation ability, cannot be ignored. The aim of this study is to develop a dual crosslinked electrospun HA nanofibrous membrane (NFM) with improved properties and prolonged degradation time as an anti-adhesion barrier film for preventing post-surgical peritoneal adhesions. The HA NFMs were ionically crosslinked with FeCl3 (HAF NFMs) and further covalently crosslinked with 1,4-butanediol diglycidyl ether (BDDE) to produce HA-Fe-BDDE (HAFB) NFMs. The membranes were characterized for their physico-chemical properties to confirm the crosslinking mechanisms through scanning electron microscopy with energy dispersive X-ray analysis, infrared spectroscopy, thermal gravimetric analysis, X-ray photoelectron spectroscopy and mechanical properties. In vitro degradation studies confirmed that only the dual crosslinked HAFB membranes demonstrated an appropriate degradation rate, which could maintain the morphology and porous structure after immersion in phosphate buffered saline and cell culture medium for up to 7 days. In vitro studies indicated that HAFB NFMs showed excellent effectiveness in preventing fibroblast penetration and attachment while preserving high biocompatibility without influencing cell proliferation. The peritoneal anti-adhesion efficacy of HAFB NFMs was tested by implanting them in the abdominal cavities of Sprague Dawley (SD) rats. The membrane could exert its barrier effect for a week to significantly reduce the development of peritoneal adhesion compared with HAF NFMs and Seprafilm®. The results show the potential of the novel dual crosslinked HA-based NFMs in prolonged adhesion prevention.

Incorporation of chitosan in biomimetic gelatin/chondroitin-6-sulfate/hyaluronan cryogel for cartilage tissue engineering.

We prepare an elastic macroporous gelatin/chondoitin-6-sulfate/hyaluronan (GCH) cryogel scaffold mimic the composition of cartilage extracellular matrix for cartilage tissue engineering. By incorporating chitosan in the cryogel to replace 20% gelatin, a GCH-chitosan cryogel was also synthesized and compared with GCH cryogel for scaffold mechanical properties and chondrocytes response. The GCH-chitosan cryogel has larger pores, higher ultimate strain (stress) and elastic modulus, and lower stress relaxation percentage than the GCH cryogel. Both cryogels show a highly elastic property with a loss tangent around 0.1, but chitosan incorporation increases the storage modulus (elasticity). Chondrocytes proliferate and redifferentiate in cryogels; chitosan diminishes cell proliferation but up-regulates glycosaminoglycans (GAGs) and type II collagen (COL II) secretion. Implantation of a chondrocytes/GCH-chitosan cryogel construct in a full-thickness articular cartilage defect regenerates cartilage with positive stainings for GAGs and COL II and an elastic modulus similar to the native cartilage.