Chemically heparinized PEEK via a green method to immobilize bone morphogenetic protein-2 (BMP-2) for enhanced osteogenic activity.

Osseointegration remains one of the major challenges in the success of bone-related implants. Recently, polyetheretherketone (PEEK) has emerged as an alternative material in orthopedic and dental applications due to its bone-mimicking mechanical properties. However, its bioinertness resulting in poor osseointegration has limited its potential application. So, the surface modification of PEEK with bone morphogenetic protein-2 (BMP-2) can be a potential approach for improving osseointegration. In this study, we proposed the chemical modification of heparin onto PEEK through an environmentally benign method to exploit the BMP-2 binding affinity of heparin. The heparin was successfully functionalized on the PEEK surface via a combination of ozone and UV treatment without using organic solvents or chemicals. Furthermore, BMP-2 was efficiently immobilized on PEEK and exhibited a sustained release of BMP-2 compared to the pristine PEEK with enhancement of bioactivity in terms of proliferation as well as osteogenic differentiation of MG-63. The significant synergistic effect of BMP-2 and heparin grafting on osteogenic differentiation of MG-63 was observed. Overall, we demonstrated a relatively safe method where no harsh chemical reagent or organic solvent was involved in the process of heparin grafting onto PEEK. The BMP-2 loaded, heparin-grafted PEEK could serve as a potential platform for osseointegration improvement of PEEK-based bone implants.

Composite system of PLCL scaffold and heparin-based hydrogel for regeneration of partial-thickness cartilage defects.

Delivering isolated chondrocytes with matrix is a promising approach to promote the cartilage repair. The present study attempted to combine the advantages of porous scaffold and hydrogel in delivering chondrocytes to partial-thickness cartilage defects. An electrospun, gelatin-incorporated PLCL scaffold mechanically similar to natural cartilage was fabricated, and chondrocytes were seeded using an injectable heparin-based hydrogel for efficient cell seeding. The scaffold/hydrogel composite showed more enhanced expression of chondrogenic genes and production of GAGs than those prepared without hydrogel. In addition, significant cartilage formation showing good integration with surrounding, similar to natural cartilage, was observed by scaffold/hydrogel composite system in partial-thickness defects of rabbit knees while no regeneration was observed in control defects. Although no exogenous chondrogenic factors were added, it was evident that the scaffold/hydrogel composite system was highly effective and better than the scaffold alone system without hydrogel for cartilage regeneration both in vitro and in vivo.

Optimizing hemocompatibility of surfactant-coated silver nanoparticles in human erythrocytes.

Several recent biological science studies have been focused on nanotechnology and nanomaterials due to their potential use in biomedicine. Drug delivery systems are an example of biomedical applications utilizing nanoparticles. Silver nanoparticles (AgNPs) can be used for these drug delivery systems. However, the effects of cytotoxicity caused by AgNPs are not fully understood. Determining the optimal characteristics to facilitate the biocompatibility of AgNPs is an important subject for application. In the present study, human erythrocytes were used as an in vitro model to examine the size, dose, and coating surfactant-dependent cytotoxicity of AgNPs. Our results demonstrated that polyvinylpyrrolidone (PVP) was a more suitable surfactant than polyethylene glycol (PEG) for AgNPs capping. In addition, we determined the appropriate particular size and dosage of AgNPs to reduce human erythrocytes hemolysis. Membrane damages including hemolysis, potassium efflux, protein leakage, and alterations in cell shape and membrane fragility were minimized with 100-nm AgNP particles. This study provides novel insights into AgNPs cytotoxicity and a basis for utilizing AgNPs for diagnostic and therapeutic applications.

The effect of gelatin incorporation into electrospun poly(L-lactide-co-epsilon-caprolactone) fibers on mechanical properties and cytocompatibility.

Very elastic poly(L-lactide-co-epsilon-caprolactone) (PLCL) (50:50) copolymer blended with gelatin was electrospun into microfibers from a hexafluoroisopropanol solution. PLCL fiber sheet exhibited the unique soft and flexible behavior while gelatin fiber was hard and brittle. As the gelatin content of PLCL/gelatin fibers increased, Young's modulus was increased, but the elongation was decreased compared to those of PLCL. However, fibers containing 10-30 wt% gelatin demonstrated an enhanced tensile strength with still high elongation to be beneficial for tissue engineering scaffolds. The cytocompatibility of electrospun fiber sheets was evaluated by fibroblasts (NIH-3T3) cell culture. The initial cell adhesion on various fibers after 5h was somewhat similar, but in the order of PLCL>PLCL70/gelatin30 approximately PLCL50/gelatin50>PLCL90/gelatin10 approximately gelatin>PLCL30/gelatin70. However, the cell proliferation exhibited a completely different and strong dependence on the fiber composition: a very high proliferation rate on PLCL90/gelatin10, followed by PLCL>gelatin>PLCL70/gelatin30. Such an enhanced effect of gelatin, especially at 10 wt% content, on strength and cytocompatibility of PLCL/gelatin fibers would be very preferable for tissue engineering scaffolds.

Removal of cetyltrimethylammonium bromide to enhance the biocompatibility of Au nanorods synthesized by a modified seed mediated growth process.

Previously, we reported the synthesis of stable gold nanorods with higher aspect ratio (approximately 15-20) and more enhanced uniformity by a modified seed mediated growth approach using a binary surfactant system consisting of CTAB and Pluronic F-127. For the in vivo application of gold nanorods, the removal of CTABs that are strongly bound on prepared Au nanorods is necessary due to their cytotoxicity. Use of heat or acid at various conditions was performed to achieve the complete removal of CTAB from the synthesized Au nanorods while maintaining their stability. Here, we report the appropriate conditions for both treatments that can remove CTAB efficiently without hampering the stability of Au nanorods. After the removal of CTAB, Pluronic F-127 was added additionally for the stabilization and further potential functionalization of Au nanorods to make useful for various applications. VIS-NIR absorption spectroscopy, SEM, TEM, and FTIR were used for characterization.

Mechanical loading-dependence of mRNA expressions of extracellular matrices of chondrocytes inoculated into elastomeric microporous poly(L-lactide-co-epsilon-caprolactone) scaffold.

The temporal response of young rabbit chondrocyte metabolism (including biosynthesis of extracellular matrix macromolecules such as collagen and aggrecan, both of which are essential components of normal cartilage tissue, and their messenger ribonucleic acid (mRNA) expression) in microporous elastomeric scaffolds made of poly(L-lactide-co-epsilon-caprolactone) subjected to different compressive regimes (loading frequency, loading duration per cycle, loading period, and continuous or intermittent compression) were studied over a 6-day culture period at 10% of compressive strain. A continuous dynamic compression improved the production of sulfated glycosaminoglycan (S-GAG), most of which was released into the culture medium upon loading. High mRNA expression of type II collagen was exhibited at a frequency of 0.1 Hz. Little frequency dependency was observed for aggrecan. An intermittent loading (24-h cycle of loading and unloading) or short loading and unloading duration per cycle-compression regime maintained high levels of mRNA expression. This strongly suggests that well-controlled mechanical conditioning regimes may control the gene expression of key metabolic substances relevant to functional cartilage tissue while the degree of release of these substances into the culture medium is minimized.

New technique of seeding chondrocytes into microporous poly(L-lactide-co-epsilon-caprolactone) sponge by cyclic compression force-induced suction.

The initial requirement for a functional engineered cartilage tissue is the effective and reproducible seeding of chondrocytes into the interior of microporous scaffolds. High seeding efficiency, high cell viability, uniform cell distribution, and short operation time are also essential. We devised a new technique of seeding rabbit chondrocytes into microporous poly(L-lactide-co-epsilon-caprolactone) (PLCL) (porosity, 71- 80%; wall thickness, 2 and 6 mm) sponges under compression force-induced suction using a custom designed loading apparatus. Cell distribution and cell viability were determined using confocal laser scanning microscopy with fluorescent dye-staining techniques. Factors that affect the quality of a cell seeded construct were studied, namely, the porosity and thickness of sponges and suction cycles. Under 1 cycle of suction, an increase in porosity promoted cell seeding efficiency (CSE; defined as the percentage of the number of cells in the sponges relative to the initial number of cells seeded), cell viability (at 1 day post seeding), and a relatively uniform cell distribution, whereas thick sponges exhibited an inhomogeneous cell distribution irrespective of incubation time. Multiple cycles of suction of 5 and 10 at 0.1 Hz significantly improved the CSE, whereas high cell viability was maintained and even spatial cell distribution was achieved in 1 week. This study revealed that our newly developed cell seeding technique with multiple cycles of suction is a promising approach to inoculating cells into microporous sponges with high CSE, high cell viability, and homogeneous cell distribution.

Mechano-active scaffold design based on microporous poly(L-lactide-co-epsilon-caprolactone) for articular cartilage tissue engineering: dependence of porosity on compression force-applied mechanical behaviors.

An essential component of functional articular cartilage tissue engineering is a mechano-active scaffold, which responds to applied compression stress and causes little permanent deformation. As the first paper of a series on mechano-active scaffold-based cartilage tissue engineering, this study focused on mechanical responses to various modes of loading of compression forces and subsequent selection of mechano-active scaffolds from the biomechanical viewpoint. Scaffolds made of elastomeric microporous poly(L-lactide-co-epsilon-caprolactone) (PLCL) with open-cell structured pores (300 approximately 500 microm) and with different porosities ranging from 71 to 86% were used. The PLCL sponges and rabbit articular cartilage tissue were subjected to compression/unloading tests (0.1 and 0.005 Hz) at 5 kPa, and stress relaxation tests at 10, 30, and 50% strain. The measurements of the maximum strain under loading and residual strain under unloading for compression tests and the maximum stress and equilibrium stress in the stress relaxation test showed that the lower the porosity, the closer the mechanical properties are to those of native cartilage tissue. Among the PLCL sponges, the sponge with 71% porosity appears to be a suitable cartilage scaffold.

Complement activation by sulfonated poly(ethylene glycol)-acrylate copolymers through alternative pathway.

Previously, novel poly(ethylene glycol) (PEG) and sulfonated PEG acrylate (PEG-SO(3)A/OA) copolymers were prepared as coating and/or blending materials for biomedical applications. Surfaces modified with copolymers exhibited increased anti-coagulation properties and decreased plasma adsorption level due to increased hydrophilic properties and reorientation characteristics of PEG/PEG-SO(3)A chains in water phase. As continuation study, anti-complement effects of PEG-SO(3)/OA copolymers were investigated in vitro, and compared with those of low-density polyethylene (LDPE) and PEG/OA. C3 activation by PEG-SO(3)/OA samples was lower than that by PEG/OA samples, which was attributed to decreased surface nucleophile level of samples. PEG-SO(3)/OA samples increased inhibition of Bb production, resulting in decreased C5 activation. Owing to reduced activations of C3 and C5, PEG-SO(3)/OA samples markedly decreased SC5b-9 levels in plasma.

A poly(lactic acid)/calcium metaphosphate composite for bone tissue engineering.

A new method to prepare PLA/CMP (poly-L-lactide/calcium metaphosphate) composite scaffolds was developed for effective bone tissue engineering. This novel sintering method is composed of pressing the mixture of PLA, CMP, and salt particles at 150 MPa for 3 min followed by heat treatment at 210 degrees C for 30 min. The scaffolds had a homogeneously interconnected porous structure without a skin layer, and they exhibited a narrower pore size distribution and higher mechanical strength in comparison with scaffolds made by a solvent casting method. The scaffolds were seeded by osteoblasts and cultured in vitro or implanted into nude mice subcutaneously for up to 5 weeks. The number of cells attached to and proliferated on the scaffolds at both in vitro and in vivo was in the order of; PLA by novel sintering < PLA/CMP by solvent casting < PLA/CMP by novel sintering. In addition, the alkaline phosphatase activity of and calcium deposition in the scaffolds explanted from mice were enhanced significantly for the scaffolds by novel sintering compared to them by solvent casting. The in vitro results agreed well with the in vivo data. Such a superior characteristic of the novel sintering method should have resulted from the fact that the CMP particles could contact directly with cells/tissues to stimulate the cell proliferation and osteogenic differentiation, while the CMP particles would be coated by polymers and hindered to interact with cells/tissues in the case of a solvent casting method. As the novel sintering method does not use any solvents it offers another advantage to avoid problems associated with solvent residue.

Mechano-active tissue engineering of vascular smooth muscle using pulsatile perfusion bioreactors and elastic PLCL scaffolds.

Blood vessels are subjected in vivo to mechanical forces in a form of radial distention, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow. Vascular smooth muscle (VSM) tissues engineered in vitro with a conventional tissue engineering technique may not be functional, because vascular smooth muscle cells (VSMCs) cultured in vitro typically revert from a contractile phenotype to a synthetic phenotype. In this study, we hypothesized that pulsatile strain and shear stress stimulate VSM tissue development and induce VSMCs to retain the differentiated phenotype in VSM engineering in vitro. To test the hypothesis, rabbit aortic smooth muscle cells (SMCs) were seeded onto rubber-like elastic, three-dimensional PLCL [poly(lactide-co-caprolactone), 50:50] scaffolds and subjected to pulsatile strain and shear stress by culturing them in pulsatile perfusion bioreactors for up to 8 weeks. As control experiments, VSMCs were cultured on PLCL scaffolds statically. The pulsatile strain and shear stress enhanced the VSMCs proliferation and collagen production. In addition, a significant cell alignment in a direction radial to the distending direction was observed in VSM tissues exposed to radial distention, which is similar to that of native VSM tissues in vivo, whereas VSMs in VSM tissues engineered in the static condition randomly aligned. Importantly, the expression of SM alpha-actin, a differentiated phenotype of SMCs, was upregulated by 2.5-fold in VSM tissues engineered under the mechano-active condition, compared to VSM tissues engineered in the static condition. This study demonstrates that tissue engineering of VSM tissues in vitro by using pulsatile perfusion bioreactors and elastic PLCL scaffolds leads to the enhancement of tissue development and the retention of differentiated cell phenotype.

Laparoscopic transabdominal preperitoneal hernioplasty of bilateral obturator hernia.

Obturator hernia is relatively rare and tends to occur in elderly, emaciated women with chronic diseases. Clinical presentations are frequently delayed and so preoperative diagnosis is difficult. Treatment is always surgical. We present a case of a 75-year-old woman with bilateral obturator hernia diagnosed by the physical examination and abdominopelvic computed tomography (CT) scan; she had no signs of bowel strangulation. We used a laparoscopic approach for correction. A transabdominal preperitoneal hernioplasty was done using a prosthetic patch of polypropylene mesh. The patient recovered very well after surgery. We suggest that a laparoscopic approach may be used as treatment, when a nonstrangulated obturator hernia is diagnosed preoperatively.

Effects of organic matrix proteins on the interfacial structure at the bone-biocompatible nacre interface in vitro.

The biocompatibility and potential osteoinductivity of nacre have favored its use as a bone-grafting material. The present study is to investigate the interfacial structure at the bone-nacre interface resulting from organic matrix proteins, which emphasizes the mechanism of bone-bonding ability and biocompatibility of the shell tissues such as nacre and biogenic calcite. To understand the interfacial reaction, the zeta potential measurements, provide for a unique method to quantify the actual state of the interface in situ, were used for synthetic and biogenic calcium carbonate suspensions with respect to pH and the organic matrix as an additive. The zeta potentials and surface charge density show that the organic matrix proteins are main charge regulators, resulting in the stabilized tissue properties as compared with synthetic crystals. Also, in forming calcium carbonate crystals with the additives, the conformation of organic matrix has an important role in the understanding of the newly formed interfacial structure. The result provides the primary role of the organic matrix proteins in controlling the formation of interfacial structure and biocompatibility with bone as well as the stability of biogenic tissues. And it gives a new insight into the usefulness of zeta potential measurement to describe the in vivo interaction between the bone and implants.

Enhanced blood compatibility of polymers grafted by sulfonated PEO via a negative cilia concept.

In our laboratory sulfonated PEO (PEO-SO(3)) was designed as a "negative cilia model" to investigate a synergistic effect of PEO and negatively charged SO(3) groups. PEO-SO(3) itself exhibited a heparin-like anticoagulant activity of 14% of free heparin. Polyurethane grafted with PEO-SO(3) (PU-PEO-SO(3)) increased the albumin adsorption to a great extent but suppressed other proteins, while PU-PEO decreased the adsorption of all the proteins. The platelet adhesion was decreased on PU-PEO but least on PU-PEO-SO(3) to demonstrate an additional effect of SO(3) groups. The enhanced blood compatibility of PU-PEO-SO(3) in the ex vivo rabbit and in vivo canine implanting tests was confirmed. Furthermore, PU-PEO-SO(3) exhibited an improved biostability and suppressed calcification in addition to the enhanced antithrombogenicity. The in vivo antithrombogenicity and biostability were improved in the order of PUPU-PEO>PU-PEO-SO(3) in spite of the possible attraction between negative SO(3) groups and positive calcium ions. The bioprosthetic tissue (BT) was grafted with H(2)N-PEO-SO(3) via glutaraldehyde (GA) residues after conventional GA fixation. BT-PEO-SO(3) also displayed the decreased calcification by in vivo animal models. The application of PEO-SO(3) was extended by designing amphiphilic copolymers containing PEO-SO(3) moiety and hydrophobic long alkyl groups as anchors. The superior effect of PEO-SO(3) groups on thromboresistance compared to PEO was confirmed also in the case of copolymers coated or blended with other polymers and the systems coupled by UV irradiation, photoreaction or gold/sulfur or silane coupling technology, and therefore it might be very useful for the medical devices.

A novel chemical modification of bioprosthetic tissues using L-arginine.

A novel chemical modification of biological tissues was developed by the direct coupling of bioactive molecule, L-arginine to bovine pericardium (BP). The modification involves pretreatment of BP using GA and followed by grafting arginine to BP by the reaction of residual aldehyde and amine group of L-arginine. BP was modified by direct coupling of bioactive molecules and the effect of L-arginine coupling on calcification and biocompatibility was evaluated in vitro and in vivo. Modified BPs were characterized by measuring shrinkage temperature, mechanical properties, digestion resistance to collagenase enzyme, in vitro plasma protein adsorption and platelet adhesion, and in vivo calcification. Thermal and mechanical properties showed that the durability of arginine treated tissue increased as compared with fresh tissue and GA treated tissue. Resistance to collagenase digestion revealed that modified tissues have greater resistance to enzyme digestion than did fresh tissue and GA treated tissue. Lower protein adsorption and platelet adhesion were observed on modified tissue than non-modified tissue. In vivo calcification study demonstrated much less calcium deposition on arginine treated BP than GA treated one. Obtained results attest to the usefulness of L-arginine treated BP for cardiovascular bioprostheses.

Fibroblast adhesion and proliferation on poly(ethylene glycol) hydrogels crosslinked by hydrolyzable polyrotaxane.

Fibroblast culture was performed to evaluate cell adhesion and proliferation on poly(ethylene glycol) (PEG) hydrogels crosslinked by a hydrolyzable polyrotaxane. The polyrotaxane consisting of alpha-cyclodextrins (alpha-CDs) and PEG terminated by benzyloxycarbonyl (Z)-L-phenylalanine (L-Phe) via ester linkage was used as a multi-functional crosslinker in the PEG hydrogels. From the results of contact angle and small angle light scattering measurements, it was suggested that the surface and bulk structure of the PEG hydrogels were heterogeneous. Fibroblast adhesion and proliferation on the hydrogels was observed. The number of fibroblast adhesion on the hydrogels crosslinked by the polyrotaxane was proportional to contact angle values and correlation length, and was significantly higher than those crosslinked by alpha-CDs in spite of similar contact angle and correlation length. These findings suggest that the cells recognize the surface heterogeneity due to the polyrotaxane structure, and the number of cell adhesion and proliferation is controllable by the polyrotaxane content in feed.

In vivo conjunctival reconstruction using modified PLGA grafts for decreased scar formation and contraction.

The in vivo reconstruction of conjunctiva was investigated by using modified poly(lactide-co-glycolide) (PLGA) 50/50 scaffolds. The porous PLGA matrices were prepared by a solvent-casting particulate-leaching method with NaCl, then modified with collagen, hyaluronic acid (HA) or/and human amniotic membrane (AM) component. The growth of corneal epithelial cells and human stromal fibroblasts on the scaffolds was investigated in vitro. All the modified PLGA scaffolds demonstrated enhanced cell adhesion and proliferation as compared to PLGA untreated, and the number of cells proliferated after 1 week was increased in the order of PLGA

In vivo biocompatibilty and degradation behavior of elastic poly(L-lactide-co-epsilon-caprolactone) scaffolds.

Tubular scaffolds were fabricated from very elastic poly(L-lactide-co-epsilon-caprolactone) (PLCL, 50:50). The scaffolds were seeded with smooth muscle cells (SMCs) and implanted in nude mice to investigate the tissue compatibility and in vivo degradation behavior. Histological examination of all the implants with haematoxylin and eosin staining, masson trichrome staining, SM alpha-actin antibody, and CM-DiI labeling confirmed that the regular morphology and biofunction of the SMCs seeded and the expression of the vascular smooth muscle matrices in PLCL scaffolds. The implanted PLCL scaffolds displayed a slow degradation on time, where caprolactone units were faster degraded than lactide did. This could be explained by the fact that amorphous regions composed of mainly CL moieties degraded earlier than hard domains where most of the LA units were located. From these results, the scaffolds applied in this study were found to exhibit excellent tissue compatibility to SMCs and might be very useful for vascular tissue engineering.

In vitro biocompatibility assessment of sulfonated polyrotaxane-immobilized polyurethane surfaces.

Sulfonated polyrotaxanes (PRx-SO(3)'s), in which sulfonated alpha-cyclodextrins (alpha-CDs) were threaded onto the poly(ethylene glycol) (PEG) segments in a PEG-b-poly(propylene glycol) (PPG)-b-PEG triblock copolymer (Pluronic) capped with benzyloxycarbonyl (Z)-L-phenylalanine (Z-L-Phe), were prepared as a novel surface-modifying biomaterial. Surface modification of the polyurethane (PU) was carried out by blending the PRx-SO(3)'s with a PU solution, followed by solution casting. The incorporated PRx-SO(3)'s led to the enhanced hydrophilicity by changing the surface properties of the PU matrix. Modified PUs showed the stable entrapment of the PRx-SO(3)'s with little extraction into water and enhanced mechanical properties after exposure to water compared to the PU control. The incorporated PRx-SO(3)'s repelled the proteins and kept them from closely approaching the surface areas, prevented platelet activation by thrombin, and effectively repelled bacteria. These results suggest that both the supramolecular structure of the polyrotaxanes and exposure of the sulfonated groups onto the surfaces contribute to these phenomena. Thus, surface modification with PRx-SO(3)'s is suggested to be useful for the fabrication of biocompatible medical devices.

Elastic biodegradable poly(glycolide-co-caprolactone) scaffold for tissue engineering.

Cyclic mechanical strain has been demonstrated to enhance the development and function of engineered smooth muscle (SM) tissues, and it would be necessary for the development of the elastic scaffolds if one wishes to engineer SM tissues under cyclic mechanical loading. This study reports on the development of an elastic scaffold fabricated from a biodegradable polymer. Biodegradable poly(glycolide-co-caprolactone) (PGCL) copolymer was synthesized from glycolide and epsilon-caprolactone in the presence of stannous octoate as catalyst. The copolymer was characterized by (1)H-NMR, gel permeation chromatography and differential scanning calorimetry. Scaffolds for tissue engineering applications were fabricated from PGCL copolymer using the solvent-casting and particle-leaching technique. The PGCL scaffolds produced in this fashion had open pore structures (average pore size = 250 microm) without the usual nonporous skin layer on external surfaces. Mechanical testing revealed that PGCL scaffolds were far more elastic than poly(lactic-co-glycolic acid) (PLGA) scaffolds fabricated using the same method. Tensile mechanical tests indicated that PGCL scaffolds could withstand an extension of 250% without cracking, which was much higher than withstood by PLGA scaffolds (10-15%). In addition, PGCL scaffolds achieved recoveries exceeding 96% at applied extensions of up to 230%, whereas PLGA scaffolds failed (cracked) at an applied strain of 20%. Dynamic mechanical tests showed that the permanent deformation of the PGCL scaffolds in a dry condition produced was less than 4% of the applied strain, when an elongation of 20% at a frequency of 1 Hz (1 cycle per second) was applied for 6 days. Moreover, PGCL scaffolds in a buffer solution also had permanent deformations less than 5% of the applied strain when an elongation of 10% at a frequency of 1 Hz was applied for 2 days. The usefulness of the PGCL scaffolds was demonstrated by engineering SM tissues in vivo. This study shows that the elastic PGCL scaffolds produced in this study could be used to engineer SM-containing tissues (e.g. blood vessels and bladders) in mechanically dynamic environments.