The application of electrospinning used in meniscus tissue engineering

Meniscus is a fibrocartilaginous organ to redistribute stress and enhance the stability of knee joint. Meniscus injury is common and still a formidable challenge to orthopedic surgeons. Surgical techniques and allograft transplantation were primary approaches to meniscus repair, but with intrinsic limitations in clinical practice. Tissue engineering is the most promising method to repair meniscus at present. Electrospinning is a method to fabricate fibers in small scale. With different materials and parameters, electrospinning materials could have different mechanical properties, porosity, and orientation, which could mimic architectural features and mechanical properties of native meniscus. Therefore, electrospinning materials could be used in meniscus regeneration and curing. This review gave a brief introduction of meniscus structure, injury, treatment and the application of electrospinning fibers in meniscus tissue engineering and curing. Besides that, we summarized materials commonly used in electrospinning to fabricate meniscus scaffolds, and discussed the form of electrospinning fibers used such as scaffold, substitute and patch. Finally, the function of electrospinning fibers, for example, carrying drugs, providing mechanical properties were described. The potential applications of electrospinning fibers in meniscus therapy were proposed.     

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.     

Heparinized PCL/keratin mats for vascular tissue engineering scaffold with potential of catalytic nitric oxide generation

Abstract

Heparins are capable of improving blood compatibility, enhancing HUVEC viability, while inhibiting HUASMC proliferation. Combination of biodegradable poly(ε-caprolactone) (PCL) with keratin and heparins would provide an anticoagulant and endothelialization supporting environment for vascular tissue engineering. Herein, PCL and keratin were first coelectrospun and then covalently conjugated with heparins. The resulting mats were surface-characterized by ATR-FTIR, SEM, WCA, and XPS. Cell viability data showed that the heparinized PCL/keratin mats could motivate the adhesion and growth of HUVEC, while inhibit HUASMC proliferation. In addition, these mats could prolong blood clotting time and reduce platelet adhesion as well as no erythrolysis. Interestingly, these mats could catalyze the NO donor in blood to release NO, which could enhance endothelial cell growth, while decrease smooth muscle cell proliferation and platelet adhesion. In summary, the heparinized mats would be a good candidate as a scaffold for vascular tissue engineering. This study is novel in that we prepared a type of heparinized tissue scaffold that could catalyze the NO donor to release NO to regulate endothelialization without angiogenesis and thrombus formation.     

Mechanical and structural response of a hybrid hydrogel based on chitosan and poly(vinyl alcohol) cross-linked with epichlorohydrin for potential use in tissue engineering

The development and characterization of a hybrid hydrogel based on chitosan (CS) and poly(vinyl alcohol) (PVA) chemically cross-linked with epichlorohydrin (ECH) is presented. The mechanical response of these hydrogels was evaluated by uniaxial tensile tests; in addition, their structural properties such as average molecular weight between cross-link points (M_crl), mesh size (D_N), and volume fraction (v_s) were determined. This was done using the equivalent polymer network theory in combination with the obtained results from tensile and swelling tests. The films showed Young’s modulus values of 11?±?2?MPa and 9?±?1?MPa for none irradiated and ultraviolet (UV) irradiated hydrogels, respectively. The cell viability was assessed using Calcein AM and Ethidium homodimer-1 assay and environmental scanning electron microscopy. The 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan thiazolyl blue formazan (MTT Formazan assay) results did not show cytotoxic effects; this was in good agreement with nuclear magnetic resonance and fourier transform infrared spectroscopies; their results did not show traces of ECH. This indicated that after the crosslinking process, there was no free ECH; furthermore, any possibility of ECH release in the construct during cell culture was discarded. The CS-PVA-ECH hybrid hydrogel allowed cell growth and extracellular matrix formation and showed adequate mechanical, structural, and biological properties for potential use in tissue engineering applications.     

Nanofibrous poly(epsilon-caprolactone)/poly(vinyl alcohol)/chitosan hybrid scaffolds for bone tissue engineering using mesenchymal stem cells

In the present study, based on a biomimetic approach, novel 3D nanofibrous hybrid scaffolds consisting of poly(epsilon-caprolactone), poly(vinyl alcohol), and chitosan were developed via a multi-jet electrospinning method. The influence of chemical, physical, and structural properties of the scaffolds on the differentiation of mesenchymal stem cells into osteoblasts, and the proliferation of the differentiated cells were investigated. Osteogenically induced cultures revealed that cells were well-attached, penetrated into the construct and were uniformly distributed. The expression of early and late phenotypic markers of osteoblastic differentiation was upregulated in the constructs cultured in osteogenic medium.     

骨组织工程支架材料的研究进展

支架材料作为骨组织工程学的关键环节，成为近年来研究的热点。就骨组织工程学不同支架材料的优缺点、研究现状及前景进行综述，希望能为今后的研究提供参考。     

计算机辅助组织工程在组织支架仿生建模和设计中的应用

计算机辅助组织工程(CATE)可以帮助进行复杂组织支架的建模,设计和制造,使很多用于改善替代材料力学及生物学性能的新方法得以实施。CATE通过获取组织的生物学、生物力学及生物化学信息,进行界面的设计、模拟和组织的制作。本文将讲述CATE在骨组织工程支架仿生设计中的应用:介绍运用CATE进行仿生建模,解剖结构重建,组织支架设计,定量CT分析,有限元分析和支架的自由挤压沉积制作。     

组织工程韧带支架材料研究进展

韧带损伤发生几率很高,并且能够导致关节不稳以及其他组织损伤进一步恶化,但在临床上却没有韧带损伤修复的有效手段。组织工程为再生韧带组织提供了一个新的途径,而运用此方法选取合适的支架材料显得尤为重要。综述了组织工程韧带支架材料的主要研究成果,着重论述了在此领域潜力巨大的天然生物材料胶原和蚕丝。虽然目前的研究取得了较大的进步,但仅处在起步阶段,距离大规模的临床应用还很远。最后指出了此领域的最终目标以及未来研究趋势。     

Design of scaffolds for blood vessel tissue engineering using a multi-layering electrospinning technique

Aiming to develop a scaffold architecture mimicking morphological and mechanically that of a blood vessel, a sequential multi-layering electrospinning (ME) was performed on a rotating mandrel-type collector. A bi-layered tubular scaffold composed of a stiff and oriented PLA outside fibrous layer and a pliable and randomly oriented PCL fibrous inner layer (PLA/PCL) was fabricated. Control over the level of fibre orientation of the different layers was achieved through the rotation speed of the collector. The structural and mechanical properties of the scaffolds were examined using scanning electron microscopy (SEM) and tensile testing. To assess their capability to support cell attachment, proliferation and migration, 3T3 mouse fibroblasts and later human venous myofibroblasts (HVS) were cultured, expanded and seeded on the scaffolds. In both cases, the cell-polymer constructs were cultured under static conditions for up to 4 weeks. Environmental-scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), histological examination and biochemical assays for cell proliferation (DNA) and extracellular matrix production (collagen and glycosaminoglycans) were performed. The findings suggest the feasibility of ME to design scaffolds with a hierarchical organization through a layer-by-layer process and control over fibre orientation. The resulting scaffolds achieved the desirable levels of pliability (elastic up to 10% strain) and proved to be capable to promote cell growth and proliferation. The electrospun PLA/PCL bi-layered tube presents appropriate characteristics to be considered a candidate scaffold for blood vessel tissue engineering.     

Nanohydroxyapatite/poly(ester urethane) scaffold for bone tissue engineering

Biodegradable viscoelastic poly(ester urethane)-based scaffolds show great promise for tissue engineering. In this study, the preparation of hydroxyapatite nanoparticles (nHA)/poly(ester urethane) composite scaffolds using a salt-leaching-phase inverse process is reported. The dispersion of nHA microaggregates in the polymer matrix were imaged by microcomputed X-ray tomography, allowing a study of the effect of the nHA mass fraction and process parameters on the inorganic phase dispersion, and ultimately the optimization of the preparation method. How the composite scaffold’s geometry and mechanical properties change with the nHA mass fraction and the process parameters were assessed. Increasing the amount of nHA particles in the composite scaffold decreased the porosity, increased the wall thickness and consequently decreased the pore size. The Young’s modulus of the poly(ester urethane) scaffold was improved by 50% by addition of 10 wt.% nHA (from 0.95 ± 0.5 to 1.26 ± 0.4 MPa), while conserving poly(ester urethane) viscoelastic properties and without significant changes in the scaffold macrostructure. Moreover, the process permitted the inclusion of nHA particles not only in the poly(ester urethane) matrix, but also at the surface of the scaffold pores, as shown by scanning electron microscopy. nHA/poly(ester urethane) composite scaffolds have great potential as osteoconductive constructs for bone tissue engineering.     

A novel strontium-doped calcium polyphosphate/erythromycin/poly(vinyl alcohol) composite for bone tissue engineering

It is our goal to develop bactericidal bone scaffolds with osteointegration potential. In this study, poly(vinyl alcohol) (PVA) coating (7%) was applied to an erythromycin (EM)-impregnated strontium-doped calcium polyphosphate (SCPP) scaffold using a simple slurry dipping method. MicroCT analysis showed that PVA coating reduced the average pore size and the percentage of pore interconnectivity to some extent. Compressive strength tests confirmed that the PVA coating significantly increased material elasticity and slightly enhanced the scaffold mechanical strength. It was also confirmed that the PVA coatings allowed a sustained EM release that is controlled by diffusion through the intact PVA hydrogel layer, irrespective of the drug solubility. PVA coating did not inhibit the EM bioactivity when the scaffolds were immersed in simulated body fluid for up to 4 weeks. EM released from SCPP-EM-PVA composite scaffolds maintained its capability of bacterial growth (S. aureus) inhibition. PVA coating is biocompatible and nontoxic to MC3T3 preosteoblast cells. Furthermore, we found that SCPP-EM-PVA composite scaffolds and their eluants remarkably inhibited RANKL-induced osteoclastogenesis in a murine RAW 264.7 macrophage cell line. Thus, this unique multifunctional bioactive composite scaffold has the potential to provide controlled delivery of relevant drugs for bone tissue engineering.     

Physically interpenetrating networks in polyurethane ionomers/poly(vinyl alcohol) blends

Three series of blends of polyurethane ionomers (PUI) with poly(vinyl alcohol) (PVA) were prepared by mixing aqueous PUI emulsions with aqueous PVA solutions and then allowing them to dry. The characterization of the blends of various compositions was carried out using infrared spectroscopy, dynamic mechanical analysis, differential scanning calorimetry and tensile-elongation testing. It was found that poly(ether-urethane) cationomer (Cat-Et)/PVA-1 and poly(ether-urethane) anionomer (An-Et)/PVA-2 blends exhibit a positive synergistic effect with respect to the tensile strength and that the amorphous phase of PVA is at least partially compatible with both the disordered hard domains and the soft domains of the ionomers. For the former blends, this behaviour is due to the formation of hydrogen bonding with the glycolate anions and ether groups, and for the latter blends to the formation of hydrogen bonds with the ether groups. For poly(ester-urethane) cationomer (Cat-Es)/PVA-3 blends, there is no hydrogen bonding between these two components and no synergistic effect in the tensile strength after blending. Physically interpenetrating polymer networks (IPNs) are expected to occur in the former two blends, but not in the latter one.     

Preparation and characterization of bionic bone structure chitosan/hydroxyapatite scaffold for bone tissue engineering

Three-dimensional oriented chitosan (CS)/hydroxyapatite (HA) scaffolds were prepared via in situ precipitation method in this research. Scanning electron microscopy (SEM) images indicated that the scaffolds with acicular nano-HA had the spoke-like, multilayer and porous structure. The SEM of osteoblasts which were polygonal or spindle-shaped on the composite scaffolds after seven-day cell culture showed that the cells grew, adhered, and spread well. The results of X-ray powder diffractometer and Fourier transform infrared spectrometer showed that the mineral particles deposited in the scaffold had phase structure similar to natural bone and confirmed that particles were exactly HA. In vitro biocompatibility evaluation indicated the composite scaffolds showed a higher degree of proliferation of MC3T3-E1 cell compared with the pure CS scaffolds and the CS/HA10 scaffold was the highest one. The CS/HA scaffold also had a higher ratio of adhesion and alkaline phosphate activity value of osteoblasts compared with the pure CS scaffold, and the ratio increased with the increase of HA content. The ALP activity value of composite scaffolds was at least six times of the pure CS scaffolds. The results suggested that the composite scaffolds possessed good biocompatibility. The compressive strength of CS/HA15 increased by 33.07% compared with the pure CS scaffold. This novel porous scaffold with three-dimensional oriented structure might have a potential application in bone tissue engineering.     

A bilayer scaffold prepared from collagen and carboxymethyl cellulose for skin tissue engineering applications

Abstract

Treatment of chronic skin wound such as diabetic ulcers, burns, pressure wounds are challenging problems in the medical area. The aim of this study was to design a bilayer skin equivalent mimicking the natural one to be used as a tissue engineered skin graft for use in the treatments of problematic wounds, and also as a model to be used in research related to skin, such as determination of the efficacy of transdermal bioactive agents on skin cells and treatment of acute skin damages that require immediate response. In this study, the top two layers of the skin were mimicked by producing a multilayer construct combining two different porous polymeric scaffolds: as the dermis layer a sodium carboxymethyl cellulose (NaCMC) hydrogel on which fibroblasts were added, and as the epidermis layer collagen (Coll) or chondroitin sulfate-incorporated collagen (CollCS) on which keratinocytes were added. The bilayer construct was designed to allow cross-talk between the two cell populations in the subsequent layers and achieves paracrine signalling. It had interconnected porosity, high water content, appropriate stability and elastic moduli. Expression of vascular endothelial growth factor (VEGF), basic-fibroblast growth factor (bFGF) and Interleukin 8 (IL-8), and the production of collagen I, collagen III, laminin and transglutaminase supported the attachment and proliferation of cells on both layers of the construct. Attachment and proliferation of fibroblasts on NaCMC were lower compared to performance of keratinocyte on collagen where keratinocytes created a dense and a stratified layer similar to epidermis. The resulting constructs succesfully mimicked in vitro the natural skin tissue. They are promising as grafts for use in the treatment of deep wounds and also as models for the study of the efficacy of bioactive agents on the skin.     

Fabrication and characterization of hydroxypropyl guar-poly (vinyl alcohol)-nano hydroxyapatite composite hydrogels for bone tissue engineering

Abstract

Biocompatible bone implants composed of natural materials are highly desirable in orthopedic reconstruction procedures. In this study, novel and ecofriendly bionanocomposite hydrogels were synthesized using a blend of hydroxypropyl guar (HPG), poly vinyl alcohol (PVA), and nano-hydroxyapatite (n-HA) under freeze-thaw and mild reaction conditions. The hydrogel materials were characterized using various techniques. TGA studies indicate that both composites, HPG/PVA and HPG/PVA/n-HA, have higher thermal stability compared to HPG alone whereas HPG/PVA/n-HA shows higher stability compared to PVA alone. The HPG/PVA hydrogel shows porous morphology as revealed by the SEM, which is suitable for bone tissue regeneration. Additionally, the hydrogels were found to be transparent and flexible in nature. In vitro biomineralization study performed in simulated body fluid shows HPG/PVA/n-HA has an apatite like structure. The hydrogel materials were employed as extracellular matrices for biocompatibility studies. In vitro cell viability studies using mouse osteoblast MC3T3 cells were performed by MTT, Trypan blue exclusion, and ethidium bromide/acridine orange staining methods. The cell viability studies reveal that composite materials support cell growth and do not show any signs of cytotoxicity compared to pristine PVA. Osteoblastic activity was confirmed by an increased alkaline phosphatase enzyme activity in MC3T3 bone cells grown on composite hydrogel matrices.     

Surface modified poly(L-lactide-co-epsilon-caprolactone) microspheres as scaffold for tissue engineering

P-15 modified poly(L-lactide-co-epsilon-caprolactone) (PLCL) microspheres were investigated as scaffolds for tissue engineering applications. PLCL copolymer was synthesized by ring opening polymerization and was composed of a soft matrix of mainly epsilon-caprolactone moieties and hard domains containing more of L-lactide units thus exhibiting a rubber like elasticity responsible for providing mechanical strength to scaffolds. Microspheres were fabricated by solvent evaporation method and surface modified with P-15, a synthetic analogue of collagen. These were then evaluated for cell adhesion, ECM formation and cell proliferation. Anchorage dependent cell lines LLCPK-1 and L6 were seeded on PLCL microspheres (unmodified surface activated microspheres) and P-15-PLCL microspheres (P-15 modified microspheres). P-15 modified microspheres showed significantly higher cell adhesion and viability than unmodified microspheres. Scanning electron microscopy also revealed copious amount of extra-cellular matrix production by P-15. Initial results of cell culture experiment on two different cell lines suggested that the growth of LLCPK-1 in a 3D environment with P-15 modified microspheres is via spreading and flattening on the surface of scaffold followed by formation of a sheet-like structure while L6 grew in the form of multilayered structures with formation of interparticulate cellular bridges. The 3D moldable nature, combined with modification of surface chemistry with cell adhesion molecules such as P-15 to enhance proliferation of epithelial cells and myoblasts, recommends further investigation of P-15-PLCL microspheres for production of an ideal scaffold for soft tissue engineering.     

Application of an elastic biodegradable poly(L-lactide-co-epsilon-caprolactone) scaffold for cartilage tissue regeneration

In cartilage tissue regeneration, it is important that an implant inserted into a defect site can maintain its mechanical integrity and endure stress loads from the body, in addition to being biocompatible and able to induce tissue growth. These factors are crucial in the design of scaffolds for cartilage tissue engineering. We developed an elastic biodegradable scaffold from poly(L-lactideco-epsilon-caprolactone) (PLCL) for application in cartilage treatment. Biodegradable PLCL co-polymer was synthesized from L-lactide and epsilon-caprolactone in the presence of stannous octoate as a catalyst. A highly elastic PLCL scaffold was fabricated by a gel-pressing method with 80% porosity and 300-500 microm pore size. The tensile mechanical and recovery tests were performed in order to examine mechanical and elastic properties of the PLCL scaffold. They could be easily twisted and bent and exhibited almost complete (over 94%) recoverable extension up to breaking point. For examining cartilaginous tissue formation, rabbit chondrocytes were seeded on scaffolds. They were then cultured in vitro for 5 weeks or implanted in nude mice subcutaneously. From in vitro and in vivo tests, the accumulation of extracellular matrix on the constructs showed that chondrogenic differentiation was sustained onto PLCL scaffolds. Histological analysis showed that cells onto PLCL scaffolds formed mature and well-developed cartilaginous tissue, as evidenced by chondrocytes within lacunae. From these results, we are confident that elastic PLCL scaffolds exhibit biocompatibility and as such would provide an environment where cartilage tissue growth is enhanced and facilitated.     

Biocompatibility of subcutaneously implanted marine macromolecules cross-linked bio-composite scaffold for cartilage tissue engineering applications

There is an intense interest in developing innovative biomaterials which support the invasion and proliferation of living cells for potential applications in tissue engineering and regenerative medicine. Present study demonstrated the in vivo biocompatibility and toxicity of a macromolecules cross-linked biocomposite scaffold composed of hydroxyapatite, alginate, chitosan and fucoidan abbreviated as HACF. The in vivo biocompatibility and toxicity of HACF scaffold were tested by comparing them with those of a biocompatible surgical metal implant (SMI) in a subcutaneous rat model. Following the implantation, animals were sacrificed and the scaffolds were resected at 1st, 4th, and 8th weeks; the surrounding tissue along with the implant was removed to evaluate its biocompatibility. The effects of implanted biomaterial scaffolds on vital organ systems such as liver, kidney, etc., have been studied by hematology and serum biochemistry. The activities of pro-inflammatory marker enzymes such as COX, 5-LOX, 15-LOX, and NOS were normal in rats implanted with HACF scaffold. Hematological parameters, antioxidant and lipid peroxidation status were also found to be normal in implanted rats same as that of control and SMI. The modulatory effect of implanted scaffold over inflammatory and stress signaling cascades were confirmed by the normalized mRNA expressions of NF-κB, TNF-α and IL-6. The histopathological analysis of liver, kidney and tissue support our results. Taken together, these results demonstrated that HACF biocomposite scaffold signifies its suitability for further research as a scaffold material for cartilage tissue engineering applications.     

Synthesis and characterization of photocrosslinkable,degradable poly(vinyl alcohol)-based tissue engineering scaffolds

Hydrogels have many advantages that make them prime candidates for tissue engineering applications: high water content, tissue-like elasticity, and relative biocompatibility. We aim to tissue engineer heart valves using a hydrogel scaffold based on poly(vinyl alcohol) (PVA), and the design parameters for a suitable tissue engineering scaffold are quite stringent. In this research, we develop degradable and photocrosslinkable poly(lactic acid)-g-PVA multifunctional macromers that can be reacted in solution to form degradable networks. The mass loss profiles and bulk properties of the resulting scaffolds are easily tailored by modifying the structure of the starting macromers. Specifically, altering the number of lactide repeat units per crosslinking side chain, percent substitution, molecular weight of PVA backbone, and macromer solution concentration, the rate of mass loss from these degradable networks is controlled. In addition, by increasing the network's hydrophobicity, valve interstitial cell adhesion is improved.