Novel crosslinked alginate/hyaluronic acid hydrogels for nerve tissue engineering

Artificial tissue engineering scaffolds can potentially provide support and guidance for the regrowth of severed axons following nerve injury. In this study, a hybrid biomaterial composed of alginate and hyaluronic acid (HA) was synthesized and characterized in terms of its suitability for covalent modification, biocompatibility for living Schwann cells and feasibility to construct three dimensional (3D) scaffolds. Carbodiimide mediated amide formation for the purpose of covalent crosslinking of the HA was carried out in the presence of calcium ions that ionically crosslink alginate. Amide formation was found to be dependent on the concentrations of carbodiimide and calcium chloride. The double-crosslinked composite hydrogels display biocompatibility that is comparable to simple HA hydrogels, allowing for Schwann cell survival and growth. No significant difference was found between composite hydrogels made from different ratios of alginate and HA. A 3D BioPlotter^TM rapid prototyping system was used to fabricate 3D scaffolds. The result indicated that combining HA with alginate facilitated the fabrication process and that 3D scaffolds with porous inner structure can be fabricated from the composite hydrogels, but not from HA alone. This information provides a basis for continuing in vitro and in vivo tests of the suitability of alginate/HA hydrogel as a biomaterial to create living cell scaffolds to support nerve regeneration.     

In Vitro Characterizations of PLLA/β-TCP Porous Matrix Materials and RMSC-PLLA-β-TCP Composite Scaffolds

To develop a novel degradable poly (L-lactic acid)/β-tricalcium phosphate (PLLA/β-TCP) bioactive materials for bone tissueengineering, β-TCP powder was produced by a new wet process. Porous scaffolds were prepared by three steps, i.e. solventcasting, compression molding and leaching stage. Factors influencing the compressive strength and the degradation behaviorof the porous scaffold, e.g. weight fraction of pore forming agent-sodium chloride (NaCl), weight ratio of PLLA: β-TCP,the particle size of β-TCP and the porosity, were discussed in details. Rat marrow stromal cells (RMSC) were incorporatedinto the composite by tissue engineering approach. Biological and osteogenesis potential of the composite scaffold weredetermined with MTT assay, alkaline phosphatase (ALP) activity and bone osteocalcin (OCN) content evaluation. Resultsshow that PLLA/β-TCP bioactive porous scaffold has good mechanical and pore structure with adjustable compressive strengthneeded for surgery. RMSCs seeding on porous PLLA/     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

Abstract

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

In vitro degradation of porous nano-hydroxyapatite/collagen/PLLA scaffold reinforced by chitin fibres

In this paper, a novel porous scaffold for bone tissue engineering was prepared with nano-hydroxyapatite/collagen/Poly-l-lactic acid (PLLA) composite reinforced by chitin fibres. To enhance the strength of the scaffold further, PLLA was linked with chitin fibres by Dicyclohexylcarbodimide (DCC). The structures of the reinforced scaffold with and without linking were characterized by Scanning Electron Microscopy (SEM). The chemical characteristics of the chitin fibres with and without linking were evaluated by Fourier-transformed infrared (FTIR) spectroscopy. The mechanical performance during degradation in vitro was investigated. The results indicated that the nano-hydroxyapatite/collagen/PLLA composite reinforced by chitin fibres with linking kept better mechanical properties than that of the composite without linking. These results denoted that the stronger interfacial bonding strength of the scaffold with linking could decrease the degradation rate in vitro. The reinforced composite with the link-treatment can be severed as a scaffold for bone tissue engineering.     

Poly(L-lactic acid) nanocylinders as nanofibrous structures for macroporous gelatin scaffolds

Electrospun Nanofiber sheets have been shown to mimic the structure of extracellular matrix (ECM). Although these nanofibers have shown great potential for use as tissue engineering scaffolds, it is difficult for the electrospun nanofiber based sheets to be shaped into the desired three-dimensional structure. In this study, poly(L-lactic acid) (PLLA), a biodegradable and biocompatible polyester, was electrospun to produce nanofibers that were treated with an amino group containing base in order to fabricate polymeric nanocylinders. The aspect ratio of the PLLA nanocylinders was tunable by varying the aminolysis time and density of the amino group containing base. The effects of changes in nanofibrous morphology of the PLLA nanocylinders/macro-porous gelatin scaffolds on cell adhesion and proliferation were evaluated. The results revealed different cell morphology, adhesion, and proliferation in the nanocylinders composite gelatin scaffold versus gelatin scaffold alone. Confocal laser scanning microscopy observation showed more spreading and a more flattened cell morphology after NIH3T3 cells were cultured on PLLA nanocylinders/gelatin scaffolds for 10 hours and 4 days. These results indicate that the gelatin/PLLA nanocylinder composite is a promising way to fabricate 3D nanofibrous scaffolds that accelerates cell adhesion and proliferation for tissue engineering.     

Poriferan chitin as a template for hydrothermal zirconia deposition

Chitin is a thermostable biopolymer found in various inorganic--organic skeletal structures of numerous invertebrates including sponges (Porifera). The occurrence of chitin within calcium- and silica-based biominerals in organisms living in extreme natural conditions has inspired development of new (extreme biomimetic) synthesis route of chitin-based hybrid materials in vitro. Here, we show for the first time that 3D-α-chitin scaffolds isolated from skeletons of the marine sponge Aplysina aerophoba can be effectively mineralized under hydrothermal conditions (150°C) using ammonium zirconium(IV) carbonate as a precursor of zirconia. Obtained chitin--ZrO₂ hybrid materials were characterized by FT-IR, SEM, HRTEM, as well as light and confocal laser microscopy. We suggest that formation of chitin--ZrO₂ hybrids occurs due to hydrogen bonds between chitin and ZrO₂.     

Composite poly-l-lactic acid/poly-(α,β)-dl-aspartic acid/collagen nanofibrous scaffolds for dermal tissue regeneration

Tissue engineering scaffolds for skin tissue regeneration is an ever expounding area of research, as the products that meet the necessary requirements are far and elite. The nanofibrous poly-l-lactic acid/poly-(α,β)-dl-aspartic acid/Collagen (PLLA/PAA/Col I&III) scaffolds were fabricated by electrospinning and characterized by SEM, contact angle and FTIR analysis for skin tissue regeneration. The cell-scaffold interactions were analyzed by cell proliferation and their morphology observed in SEM. The results showed that the cell proliferation was significantly increased (p ≤ 0.05) in PLLA/PAA/Col I&III scaffolds compared to PLLA and PLLA/PAA nanofibrous scaffolds. The abundance and accessibility of adipose derived stem cells (ADSCs) may prove to be novel cell therapeutics for dermal tissue regeneration. The differentiation of ADSCs was confirmed using collagen expression and their morphology by CMFDA dye extrusion technique. The current study focuses on the application of PLLA/PAA/Col I&III nanofibrous scaffolds for skin tissue engineering and their potential use as substrate for the culture and differentiation of ADSCs. The objective for inclusion of a novel cell binding moiety like PAA was to replace damaged extracellular matrix and to guide new cells directly into the wound bed with enhanced proliferation and overall organization. This combinatorial epitome of PLLA/PAA/Col I&III nanofibrous scaffold with stem cell therapy to induce the necessary paracrine signalling effect would favour faster regeneration of the damaged skin tissues.     

Needle-like nano hydroxyapatite/poly(l-lactide acid) composite scaffold for bone tissue engineering application

In this paper, a new nano-hydroxyapatite / poly (l-lactide acid) (nHAP/PLLA) composite scaffold comprising needle-like nHAP particles was prepared. In the first step, the identification and morphology of chemically synthesized HAP particles were determined by XRD, EDX, FTIR and SEM analyses. The needle-like nHAP particles with an average size of approximately 30–60 nm in width and 100–400 nm in length were found similar to needle-like bone nano apatites in terms of chemical composition and morphology. In the second step, nHAP and micro-sized HAP (mHAP) particles were used to fabricate HAP filled PLLA (HAP/PLLA) composites scaffolds using solid–liquid phase separation method. The porosity of scaffolds was up to 85%, and their average macropore diameter was in the range of 64–175 µm. FTIR and XRD analyses showed the presence of molecular interactions and chemical linkages between HAP particles and PLLA matrix. The compressive strength of nanocomposite scaffolds could high up to 8.46 MPa while those of pure PLLA and microcomposite scaffolds were 1.79 and 4.61 MPa, respectively. The cell affinity and cytocompatibility of the nanocomposite scaffold were found to be higher than those of pure PLLA and microcomposite scaffolds. Based on the results, the newly developed nHAP/PLLA composite scaffold is comparable with cancellous bone in terms of microstructure and mechanical strength, so it may be a suitable alternative for bone tissue engineering applications.     

PLGA组织工程支架材料在SBF中的降解性能和生物矿化性能

本文采用pH值测量、特性粘度、失重、DSC和电子探针的研究方法,研究了PLGA组织工程支架在模拟体液中的降解性能和生物矿化性能。研究发现随着在SBF中浸泡时间的增长,PLGA支架材料的分子量不断下降;浸泡在SBF中的PLGA组织工程支架材料的重量由沉积进程和降解进程共同决定;DSC测试显示,浸泡在SBF中的PLGA组织工程支架材料的羟基乙酸单元(GA)相对于乳酸单元(LA)更易降解;电子探针测试显示,浸泡在SBF中的PLGA组织工程支架材料表面有磷酸盐沉积物产生。     

Improvement of cytocompatibility of electrospinning PLLA microfibers by blending PVP

In this study, microfiber films were used as scaffolds for the purpose of vascular tissue engineering. The microfiber films were prepared by electrospinning of poly (l-lactide) (PLLA) and polyvinyl pyrrolidone (PVP). PLLA and PVP with different ratios were blended with dichloromethane as a spinning solvent at room temperature. The properties of the composite microfiber films were investigated by differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and contact angle measurement. The SEM images showed that the morphology of the microfiber films was mainly affected by the weight ratios of PLLA/PVP. The DSC results demonstrated that PLLA and PVP mixed uniformly. And the hydrophilicity of the films measured increased along with the decrease of the PLLA/PVP ratio. Vascular smooth muscle cells (VSMCs) were used to test the cytocompatibility. Cell morphology and cell proliferation were measured by SEM, laser scanning confocal microscopy (LSCM) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay after 2, 4, 6 days of culture. The results indicated that the cell morphology and proliferation on the composite films were better than that on the pure PLLA film. Furthermore, morphology and proliferation of VSMCs became better with decreasing of the weight ratio of PLLA/PVP. In addition, adhesion of platelet on the films was observed by SEM. The SEM images showed that the number of adhered platelets decreased with increment of PVP content in the films. The electrospinning microfiber composite films of PLLA and PVP would have potential use as the scaffolds for vascular tissue engineering.     

The effect of selected alloying element additions on properties of Mg-based alloy as bioimplants: A literature review

This review investigates the current application limitations of Mg and Mg alloys. The key issues hindering the application of biodegradable Mg alloys as implants are their fast degradation rate and biological consideration. We have discussed the effect of some selected alloying element additions on the properties of the Mg-based alloy, especially the nutrient elements in human (Zn, Mn, Ca, Sr). Different grain sizes, phase constituents and distributions consequently influence the mechanical properties of the Mg alloys. Solution strengthening and precipitation strengthening are enhanced by the addition of alloying elements, generally improving the mechanical properties. Besides, the hot working process can also improve the mechanical properties. Combination of different processing steps is suggested to be adopted in the fabrication of Mg-based alloys. Corrosion properties of these Mg-based alloys have been measured in vitro and in vivo. The degradation mechanism is also discussed in terms of corrosion types, rates, by-products and response of the surrounding tissues. Moreover, the clinical response and requirements of degradable implants are presented, especially for the nutrient elements (Ca, Mn, Zn, Sr). This review provides information related to different Mg alloying elements and presents the promising candidates for an ideal implant.     

Long-term viability of coronary artery smooth muscle cells on poly(L-lactide-co-epsilon-caprolactone) nanofibrous scaffold indicates its potential for blood vessel tissue engineering.

Biodegradable polymer nanofibres have been extensively studied as cell culture scaffolds in tissue engineering. However, long-term in vitro studies of cell-nanofibre interactions were rarely reported and successful organ regeneration using tissue engineering techniques may take months (e.g. blood vessel tissue engineering). Understanding the long-term interaction between cells and nanofibrous scaffolds (NFS) is crucial in material selection, design and processing of the tissue engineering scaffolds. In this study, poly(L-lactide-co-epsilon-caprolactone) [P(LLA-CL)] (70:30) copolymer NFS were produced by electrospinning. Porcine coronary artery smooth muscle cells (PCASMCs) were seeded and cultured on the scaffold to evaluate cell-nanofibre interactions for up to 105 days. A favourable interaction between this scaffold and PCASMCs was demonstrated by cell viability assay, scanning electron microscopy, histological staining and extracellular matrix (ECM) secretion. Degradation behaviours of the scaffolds with or without PCASMC culture were determined by mechanical testing and gel permeation chromatography (GPC). The results showed that the PCASMCs attached and proliferated well on the P(LLA-CL) NFS. Large amount of ECM protein secretion was observed after 50 days of culture. Multilayers of aligned oriented PCASMCs were formed on the scaffold after two months of in vitro culture. In the degradation study, the PCASMCs were not shown to significantly increase the degradation rate of the scaffolds for up to 105 days of culture. The in vitro degradation time of the scaffold could be as long as eight months by extrapolating the results from GPC. These observations further supported the potential use of the P(LLA-CL) nanofibre in blood vessel tissue engineering.     

Formation of bone-like apatite on poly(L-lactide) to improve osteoblast-like compatibility in vitro and in vivo

The biomimetic apatite coating process was adopted to modify poly(L-lactide) (PLLA) surfaces with osteoblasts-like cell compatibility. The apatite coating was formed on the pre-hydrolyzed PLLA film and scaffold surfaces by incubating in simulated body fluid (SBF). Scanning electron microscopy and energy dispersive X-ray analyzer were utilized to characterize the composition and the structure of the apatite coating. The cytocompatibility of the modified PLLA films was investigated by testing osteoblast-like attachment, proliferation, alkaline phosphatase (ALP) activity, and cell cycle. Subsequently, the modified PLLA scaffolds were co-cultured with the osteoblasts-like in vitro and subcutaneously implanted into nude mice. The experimental results showed that the formed apatite had a nano-sized particle and matrix configuration. The surface modification of PLLA with apatite coating significantly promoted osteoblast-like compatibility. After a four-week culture in vivo, no significant inflammatory signs were observed in the implanted regions and osteoblast-like congeries with bone-like structure began to form in the scaffolds. The positive results of this study suggest a good way to produce desirable PLLA biomaterials for bone tissue engineering.     

Selective laser sintering of porous tissue engineering scaffolds from poly(<Emphasis Type="SmallCaps">l</Emphasis>-lactide)/carbonated hydroxyapatite nanocomposite microspheres

This study focuses on the use of bio-nanocomposite microspheres, consisting of carbonated hydroxyapatite (CHAp) nanospheres within a poly(L: -lactide) (PLLA) matrix, to produce tissue engineering (TE) scaffolds using a modified selective laser sintering (SLS) machine. PLLA microspheres and PLLA/CHAp nanocomposite microspheres were prepared by emulsion techniques. The resultant microspheres had a size range of 5-30 mum, suitable for the SLS process. Microstructural analyses revealed that the CHAp nanospheres were embedded throughout the PLLA microsphere, forming a nanocomposite structure. A custom-made miniature sintering platform was installed in a commercial Sinterstation((R)) 2000 SLS machine. This platform allowed the use of small quantities of biomaterials for TE scaffold production. The effects of laser power; scan spacing and part bed temperature were investigated and optimized. Finally, porous scaffolds were successfully fabricated from the PLLA microspheres and PLLA/CHAp nanocomposite microspheres. In particular, the PLLA/CHAp nanocomposite microspheres appeared to be promising for porous bone TE scaffold production using the SLS technique.     

Development of cryogenic prototyping for tissue engineering

A major challenge in tissue engineering has been the creation of scaffolds with controlled complex geometries. Rapid prototyping (RP) has the ability to produce complex three-dimensional structures with precise control of pore size, geometry and connectivity. In this paper, a novel technique utilising RP technology for the fabrication of tissue engineering scaffolds is presented. The main advantage of this cryogenic prototyping (CP) technique is the low operating temperatures which will allow the processing of temperature sensitive and bioactive components. Microstructure of CP Chitosan scaffolds fabricated can be controlled by processing parameters, such as the processing temperature. The macrostructure of the scaffolds is controlled by 3D computer aided design (CAD). In addition, in vitro studies with Chitosan CP scaffolds have shown that the scaffold designs are useful in promoting cell infiltration and alignment. Preliminary in vivo studies show encouraging results of cellular infiltration as well as vascularisation.     

Accurate micro-computed tomography imaging of pore spaces in collagen-based scaffold

In this work we have used X-ray micro-computed tomography (μCT) as a method to observe the morphology of 3D porous pure collagen and collagen-composite scaffolds useful in tissue engineering. Two aspects of visualizations were taken into consideration: improvement of the scan and investigation of its sensitivity to the scan parameters. Due to the low material density some parts of collagen scaffolds are invisible in a μCT scan. Therefore, here we present different contrast agents, which increase the contrast of the scanned biopolymeric sample for μCT visualization. The increase of contrast of collagenous scaffolds was performed with ceramic hydroxyapatite microparticles (HAp), silver ions (Ag⁺) and silver nanoparticles (Ag-NPs). Since a relatively small change in imaging parameters (e.g. in 3D volume rendering, threshold value and μCT acquisition conditions) leads to a completely different visualized pattern, we have optimized these parameters to obtain the most realistic picture for visual and qualitative evaluation of the biopolymeric scaffold. Moreover, scaffold images were stereoscopically visualized in order to better see the 3D biopolymer composite scaffold morphology. However, the optimized visualization has some discontinuities in zoomed view, which can be problematic for further analysis of interconnected pores by commonly used numerical methods. Therefore, we applied the locally adaptive method to solve discontinuities issue. The combination of contrast agent and imaging techniques presented in this paper help us to better understand the structure and morphology of the biopolymeric scaffold that is crucial in the design of new biomaterials useful in tissue engineering.     

Compressive mechanical properties and deformation behavior of porous polymer blends of poly(ε-caprolactone) and poly(l-lactic acid)

Porous biodegradable polymeric scaffolds are developed by physically blending two different kinds of biodegradable polymers, PCL, and PLLA, for application in tissue engineering. The main objective of the development of this material is to control the mechanical properties, such as, elastic modulus and strength. The results from mechanical testing showed that the compressive mechanical properties of PCL/PLLA scaffold can be varied by changing the blend ratio. It also showed that these properties can be well predicted by the rule of mixture. The primary deformation mechanism of the scaffolds was found to be localized buckling of struts surrounding the pores. Localized ductile failure caused by PCL phase tends to be suppressed with increasing PLLA content. The immiscibility of PCL and PLLA caused the phase-separation morphology that strongly affected the macroscopic mechanical properties and the microscopic deformation behavior.     

Morphology and degradation properties of PCL/HYAFF11® composite scaffolds with multi-scale degradation rate

The analysis of scaffold degradation is a promising strategy for understanding the dynamic changes in texture and pore morphology which accompany polymer resorption, and for collecting same fundamental indicators regarding the potential fate of the scaffold in the biological environment. In this study, we investigate the morphology and degradation properties of three composite scaffolds based on poly(ε-caprolactone) (PCL) embedded with benzyl ester of hyaluronic acid (HYAFF11®) phases, and, in turn, different reinforcement systems – i.e., calcium phosphate particles or continuous poly(lactic acid) (PLA) fibres. Scanning electron microscopy (SEM) and μ-tomography supported by digital image analysis enabled a not invasive investigation of the scaffold morphology, providing a quantitative assessment of porosity (which ranged from 63.1 to 82.8), pore sizes (which varied from 170.5 to 230.4 μm) and pore interconnectivity. Thermal analyses (DSC and TGA) and Raman spectroscopy demonstrated the multi-scale degradation of the composite with highly tailoring degradation kinetics depending on the component material phases and scaffold architecture changes, due to their conditioning in simulated in vivo environment (i.e., SBF solution). These results demonstrate that the judicious mixing of materials with faster (i.e., HYAFF11) and slower (i.e., PLA and PCL) degradation kinetics, different size and shape (i.e., domains, particles or long fibres), certainly concurs to design a smart composite scaffold with time-controlled degradation which can support the regeneration of a large variety of tissues, from the cartilage to the bone.     

CPP/PLLA软骨组织工程支架复合材料初步研究

采用溶媒投放、颗粒滤取技术制备出CPP/PLLA软骨组织工程支架复合材料,测试了该复合材料的物理力学性能和降解性能。研究结果表明,CPP/PLLA软骨组织工程支架复合材料具有高的孔隙率(90%)、良好的生物降解性能和物理力学性能,以及三维连通、微孔、网状微观结构,故该复合材料有希望成为软骨组织工程支架材料之一。