Growth of outgrowth endothelial cells on aligned PLLA nanofibrous scaffolds

Tissue engineering holds great promise in providing vascular grafts as substitutes for damaged small-diameter blood vessels. Two of the key factors in vascular tissue engineering are biocompatible scaffolds that mimic the effects of extracellular matrix and the source of seeding cells. Synthetic poly-l-lactic acid (PLLA) nanofibers has been shown to be excellent scaffolds for tissue engineering. Outgrowth endothelial cells (OECs) isolated from human peripheral blood could also be expanded in vitro and stably maintain the differentiated phenotypes and could be used as the seeding cells for engineering autologous vascular crafts. Here we tested the possibility of combining these two together. We found that PLLA nanofibers are not only biocompatible with OECs originally isolated from rabbit peripheral blood, the aligned PLLA fibers actually promoted and guided their sustained proliferation. These results suggest that aligned PLLA could be excellent both as the scaffolds and as a promoter of cell growth during vascular tissue engineering.     

Scaffolds with a standardized macro-architecture fabricated from several calcium phosphate ceramics using an indirect rapid prototyping technique

Calcium phosphate ceramics, commonly applied as bone graft substitutes, are a natural choice of scaffolding material for bone tissue engineering. Evidence shows that the chemical composition, macroporosity and microporosity of these ceramics influences their behavior as bone graft substitutes and bone tissue engineering scaffolds but little has been done to optimize these parameters. One method of optimization is to place focus on a particular parameter by normalizing the influence, as much as possible, of confounding parameters. This is difficult to accomplish with traditional fabrication techniques. In this study we describe a design based rapid prototyping method of manufacturing scaffolds with virtually identical macroporous architectures from different calcium phosphate ceramic compositions. Beta-tricalcium phosphate, hydroxyapatite (at two sintering temperatures) and biphasic calcium phosphate scaffolds were manufactured. The macro- and micro-architectures of the scaffolds were characterized as well as the influence of the manufacturing method on the chemistries of the calcium phosphate compositions. The structural characteristics of the resulting scaffolds were remarkably similar. The manufacturing process had little influence on the composition of the materials except for the consistent but small addition of, or increase in, a beta-tricalcium phosphate phase. Among other applications, scaffolds produced by the method described provide a means of examining the influence of different calcium phosphate compositions while confidently excluding the influence of the macroporous structure of the scaffolds.     

Fabrication of PU/PEGMA crosslinked hybrid scaffolds by in situ UV photopolymerization favoring human endothelial cells growth for vascular tissue engineering

Poly(ethylene glycol) methacrylate (PEGMA) was introduced into a polyurethane (PU) solution in order to prepare electrospun scaffold with improving the biocompatibility by electrospinning technology for potential application as small diameter vascular scaffolds. Crosslinked electrospun PU/PEGMA hybrid nanofibers were fabricated by a reactive electrospinning process with N,N'-methylenebisacrylamide as crosslinker and benzophenone as photoinitiator. The photoinduced polymerization and crosslinking reaction took place simultaneously during the electrospinning process. The electrospinning solutions with various weight ratios of PU/PEGMA were successfully electrospun. No significant difference in the scaffold morphology was found by SEM when PEGMA content was <20 wt%. The crosslinked fibrous scaffolds of PU/PEGMA exhibited higher mechanical strength than the pure PU scaffold. The hydrophilicity of scaffolds was controlled by varying the PU/PEGMA weight ratio. The tissue compatibility of electrospun nanofibrous scaffolds were tested using human umbilical vein endothelial cells (HUVECs). Cell morphology and cell proliferation were measured by SEM, fluorescence microscopy and thiazolyl blue assay (MTT) after 1, 3, 7 days of culture. The results indicated that the cell morphology and proliferation on the crosslinked PU/PEGMA scaffolds were better than that on the pure PU scaffold. Furthermore, the appropriate hydrophilic surface with water contact angle in the range of 55-75° was favorable of improvement the HUVECs adhesion and proliferation. Cells seeded on the crosslinked PU/PEGMA (80/20) scaffolds infiltrated into the scaffolds after 7 days of growth. These results indicated the crosslinked electrospun PU/PEGMA nanofibrous scaffolds were potential substitutes for artificial vascular scaffolds.     

Processing Methodologies for Polycaprolactone-Hydroxyapatite Composites: A Review

Biodegradable implants have shown great promise for the repair of bone defects and have been commonly used as bone substitutes, which traditionally would be treated using metallic implants. The need for a second surgery exacerbated by the stress shielding effect caused by an implant has led researchers to consider more effective, synthetic biodegradable graft substitutes. The hierarchical structures commonly designed are inspired by nature in human bones, which consist of minerals such as hydroxyapatite, a form of calcium phosphate and protein fiber. The bone graft bio-substitutes should possess a combination of properties for the purpose of facilitating cell growth and adhesion, a high degree of porosity, which would facilitate the transfer of nutrients and excretion of the waste products, and the scaffold should have high tensile strength and high toughness in order to be consistent with human tissues. Blending of polycaprolactone and hydroxyapatite has demonstrated great potential as bone substitutes. It is essential to identify a standardized processing methodology for the composite, which would result in optimum mechanical property for the biocomposite. In this study, biocomposites made of polycaprolactone (PCL) and hydroxyapatite (HAP) are reviewed for their applications in bone tissue engineering. The processing methodologies are discussed for the purpose of obtaining the porosity and pore size required in an ideal tissue scaffold. The properties of the composite can be varied based on the change in pore size, porosity, and processing methodology. This paper reviews and evaluates the methods to produce the hydroxyapatite-polycaprolactone scaffolds.     

Development and characterisation of a collagen nano-hydroxyapatite composite scaffold for bone tissue engineering

Bone regeneration requires scaffolds that possess suitable mechanical and biological properties. This study sought to develop a novel collagen-nHA biocomposite scaffold via two new methods. Firstly a stable nHA suspension was produced and added to a collagen slurry (suspension method), and secondly, porous collagen scaffolds were immersed in nHA suspension after freeze-drying (immersion method). Significantly stronger constructs were produced using both methods compared to collagen only scaffolds, with a high porosity maintained (>98.9%). It was found that Coll-nHA composite scaffolds produced by the suspension method were up to 18 times stiffer than the collagen control (5.50 ± 1.70 kPa vs. 0.30 ± 0.09 kPa). The suspension method was also more reproducible, and the quantity of nHA incorporated could be varied with greater ease than with the immersion technique. In addition, Coll-nHA composites display excellent biological activity, demonstrating their potential as bone graft substitutes in orthopaedic regenerative medicine.     

Hydrogel/bioactive glass composites for bone regeneration applications: Synthesis and characterisation

Due to the deficiencies of current commercially available biological bone grafts, alternative bone graft substitutes have come to the forefront of tissue engineering in recent times. The main challenge for scientists in manufacturing bone graft substitutes is to obtain a scaffold that has sufficient mechanical strength and bioactive properties to promote formation of new tissue. The ability to synthesise hydrogel based composite scaffolds using photopolymerisation has been demonstrated in this study. The prepared hydrogel based composites were characterised using techniques including Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), Energy-dispersive X-ray spectrometry (EDX), rheological studies and compression testing. In addition, gel fraction, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), porosity and swelling studies of the composites were carried out. It was found that these novel hydrogel bioglass composite formulations did not display the inherent brittleness that is typically associated with bioactive glass based bone graft materials and exhibited enhanced biomechanical properties compared to the polyethylene glycol hydrogel scaffolds along. Together, the combination of enhanced mechanical properties and the deposition of apatite on the surface of these hydrogel based composites make them an ideal candidate as bone graft substitutes in cancellous bone defects or low load bearing applications.     

Bone‐Like Elastomer‐Toughened Scaffolds with Degradability Kinetics Matching Healing Rates of Injured Bone

Biomaterial scaffolds provide a potentially powerful means of creating precisely engineered bone tissue substitutes with appropriate architecture and mechanical properties. Despite many efforts, there are few satisfactory products available for clinical use, and significant challenges remain in the design of smart constructs, especially for mechanically functional scaffolds. For successful long‐term repair of bone, a scaffold must be strong yet have degradation kinetics matching the healing rate of remodeling bone. Here we report a new family of elastomer‐toughened composite scaffolds fabricated from poly(glycerol sebacate) and Bioglass®. These synthetic scaffolds have very similar mechanical properties to that of cancellous bone of the same porosity, and exhibit a mechanically steady state over an extended period in a physiologic environment. The second feature is of great importance to bone tissue engineering, where a lag phase of degradation following implantation is highly desirable in order to provide support to the damaged or fragmented bone tissue. This work shows that a mechanically smart construct with the three‐stage profile (lag, log, and plateau phases) of ideal degradation kinetics in mechanical function is achievable with synthetic biomaterials.     

Alginate based scaffolds for bone tissue engineering

The design and production of scaffolds for bone tissue regeneration is yet unable to completely reproduce the native bone properties. In the present study new alginate microparticle and microfiber aggregated scaffolds were produced to be applied in this area of regenerative medicine.The scaffolds' mechanical properties were characterized by thermo mechanical assays. Their morphological characteristics were evaluated by isothermal nitrogen adsorption and scanning electron microscopy. The density of both types of scaffolds was determined by helium pycnometry and mercury intrusion porosimetry. Furthermore, scaffolds' cytotoxic profiles were evaluated in vitro by seeding human osteoblast cells in their presence.The results obtained showed that scaffolds have good mechanical and morphological properties compatible with their application as bone substitutes. Moreover, scaffold's biocompatibility was confirmed by the observation of cell adhesion and proliferation after 5 days of being seeded in their presence and by non-radioactive assays.     

Electrospun composite nanofibers for tissue regeneration

Nanotechnology assists in the development of biocomposite nanofibrous scaffolds that can react positively to changes in the immediate cellular environment and stimulate specific regenerative events at molecular level to generate healthy tissues. Recently, electrospinning has gained huge momentum with greater accessibility of fabrication of composite, controlled and oriented nanofibers with sufficient porosity required for effective tissue regeneration. Current developments include the fabrication of nanofibrous scaffolds which can provide chemical, mechanical and biological signals to respond to the environmental stimuli. These nanofibers are fabricated by simple coating, blending of polymers/bioactive molecules or by surface modification methods. For obtaining optimized surface functionality, with specially designed architectures for the nanofibers (multi-layered, core-shell, aligned), electrospinning process has been modified and simultaneous 'electrospin-electrospraying' process is one of the most lately introduced technique in this perspective. Properties such as porosity, biodegradation and mechanical properties of composite electrospun nanofibers along with their utilization for nerve, cardiac, bone, skin, vascular and cartilage tissue engineering are discussed in this review. In order to locally deliver electrical stimulus and provide a physical template for cell proliferations, and to gain an external control on the level and duration of stimulation, electrically conducting polymeric nanofibers are also fabricated by electrospinning. Electrospun polypyrrole (PPy) and polyaniline (PAN) based scaffolds are the most extensively studied composite substrates for nerve and cardiac tissue engineering with or without electrical stimulations, and are discussed here. However, the major focus of ongoing and future research in regenerative medicine is to effectively exploit the pluripotent potential of Mesenchymal Stem Cell (MSC) differentiation on composite nanofibrous scaffolds for repair of organs.     

Characterization and osteoblast-like cell compatibility of porous scaffolds: bovine hydroxyapatite and novel hydroxyapatite artificial bone

Three different porous scaffolds were tested. The first two were prepared by sintering bovine bone. The third scaffold was prepared using three-dimensional gel-lamination, a new rapid prototyping method, and was named as hydroxyapatite artificial bone. X-ray diffraction and Fourier transform infrared spectroscopy analysis confirmed that the samples were mainly highly crystalline hydroxyapatite ceramics. Scanning electron microscopy and mercury intrusion porosimetry measurement showed that the pores were interconnected and pore sizes ranged from several microns to hundreds of microns. Mouse osteoblast-like cells grown on the three scaffolds retained their characteristic morphology. Cell proliferation and differentiation, analyzed by methylthiazol tetrazolium (MTT) and alkaline phosphatase activity assays, were significantly higher on the hydroxyapatite artificial bone than on the other two scaffolds tested. All the scaffolds provided good attachment, proliferation and differentiation of bone cells. These results indicate that the scaffolds have a favorable interaction with cells, they support cell growth and functions, and therefore these scaffolds may have great potential as bone substitutes. The three-dimensional gel-lamination method is proven to be an attractive process to design and fabricate bone scaffolds with favorable properties, and therefore, has promising potential for bone repair applications.     

Biocompatibility evaluation of protein-incorporated electrospun polyurethane-based scaffolds with smooth muscle cells for vascular tissue engineering

Nanotechnology has enabled the engineering of a variety of materials to meet the current challenges and needs in vascular tissue regeneration. In this study, four different kinds of native proteins namely collagen, gelatin, fibrinogen, and bovine serum albumin were incorporated with polyurethane (PU) and electropsun to obtain composite PU/protein nanofibers. SEM studies showed that the fiber diameters of PU/protein scaffolds ranged from 245 to 273 nm, mimicking the nanoscale dimensions of native ECM. Human aortic smooth muscle cells (SMCs) were cultured on the electrospun nanofibers, and the ability of the cells to proliferate on different scaffolds was evaluated via a cell proliferation assay. Cell proliferation on PU/Coll nanofibers was found the highest compared to other electrospun scaffolds and it was 42 % higher than the proliferation on PU/Fib nanofibers after 12 days of cell culture. The cell–biomaterial interaction studies by SEM confirmed that SMCs adhered to PU/Coll and PU/Gel nanofibers, with high cell substrate coverage, and both the scaffolds promoted cell alignment. The functionality of the cells was further demonstrated by immunocytochemical analysis, where the SMCs on PU/Coll and PU/Gel nanofibers expressed higher density of SMC proteins such as alpha smooth muscle actin and smooth muscle myosin heavy chain. Cells expressed biological markers of SMCs including aligned spindle-like morphology on both PU/Coll and PU/Gel with actin filament organizations, better than PU/Fib and PU/BSA scaffolds. Our studies demonstrate the potential of randomly oriented elastomeric composite scaffolds for engineering of vascular tissues causing cell alignment.     

Analysis of OPLA scaffolds for bone engineering constructs using human jaw periosteal cells

For bone regeneration constructs using human jaw periosteal cells (JPC) the extent of osteoinductive ability of different three-dimensional scaffolds is not yet established. We analyzed open-cell polylactic acid (OPLA) scaffolds for their suitability as bone engineering constructs using human JPC. Cell adhesion and spreading was visualized on the surface of scaffolds by scanning electron microscopy. JPC proliferation within OPLA scaffolds was compared with proliferation within collagen and calcium phosphate scaffolds. We found a significant increase of proliferation rates in OPLA scaffolds versus Coll/CaP scaffolds at three time points. Live-measurements of oxygen consumption within the cell-seeded scaffolds indicate that the in vitro culturing time should not exceed 12–15 days. OPLA scaffolds, which were turned out to be the most beneficial for JPC growth, were chosen for osteogenic differentiation experiments with or without BMP-2. Gene expression analyses demonstrated induction of several osteogenic genes (alkaline phosphatase, osterix, Runx-2 and insulin-like growth factor) within the 3D-scaffolds after 12 days of in vitro culturing. Element analysis by EDX spectrometry of arising nodules during osteogenesis demonstrated that JPC growing within OPLA scaffolds are able to form CaP particles. We conclude that OPLA scaffolds provide a promising environment for bone substitutes using human JPC.     

A generalized self consistent model for effective elastic moduli of human dentine

Closed-form expressions are presented for effective material properties of human dentine in this paper. The derivation is based a Generalized Self Consistent Method and the strain energy principle. The Generalized Self Consistent Model for cell model of fiber-reinforced composites is extended to the case of hollow cylinder model and the corresponding cell model is chosen to consist of a circular hollow cylinder filled with liquid or gas phase, which surrounded by a circular cylindrical shell of matrix phase. Each layer of cylindrical shell is here considered as a kind of composite consisting of collagen fibrils, with mineralized hydroxyapatite, loosely connected to their neighbours, and water (or gas in the case of dry dentine composite). Using the cell model, the effect of Poisson’s ratio and volume fraction of intertubular dentine on effective mechanical constants is analyzed. Results obtained from the proposed model are compared with those from other models such as nano-indentation method.     

Three Phase Composite Cylinder Assemblage Model for Analyzing the Elastic Behavior of MWCNT-Reinforced Polymers

Evolution of computational modeling and simulation has given more emphasis on the research activities related to carbon nanotube (CNT) reinforced polymer composites recently. This paper presents the composite cylinder assemblage (CCA) approach based on continuum mechanics for investigating the elastic properties of a polymer resin reinforced by multi-walled carbon nanotubes (MWCNTs). A three-phase cylindrical representative volume element (RVE) model is employed based on CCA technique to elucidate the effects of inter layers, chirality, interspacing, volume fraction of MWCNT, interphase properties and temperature conditions on the elastic modulus of the composite. The interface region between CNT and polymer matrix is modeled as the third phase with varying material properties. The constitutive relations for each material system have been derived based on solid mechanics and proper interfacial traction continuity conditions are imposed. The predicted results from the CCA approach are in well agreement with RVE-based finite element model. The outcomes reveal that temperature softening effect becomes more pronounced at higher volume fractions of CNTs.     

Three dimensional printed macroporous polylactic acid/hydroxyapatite composite scaffolds for promoting bone formation in a critical-size rat calvarial defect model

We have explored the applicability of printed scaffold by comparing osteogenic ability and biodegradation property of three resorbable biomaterials. A polylactic acid/hydroxyapatite (PLA/HA) composite with a pore size of 500 μm and 60% porosity was fabricated by three-dimensional printing. Three-dimensional printed PLA/HA, β-tricalcium phosphate (β-TCP) and partially demineralized bone matrix (DBM) seeded with bone marrow stromal cells (BMSCs) were evaluated by cell adhesion, proliferation, alkaline phosphatase activity and osteogenic gene expression of osteopontin (OPN) and collagen type I (COL-1). Moreover, the biocompatibility, bone repairing capacity and degradation in three different bone substitute materials were estimated using a critical-size rat calvarial defect model in vivo. The defects were evaluated by micro-computed tomography and histological analysis at four and eight weeks after surgery, respectively. The results showed that each of the studied scaffolds had its own specific merits and drawbacks. Three-dimensional printed PLA/HA scaffolds possessed good biocompatibility and stimulated BMSC cell proliferation and differentiation to osteogenic cells. The outcomes in vivo revealed that 3D printed PLA/HA scaffolds had good osteogenic capability and biodegradation activity with no difference in inflammation reaction. Therefore, 3D printed PLA/HA scaffolds have potential applications in bone tissue engineering and may be used as graft substitutes in reconstructive surgery.     

Porous scaffold design for tissue engineering

A paradigm shift is taking place in medicine from using synthetic implants and tissue grafts to a tissue engineering approach that uses degradable porous material scaffolds integrated with biological cells or molecules to regenerate tissues. This new paradigm requires scaffolds that balance temporary mechanical function with mass transport to aid biological delivery and tissue regeneration. Little is known quantitatively about this balance as early scaffolds were not fabricated with precise porous architecture. Recent advances in both computational topology design (CTD) and solid free-form fabrication (SFF) have made it possible to create scaffolds with controlled architecture. This paper reviews the integration of CTD with SFF to build designer tissue-engineering scaffolds. It also details the mechanical properties and tissue regeneration achieved using designer scaffolds. Finally, future directions are suggested for using designer scaffolds with in vivo experimentation to optimize tissue-engineering treatments, and coupling designer scaffolds with cell printing to create designer material/biofactor hybrids.     

Poly (<Emphasis Type="SmallCaps">d</Emphasis>,<Emphasis Type="SmallCaps">l</Emphasis>-lactide)/nano-hydroxyapatite composite scaffolds for bone tissue engineering and biocompatibility evaluation

Biodegradable polymer/bioceramic composite scaffolds can overcome the limitations of conventional ceramic bone substitutes such as brittleness and difficulty in shaping. However, conventional methods for fabricating polymer/bioceramic composite scaffolds often use organic solvents (e.g., the solvent casting and particulate leaching (SC/PL) method), which might be harmful to cells or tissues. In this study, Poly (d,l-lactide)/nano-hydroxyapatite (PDLLA/NHA) composites were prepared by in-situ polymerization, and highly porous scaffolds were fabricated using a novel method, supercritical CO₂/salt-leaching method (SC CO₂/SL). The materials and scaffolds were investigated by scanning electronic microscopy (SEM), transmission electronic microscopy (TEM) and gel permeation chromatography (GPC). GPC showed that the molecular weight of composites decreased with increase of NHA content. However, the water absorption and compressive strength increased dramatically. The SEM micrographs showed that the scaffolds with pore size about 250 μm were obtained by controlling parameters of SC CO₂/SL. The biocompatibility of PDLLA/NHA porous scaffolds were evaluated in vitro and in vivo. The evaluation on the cytotoxicity were carried out by cell relative growth rate (RGR) method and cell direct contact method. The cytotoxicity of these scaffolds was in grade I according to ISO 10993-1. There was no toxicosis and death cases observed in acute systemic toxicity test. And histological observation of the tissue response (1 and 9 weeks after the implantation) showed that there are still some slight inflammation responses.     

Nanotechnological strategies for engineering complex tissues

Tissue engineering aims at developing functional substitutes for damaged tissues and organs. Before transplantation, cells are generally seeded on biomaterial scaffolds that recapitulate the extracellular matrix and provide cells with information that is important for tissue development. Here we review the nanocomposite nature of the extracellular matrix, describe the design considerations for different tissues and discuss the impact of nanostructures on the properties of scaffolds and their uses in monitoring the behaviour of engineered tissues. We also examine the different nanodevices used to trigger certain processes for tissue development, and offer our view on the principal challenges and prospects of applying nanotechnology in tissue engineering.     

Ultrafine fibrous gelatin scaffolds with deep cell infiltration mimicking 3D ECMs for soft tissue repair

In this research, ultrafine fibrous scaffolds with deep cell infiltration and sufficient water stability have been developed from gelatin, aiming to mimic the extracellular matrices (ECMs) as three dimensional (3D) stromas for soft tissue repair. The ultrafine fibrous scaffolds produced from the current technologies of electrospinning and phase separation are either lack of 3D oriented fibrous structure or too compact to be penetrated by cells. Whilst electrospun scaffolds are able to emulate two dimensional (2D) ECMs, they cannot mimic the 3D ECM stroma. In this work, ultralow concentration phase separation (ULCPS) has been developed to fabricate gelatin scaffolds with 3D randomly oriented ultrafine fibers and loose structures. Besides, a non-toxic citric acid crosslinking system has been established for the ULCPS method. This system could endow the scaffolds with sufficient water stability, while maintain the fibrous structures of scaffolds. Comparing with electrospun scaffolds, the ULCPS scaffolds showed improved cytocompatibility and more importantly, cell infiltration. This research has proved the possibility of using gelatin ULCPS scaffolds as the substitutes of 3D ECMs.     

Feasibility, tailoring and properties of polyurethane/bioactive glass composite scaffolds for tissue engineering

This research work aims to propose highly porous polymer/bioactive glass composites as potential scaffolds for hard-tissue and soft-tissue engineering. The scaffolds were prepared by impregnating an open-cells polyurethane sponge with melt-derived particles of a bioactive glass belonging to the SiO₂–P₂O₅–CaO–MgO–Na₂O–K₂O system (CEL2). Both the starting materials and the composite scaffolds were investigated from a morphological and structural viewpoint by X-ray diffraction analysis and scanning electron microscopy. Tensile mechanical tests, carried out according to international ISO and ASTM standards, were performed by using properly tailored specimens. In vitro tests by soaking the scaffolds in simulated body fluid (SBF) were also carried out to assess the bioactivity of the porous composites. It was found that the composite scaffolds were highly bioactive as after 7 days of soaking in SBF a HA layer grew on their surface. The obtained polyurethane/CEL2 composite scaffolds are promising candidates for tissue engineering applications.