Fabrication and cell affinity of biomimetic structured PLGA/articular cartilage ECM composite scaffold

An ideal scaffold for cartilage tissue engineering should be biomimetic in not only mechanical property and biochemical composition, but also the morphological structure. In this research, we fabricated a composite scaffold with oriented structure to mimic cartilage physiological morphology, where natural nanofibrous articular cartilage extracellular matrix (ACECM) was used to mimic the biochemical composition, and synthetic PLGA was used to enhance the mechanical strength of ACECM. The composite scaffold has well oriented structure and more than 89% of porosity as well as about 107 μm of average pore diameter. The composite scaffold was compared with ACECM and PLGA scaffolds. Cell proliferation test showed that the number of MSCs in ACECM and composite scaffolds was noticeably bigger than that in PLGA scaffold, which was coincident with results of SEM observation and cell viability staining. The water absorption of ACECM and composite scaffolds were 22.1 and 10.2 times respectively, which was much higher than that of PLGA scaffolds (3.8 times). The compressive modulus of composite scaffold in hydrous status was 1.03 MPa, which was near 10 times higher than that of hydrous ACECM scaffold. The aforementioned results suggested that the composite scaffold has the potential for application in cartilage tissue engineering.     

A novel surface modification on calcium polyphosphate scaffold for articular cartilage tissue engineering

The surface of porous three-dimensional (3D) calcium polyphosphate (CPP) scaffold was modified by treatment of quenching-after-sintering in the fabrication process. Scanning electron microscopic examination and degradation tests confirmed a new type of surface modification. A rotary-shaking culture was compared to that of a stationary culture and the results showed that rotary shaking led to enhanced extracellular matrices (ECM) secretion of both proteoglycans and collagen. Rotary-shaking cultured results showed that the quenching-treated CPP scaffold produced a better cartilage tissue, with both proteoglycans and collagen secretions enhanced, than the air-cooled-after-sintering scaffolds. Moreover, β-CPP scaffolds were better for the ECM secretion of both proteoglycans and collagen than the β-CPP + γ-CPP multiphase scaffold. However, the multiphase scaffold led to higher growth rate than that of β-CPP scaffold; the quenching-after-sintering treatment reversed this. In addition, the ECM secretions of both proteoglycans and collagen in the quenching-treated β-CPP scaffold were higher than those in the air-cooled one. Thus, the novel treatment of quenching-after-sintering has shown merits to the porous 3D CPP scaffolds for articular cartilage tissue engineering.     

The influence of surface nanoroughness of electrospun PLGA nanofibrous scaffold on nerve cell adhesion and proliferation

Electrospun nanofibrous scaffolds in neural tissue engineering provide an alternative approach for neural regeneration. Since the topography of a surface affects the microscopic behaviour of material; the creation of nanoscale surface features, which mimic the natural roughness of live tissue, on polymer surfaces can promote an appropriate cell growth and proliferation. In this research, a unique PLGA nanofibrous structure was fabricated without any post-electrospinning treatment. Scaffolds were prepared in two general groups: cylindrical and ribbon-shaped electrospun fibres, with smooth and rough (porous and grooved) surfaces. The experiments about nerve cell culture have demonstrated that the nanoroughness of PLGA electrospun scaffolds can increase the cell growing rate to 50 % in comparison with smooth and conventional electrospun scaffolds. SEM and AFM images and MTT assay results have shown that the roughened cylindrical scaffolds enhance the nerve growth and proliferation compared to smooth and ribbon-shaped nanofibrous scaffolds. A linear interaction has been found between cell proliferation and surface features. This helps to approximate MTT assay results by roughness parameters.     

Effect of crosslinking temperature on compression strength of gelatin scaffold for articular cartilage tissue engineering

A crosslinking method with directly crosslinking the gelatin gel by genipin was performed at 10–25 °C. The microstructure of the gelatin scaffold can be modulated by the crosslinking temperature. During repeated compression–swelling test, a plateau stress occurred. This plateau stress was relatively constant during the cyclic test. The cartilage tissue developed in the scaffold can largely enhance the compression strength of the scaffold and the scaffold with largest pore (350–500 μm, 25 °C-crosslinked) developed largest amount of the cartilage tissue after cell culture. With or without cell culture, the scaffold with smallest pore (50–150 μm, 10 °C-crosslinked) showed the highest compression strength. The pure gelatin scaffold can undergo reversible deformation for the motion of the articular cartilage.     

A novel osteochondral scaffold of ceramic–gelatin assembly for articular cartilage repair

A novel ceramic–gelatin assembly (CGA) has been designed as an osteochondral scaffold for articular cartilage repair. The CGA scaffold consists of four layers, that is, a porous ceramic layer as osseous component and also as anchor, a dense ceramic layer to prevent blood vessel penetration and also to stand shear stress, a porous ceramic layer for fixation of bone to cartilage, i.e. for joining the ceramic part to the porous gelatin layer, the latter being used as cartilaginous component. The joining was done by the infiltration of gelatin solution into the porous ceramic layer, gelling and crosslinking. This CGA scaffold can offer solutions to the so-far not satisfactorily resolved issues of the osteochondral scaffold, i.e. anchoring, blood vessel penetration, shear stress distribution during articular joint motion, and enough strength to join the cartilaginous component to the osseous component to prevent delamination. This novel scaffold was tested by in vitro cell culture with Wistar rat's joint chondrocytes. DNA assay, GAGs assay, RT-PCR, and histological evaluations with hematoxylin–eosin and Safranin-O staining were carried out to show that cartilage tissue can be developed in four weeks.     

Development of artificial articular cartilage

The present study describes the development of artificial articular cartilage on the basis of mimicking structural gel properties and mechanical gel properties of natural articular cartilage. It is synthesized from PVA/Si nanocomposite containing 20% Tetra ethoxy silane (TEOS) by sol-gel method. Mechanical strength of Poly(vinyl alcohol), PVA is improved up to 35 MPa. Manufacturing method is adopted considering colloidal stability of nano silica particle in PVA sol at specific pH = 1. An adhesive is also prepared from PVA/Si nanocomposite containing 40% TEOS for firm attachment of artificial articular cartilage on underlying bone with high bond strength.     

Dynamic nanoindentation of articular porcine cartilage

Articular cartilage is a poroelastic (biphasic) material with a complex deformation behavior, which can be considered elastic–viscoelastic. In this article, articular porcine cartilage is tested in vitro using dynamic nanoindentation and is analyzed using the frequency domain. The testing and data analysis are presented as a function of the strain rate and frequency, which allows for the results to be compared for various load amplitudes over the same frequency range. In addition, a new approach to correct the contact area during dynamic nanoindentation is presented and the effects of sample freezing on the mechanical properties are also discussed.     

Conducting cryogel scaffold as a potential biomaterial for cell stimulation and proliferation

The aim of the study was to demonstrate the potential of the cryogelation technique for the synthesis of the conducting cryogel scaffolds which would encompass the advantages of the cryogel matrix, like the mechanical strength and interconnected porous network as well as the conductive properties of the incorporated conducting polymeric material, polypyrrole. The cryogels were synthesized using different combinations of oxidizing agents and surfactants like, sodium dodecyl sulfate (SDS)/ammonium persulfate (APS), SDS/iron chloride (FeCl₃), cetyl trimethyl ammonium bromide (CTAB)/APS, and CTAB/FeCl₃. The synthesized gels were characterized by scanning electron microscopic analysis for morphology, Fourier transform infrared spectroscopy for analyzing the presence of the polypyrrole (0.5–4 %) as nano-fillers in the gel. It was observed that the presence of these nano-fillers increased the swelling ratio by approximately 50 %. The synthesized conducting cryogels displayed high stress bearing capacity without being deformed as analysed by rheological measurements. The degradation studies showed 12–15 % degradation in 4 weeks time. In vitro studies with conducting and non-conducting cryogel scaffold were carried out to optimize the stimulation conditions for the two cell lines, neuro2a and cardiac muscle C2C12. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed approximately 25 and 15 % increase in the cell proliferation rate for neuro2a and C2C12 cell line, respectively. This was observed at a specific voltage of 100 mV and 2 V, for a specified duration of 2 h and 1 min, respectively for the conducting scaffold as compared to the control. This can play an important role in tissue engineering applications for cell lines where acquiring a high cell number and functionality is desired.     

Analysis of friction between articular cartilage and polyvinyl alcohol hydrogel artificial cartilage

Many biomaterials are being used to repair damaged articular cartilage. In particular, poly vinyl alcohol hydrogel has similar mechanical properties to natural cartilage under compressive and shearing loading. Here, three-factor and two-level friction experiments and long-term tests were conducted to better evaluate its tribological properties. The friction coefficient between articular cartilage and the poly vinyl alcohol hydrogel depended primarily on the three factors of load, speed, and lubrication. When the speed increased from 10 to 20 mm/s under a load of 10 N, the friction coefficient increased from 0.12 to 0.147. When the lubricant was changed from Ringer’s solution to a hyaluronic acid solution, the friction coefficient decreased to 0.084 with loads as high as 22 N. The poly vinyl alcohol hydrogel was severely damaged and lost its top surface layers, which were transferred to the articular cartilage surface. Wear was observed in the surface morphologies, which indicated the occurrence of surface adhesion of bovine cartilage. Surface fatigue and adhesive wear was the dominant wear mechanism.     

An evaluation of human articular cartilage on femoral head

In this study, we investigated stress relaxation behavior of the human articular cartilage on femoral head. Articular cartilage is a white dense connective tissue that covers the bone ends within diarthrodial joints and works as a weight-transmitting and energy-absorbing material. Human articular cartilage on femoral head was used as test material. Relaxation tests were carried out by using the indentation technique via Instron Universal Testing Machine. Test materials were investigated in an isotonic salt solution at 37 °C. To keep the temperature constant, two vessels being in each other were utilized. Thus, hot water was circulated in the outer vessel and isotonic salt solution was kept in the inner vessel. Experimental results showed that there is a remarkable difference between normal and degenerated cartilage for the same age and sex. It was observed that the relaxation percent of normal cartilage as a function of relaxation time is much higher than that of degenerated cartilage.     

The influence of implant surface properties on cell adhesion and proliferation

Interactions of the foreign material of implant and the living tissue on the cell level can cause prolonged healing or, worse, loss of the implant. The cell response to the presence of some implant materials was studied under in vitro conditions. The influence of physicochemical surface parameters on the response of the cells in the immediate vicinity of implants, namely on adhesion, proliferation and synthetic activity of fibroblasts, and on the blood coagulation were compared. The direct contact of tested materials (titanium and Ti6Al4V alloy with various surface treatments, Cr Co Mo alloy, hydroxyapatite-coated titanium, zirconium oxide ceramics, polyethylene and carbon composite) on cell spreading was monitored and the presence of TNF-α and IL-8 was evaluated in the cultivation medium. The formation of blood clots was investigated on samples immersed in a well with freshly drawn whole rabbit blood using a scanning electron microscope. The surface free energy was estimated using the measurement of static contact angle. Both the advancing and receding contact angles were measured by the dynamic Wilhemy plate method. Two main groups with extremes in cell viability were established. In the first group the increased polar component of surface free energy, the highest cell density, the lowest inflammatory cytokine production, but no fibres in the clotting blood were found. On the contrary, the second group of materials with a very low polar component of the surface free energy showed distinctly higher expression of inflammatory mediators, low cell proliferation, but faster formation of fibres in the blood coagulum.     

Control of cell proliferation by a porous chitosan scaffold with multiple releasing capabilities

Abstract

The aim of this study was to develop a porous chitosan scaffold with long-acting drug release as an artificial dressing to promote skin wound healing. The dressing was fabricated by pre-freezing at different temperatures (?20 and ?80 °C) for different periods of time, followed by freeze-drying to form porous chitosan scaffolds with different pore sizes. The chitosan scaffolds were then used to investigate the effect of the controlled release of fibroblast growth factor-basic (bFGF) and transforming growth factor-β1 (TGFβ1) on mouse fibroblast cells (L929) and bovine carotid endothelial cells (BEC). The biocompatibility of the prepared chitosan scaffold was confirmed with WST-1 proliferation and viability assay, which demonstrated that the material is suitable for cell growth. The results of this study show that the pore sizes of the porous scaffolds prepared by freeze-drying can change depending on the pre-freezing temperature and time via the formation of ice crystals. In this study, the scaffolds with the largest pore size were found to be 153 ± 32 μm and scaffolds with the smallest pores to be 34 ± 9 μm. Through cell culture analysis, it was found that the concentration that increased proliferation of L929 cells for bFGF was 0.005 to 0.1 ng/mL, and the concentration for TGFβ1 was 0.005 to 1 ng/mL. The cell culture of the chitosan scaffold and growth factors shows that 3.75 ng of bFGF in scaffolds with pore sizes of 153 ± 32 μm can promote L929 cell proliferation, while 400 pg of TGFβ1 in scaffolds with pore size of 34 ± 9 μm can enhance the proliferation of L929 cells, but also inhibit BEC proliferation. It is proposed that the prepared chitosan scaffolds can form a multi-drug (bFGF and TGFβ1) release dressing that has the ability to control wound healing via regulating the proliferation of different cell types.     

A hydrophobically-modified alginate gel system: utility in the repair of articular cartilage defects

Alginate is a family of natural polysaccharides, widely used in industry and medicine for many applications, with its non toxic nature, gentle sol/gel transition procedure and low cost, alginate inferior biomechanical properties have limited its utility especially in tissue engineering. Additionally, ionically cross-linked alginate hydrogels generally lose most of their initial mechanical and swelling properties within a few hours in physiological solution. In order to overcome these limitations, the referenced alginate was treated by covalent fixation of octadecyl chains onto the polysaccharide backbone by esterification. In semi dilute solution, intermolecular hydrophobic interactions of long alkyl chains result in the formation of physical hydrogels, which can then be reinforced by the addition of calcium chloride. FTIR studies clearly showed the presence of ester bonds at 1612 and 1730?cm(-1) indicating that the alkyl groups are incorporated in the backbone of resulting polymer. The endothermic peak and exothermic peak present in the DSC thermogram of Alg-C18 had shifted to lower temperatures comparing to native alginate (from 106 to 83°C and from 250 to 245°C, respectively) due to the esterification reaction that leads to high hydrophobic nature of the modified sample. From rheological experiments, it can be inferred that the combination of both calcium bridges and intermolecular hydrophobic interaction in the treated alginate leads to enhanced gel strength accompanied by more stable structure in physiological solution comparing to native sodium alginate hydrogel. Finally, the modified alginate tended to have no toxic effects on mesenchymal stem cell culture, rather it supported MSC chondrogenic differentiation.     

High resolution X-ray tomography – three-dimensional characterisation of cell–scaffold constructs for cartilage tissue engineering

Abstract

Synchrotron radiation based microcomputed tomography (SR-μCT) has become a valuable tool for the structural analysis of different types of biomaterials. This methodology allows the non-destructive investigation of specimens in their three-dimensional context. In the present paper, articular cartilage is taken as an exemplary tissue to demonstrate the suitability of the SR-μCT method for the investigation of biomaterials for different tissue engineering approaches. Thus, a biodegradable scaffold for cartilage tissue engineering in different modifications was analysed. Using enhanced phase contrast imaging, it was possible to demonstrate single cells without further metal staining. The three-dimensional data acquired for each investigated sample allowed qualitative and quantitative analyses without irreversibly damaging the samples. The use of the phase contrast mode enables the analysis of single cells within a scaffold material even under mechanical stimulation. This opens up innovative perspectives for the future study of the behaviour of cells in their three-dimensional environment and the non-destructive study of morphogenesis in cell–scaffold constructs.     

Impact testing to determine the mechanical properties of articular cartilage in isolation and on bone

The biomechanical response of cartilage to impact loads, both in isolation and in situ on its bone substrate, has been little studied despite the common occurrence of osteoarthritis subsequent to cartilage injury. An instrumented drop tower was used to apply controlled impact loads of different energies to explants of bovine articular cartilage. Results were compared with a conventional slow stress-strain test. The effects of the underlying bone were investigated by progressively shortening a core of bone removed with the cartilage, and by gluing cartilage samples to substrates of different moduli. The maximum dynamic modulus of isolated samples of bovine articular cartilage, at strain rates between 1100 and 1500 s⁻¹, was approximately two orders of magnitude larger than the quasistatic modulus and varied non-linearly with applied stress. When attached to a substrate of higher modulus, increasing the thickness of the substrate increased the effective modulus of the combination until a steady value was achieved. A lower modulus substrate reduced the effective modulus of the combination. Severe impacts resulted in damage to the bone rather than to the cartilage. The modulus of cartilage rises rapidly and non-linearly with strain rate, giving the tissue a remarkable ability to withstand impact loads. The presence of cartilage attenuated the peak force experienced by the bone and spread the impact loading period over a longer time.     

Crystal aggregates in articular cartilage as observed in the SEM

Scanning electron microscopy, in conjunction with freeze-fracturing and freeze-drying preparation techniques, was used to characterize the morphology and distribution of crystal deposits foundin situ in the articular cartilage of threepost mortem human knee joints. Energy dispersive analysis and X-ray diffraction were used to analyse the chemical composition of the individual crystals. Results included a unique observation of an aggregate of bow-shaped monosodium urate monohydrate crystals within the mid-zone of the articular cartilage, and two types of a large aggregate of calcium pyrophosphate dihydrate crystals distributed throughout the thickness of the cartilage. These results provided evidence of an organized crystal distribution and therefore supported the idea that local growth factors are important.     

A study of crystalline biomaterials for articular cartilage bioengineering

This study examines the suitability of marine origin coral species, Porites lutea (POR) and the hydrozoan Millepora dichotoma (MIL), for use as novel three dimensional growth matrices in the field of articular cartilage tissue engineering. Therefore, mesenchymal stem cells (MSCs) and chondrocytes were grown on the skeletal material obtained from each of these two organisms to investigate their potential use as three dimensional scaffolding for cartilage tissue growth. Chondrogenic induction of MSCs was achieved by addition of transforming growth factor-β1 (TGF-β1) and insulin growth factor-I (IGF-I). Cell adherence, proliferation, differentiation and tissue development were investigated through six weeks of culture. Cartilage tissue growth and chondrocytic phenotype maintenance of each cell type were examined by cell morphology, histochemical analyses, expression of collagen type II and quantitative measures of glycosaminoglycan (GAG) content. The MSCs and the chondrocytes were shown good adherence to the scaffolds and maintenance of the chondrocytic phenotype in the initial stages of culture. However after two weeks of culture on MIL and three weeks on POR these cultures began to exhibit signs of further differentiation and phenotypic loss. The shown results indicated that POR was a better substrate for chondrocytes phenotype maintenance than MIL. We believe that surface modification of POR combined with mechanical stimuli will provide a suitable environment for chondrogenic phenotype maintenance. Further investigation of POR and other novel coralline biomatrices is indicated and warranted in the field of cartilage tissue engineering applications.