In vitro and in vivo evaluation of chitosan–gelatin scaffolds for cartilage tissue engineering

Chitosan–gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan–gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1_WSC) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1_WSC promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5 months. The amount of glycosaminoglycans in C1G1_WSC constructs (0.187 ± 0.095 μg/mg tissue) harvested from the animals after 6.5 months was 14 wt.% of that in normal cartilage (1.329 ± 0.660 μg/mg tissue). The average compressive modulus of regenerated tissue at 6.5 months was about 0.539 MPa, which approached to that of normal cartilage (0.735 MPa), while that in the blank control (3.881 MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1_WSC constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1_WSC scaffolds may enhance the cartilage regeneration in vitro and in vivo.     

Supermacroprous chitosan–agarose–gelatin cryogels: in vitro characterization and in vivo assessment for cartilage tissue engineering

The study focuses on the synthesis of a novel polymeric scaffold having good porosity and mechanical characteristics synthesized by using natural polymers and their optimization for application in cartilage tissue engineering. The scaffolds were synthesized via cryogelation technology using an optimized ratio of the polymer solutions (chitosan, agarose and gelatin) and cross-linker followed by the incubation at sub-zero temperature (−12°C). Microstructure examination of the chitosan–agarose–gelatine (CAG) cryogels was done using scanning electron microscopy (SEM) and fluorescent microscopy. Mechanical analysis, such as the unconfined compression test, demonstrated that cryogels with varying chitosan concentrations, i.e. 0.5–1% have a high compression modulus. In addition, fatigue tests revealed that scaffolds are suitable for bioreactor studies where gels are subjected to continuous cyclic strain. In order to confirm the stability, cryogels were subjected to high frequency (5 Hz) with 30 per cent compression of their original length up to 1 × 10⁵ cycles, gels did not show any significant changes in their mass and dimensions during the experiment. These cryogels have exhibited degradation capacity under aseptic conditions. CAG cryogels showed good cell adhesion of primary goat chondrocytes examined by SEM. Cytotoxicity of the material was checked by MTT assay and results confirmed the biocompatibility of the material. In vivo biocompatibility of the scaffolds was checked by the implantation of the scaffolds in laboratory animals. These results suggest the potential of CAG cryogels as a good three-dimensional scaffold for cartilage tissue engineering.     

Injectable calcium phosphate–alginate–chitosan microencapsulated MC3T3-E1 cell paste for bone tissue engineering in vivo

Osteoblasts or stem cells have been delivered into injectable calcium phosphate cement (CPC) to improve its effectiveness and biological function. However, the osteogenic potential of the new construct in vivo has been rarely reported, and there are no reports on alginate–chitosan microencapsulated osteoblasts mixed with CPC. This study aimed to develop alginate–chitosan microencapsulated mouse osteoblast MC3T3-E1 cells (AC-cells), evaluate the osteogenic potential of a calcium phosphate cement complex with these AC-cells (CPC-AC-cell), and trace the implanted MC3T3-E1 cells in vivo. MC3T3-E1 cells were embedded in alginate microcapsules, cultured in osteogenic medium for 7 days, and then covered with chitosan before mixing with a paste of β-tricalcium phosphate/calcium phosphate cement (β-TCP/CPC). The construct was injected into the dorsal subcutaneous area of nude mice. Lamellar-bone-like mineralization, newly formed collagen and angiogenesis were observed at 4 weeks. At 8 weeks, areas of newly formed collagen expanded; further absorption of β-TCP/CPC and osteoid-like structures could be seen. Cell tracing in vivo showed that implanted MC3T3-E1 cells were clearly visible at 2 weeks. These in vivo results indicate that the novel injectable CPC-AC-cell construct is promising for bone tissue engineering applications.     

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.     

Cell-laden photocrosslinked GelMA–DexMA copolymer hydrogels with tunable mechanical properties for tissue engineering

To effectively repair or replace damaged tissues, it is necessary to design three dimensional (3D) extracellular matrix (ECM) mimicking scaffolds with tunable biomechanical properties close to the desired tissue application. In the present work, gelatin methacrylate (GelMA) and dextran glycidyl methacrylate (DexMA) with tunable mechanical and biological properties were utilized to prepared novel bicomponent polymeric hydrogels by cross-linking polymerization using photoinitiation. We controlled the degree of substitution (DS) of glycidyl methacrylate in DexMA so that they could obtain relevant mechanical properties. The results indicated that copolymer hydrogels demonstrated a lower swelling ratio and higher compressive modulus as compared to the GelMA. Moreover, all of the hydrogels exhibited a honeycomb-like architecture, the pore sizes decreased as DS increased, and NIH-3T3 fibroblasts encapsulated in these hydrogels all exhibited excellent viability. These characteristics suggest a class of photocrosslinkable, tunable mechanically copolymer hydrogels that may find potential application in tissue engineering and regenerative medicine applications.     

Tissue differentiation in an in vivo bioreactor: in silico investigations of scaffold stiffness

Scaffold design remains a main challenge in tissue engineering due to the large number of requirements that need to be met in order to create functional tissues in vivo. Computer simulations of tissue differentiation within scaffolds could serve as a powerful tool in elucidating the design requirements for scaffolds in tissue engineering. In this study, a lattice-based model of a 3D porous scaffold construct derived from micro CT and a mechano-biological simulation of a bone chamber experiment were combined to investigate the effect of scaffold stiffness on tissue differentiation inside the chamber. The results indicate that higher scaffold stiffness, holding pore structure constant, enhances bone formation. This study demonstrates that a lattice approach is very suitable for modelling scaffolds in mechano-biological simulations, since it can accurately represent the micro-porous geometries of scaffolds in a 3D environment and reduce computational costs at the same time.     

Evaluation of 3D nano–macro porous bioactive glass scaffold for hard tissue engineering

Recently, nano–macro dual-porous, three-dimensional (3D) glass structures were developed for use as bioscaffolds for hard tissue regeneration, but there have been concerns regarding the interconnectivity and homogeneity of nanopores in the scaffolds, as well as the cytotoxicity of the environment deep inside due to limited fluid access. Therefore, mercury porosimetry, nitrogen absorption, and TEM have been used to characterize nanopore network of the scaffolds. In parallel, viability of MG 63 human osteosarcoma cells seeded on scaffold surface was investigated by fluorescence, confocal and electron microscopy methods. The results show that cells attach, migrate and penetrate inside the glass scaffold with high proliferation and viability rate. Additionally, scaffolds were implanted under the skin of a male New Zealand rabbit for in vivo animal test. Initial observations show the formation of new tissue with blood vessels and collagen fibers deep inside the implanted scaffolds with no obvious inflammatory reaction. Thus, the new nano–macro dual-porous glass structure could be a promising bioscaffold for use in regenerative medicine and tissue engineering for bone regeneration.     

Evaluation of polyethersulfone highflux hemodialysis membrane in vitro and in vivo

A new hemodialysis membrane manufactured by a blend of polyethersulfone (PES) and polyvinylpyrrolidone (PVP) was evaluated in vitro and in vivo. Goat was selected as the experimental animal. The clearance and the reduction ratio after the hemodialysis of small molecules (urea, creatinine, phosphate) for the PES membrane were higher in vitro than that in vivo. The reduction ratio of β₂-microglobulin was about 50% after the treatment for 4 h. The biocompatibility profiles of the membranes indicated slight neutropenia and platelet adhesion at the initial stage of the hemodialysis. Electrolyte, blood gas, and blood biochemistry were also analyzed before and after the treatment. The results indicated that PES hollow fiber membrane had a potential widely use for hemodialysis.     

Fabrication and evaluation of biomimetic scaffolds by using collagen–alginate fibrillar gels for potential tissue engineering applications

Pore architecture and its stable functionality under cell culturing of three dimensional (3D) scaffolds are of great importance for tissue engineering purposes. In this study, alginate was incorporated with collagen to fabricate collagen–alginate composite scaffolds with different collagen/alginate ratios by lyophilizing the respective composite gels formed via collagen fibrillogenesis in vitro and then chemically crosslinking. The effects of alginate amount and crosslinking treatment on pore architecture, swelling behavior, enzymatic degradation and tensile property of composite scaffolds were systematically investigated. The relevant results indicated that the present strategy was simple but efficient to fabricate highly interconnected strong biomimetic 3D scaffolds with nanofibrous surface. NIH3T3 cells were used as a model cell to evaluate the cytocompatibility, attachment to the nanofibrous surface and porous architectural stability in terms of cell proliferation and infiltration within the crosslinked scaffolds. Compared with the mechanically weakest crosslinked collagen sponges, the cell-cultured composite scaffolds presented a good porous architecture, thus permitting cell proliferation on the top surface as well as infiltration into the inner part of 3D composite scaffolds. These composite scaffolds with pore size ranging from 150 to 300 μm, over 90% porosity, tuned biodegradability and water-uptake capability are promising for tissue engineering applications.     

A polylactide/fibrin gel composite scaffold for cartilage tissue engineering: fabrication and an in vitro evaluation

A composite scaffold for cartilage tissue engineering was fabricated by filling a porous poly (l-lactide) (PLLA) scaffold with fibrin gel. The porous PLLA scaffold prepared by a method of thermally induced phase separation has an average pore diameter of 200 μm and a porosity of 93%. Incorporation of fibrin gel into the scaffold was achieved by dropping a fibrinogen and thrombin mixture solution onto the scaffold. For a couple of minutes the fibrin gel was in situ formed within the scaffold. The filling efficiency was decreased along with the increase of the fibrinogen concentration. After fibrin gel filling, the compressive modulus and the yield stress increased from 5.94 MPa and 0.37 MPa (control PLLA scaffold in a hydrated state) to 7.21 MPa and 0.53 MPa, respectively. While the fibrin gel lost its weight in phosphate buffered saline up to ~50% within 3 days, 85% and 70% of the fibrin gel weight in the composite scaffold was remained within 3 and 35 days, respectively. A consistent significant higher level of rabbit auricular chondrocyte viability, cell number and glycosaminoglycan was measured in the composite scaffold than that in the control PLLA scaffold. Rabbit auricular chondrocytes with round morphology were also observed in the composite scaffold by confocal microscopy and scanning electron microscopy. Altogether with the features of better strength and cytocompatibility, this type of composite scaffold may have better performance as a matrix for cartilage tissue engineering.     

Micro- and nanotechnology in cardiovascular tissue engineering

While in nature the formation of complex tissues is gradually shaped by the long journey of development, in tissue engineering constructing complex tissues relies heavily on our ability to directly manipulate and control the micro-cellular environment in?vitro. Not surprisingly, advancements in both microfabrication and nanofabrication have powered the field of tissue engineering in many aspects. Focusing on cardiac tissue engineering, this paper highlights the applications of fabrication techniques in various aspects of tissue engineering research: (1)?cell responses to micro-?and nanopatterned topographical cues, (2)?cell responses to patterned biochemical cues, (3)?controlled 3D scaffolds, (4)?patterned tissue vascularization and (5)?electromechanical regulation of tissue assembly and function.     

Hyaluronan-based heparin-incorporated hydrogels for generation of axially vascularized bioartificial bone tissues: in vitro and in vivo evaluation in a PLDLLA–TCP–PCL-composite system

Smart matrices are required in bone tissue-engineered grafts that provide an optimal environment for cells and retain osteo-inductive factors for sustained biological activity. We hypothesized that a slow-degrading heparin-incorporated hyaluronan (HA) hydrogel can preserve BMP-2; while an arterio–venous (A–V) loop can support axial vascularization to provide nutrition for a bio-artificial bone graft. HA was evaluated for osteoblast growth and BMP-2 release. Porous PLDLLA–TCP–PCL scaffolds were produced by rapid prototyping technology and applied in vivo along with HA-hydrogel, loaded with either primary osteoblasts or BMP-2. A microsurgically created A–V loop was placed around the scaffold, encased in an isolation chamber in Lewis rats. HA-hydrogel supported growth of osteoblasts over 8 weeks and allowed sustained release of BMP-2 over 35 days. The A–V loop provided an angiogenic stimulus with the formation of vascularized tissue in the scaffolds. Bone-specific genes were detected by real time RT-PCR after 8 weeks. However, no significant amount of bone was observed histologically. The heterotopic isolation chamber in combination with absent biomechanical stimulation might explain the insufficient bone formation despite adequate expression of bone-related genes. Optimization of the interplay of osteogenic cells and osteo-inductive factors might eventually generate sufficient amounts of axially vascularized bone grafts for reconstructive surgery.     

Fabrication of chitosan/poly(ε-caprolactone) composite hydrogels for tissue engineering applications

The aim of this study was to fabricate three-dimensional (3D) porous chitosan/poly(ε-caprolactone) (PCL) hydrogels with improved mechanical properties for tissue engineering applications. A modified emulsion lyophilisation technique was developed to produce 3D chitosan/PCL hydrogels. The addition of 25 and 50 wt% of PCL into chitosan substantially enhanced the compressive strength of composite hydrogel 160 and 290%, respectively, compared to pure chitosan hydrogel. The result of ATR–FTIR imaging corroborated that PCL and chitosan were well mixed and physically co-existed in the composite structures. The composite hydrogels were constructed of homogenous structure with average pore size of 59.7 ± 14 μm and finer pores with average size of 4.4 ± 2 μm on the wall of these larger pores. The SEM and confocal laser scanning microscopy images confirmed that fibroblast cells were attached and proliferated on the 3D structure of these composite hydrogels. The composite hydrogels acquired in this study possessed homogeneous porous structure with improved mechanical strength and integrity. They may have a high potential for the production of 3D hydrogels for tissue engineering applications.     

Novel mineralized heparin–gelatin nanoparticles for potential application in tissue engineering of bone

Nanoparticles (NPs) were prepared from succinylated gelatin (s-GL) cross-linked with aldehyde heparin (a-HEP) and used subsequently as a nano-template for the mineralization of hydroxyapatite (HAP). Gelatin was functionalized with succinyl groups that made it soluble at room temperature. Heparin was oxidized to generate aldehyde groups and then used as a cross-linker that can react with s-GL to form NPs via Schiff’s base linkage. The polymer concentrations, feed molar ratios and pH conditions were varied to fabricate NPs suspension. NPs were obtained with a spheroid shape of an average size of 196 nm at pH 2.5 and 202 nm at pH 7.4. These NPs had a positive zeta potential of 7.3 ± 3.0 mV and a narrow distribution with PDI 0.123 at pH 2.5, while they had a negative zeta potential of ?2.6 ± 0.3 mV and formed aggregates (PDI 0.257) at pH 7.4. The NPs prepared at pH 2.5 with a mean particle size of 196 nm were further used for mineralization studies. The mineralization process was mediated by solution without calcination at 37 °C. The HAP formed on NPs was analyzed by Fourier transform infrared spectroscopy and X-ray diffraction. HAP coated s-GL/a-HEP NPs developed in this study may be used in future as osteoinductive fillers enhancing the mechanical properties of injectable hydrogel or use as potential multifunctional device for nanotherapeutic approaches.     

Human tissue allograft processing: impact on in vitro and in vivo biocompatibility

This work investigates the impact of chemical and physical treatments on biocompatibility for human bone/tendon tissues. Nontreated and treated tissues were compared. In vitro testing assessed indirect and direct cytotoxicity. Tissues were subcutaneously implanted in rats to assess the immunological, recolonization, and revascularization processes at 2–4 weeks postimplantation. No significant cytotoxicity was found for freeze-dried treated bones and tendons in comparison to control. The cellular adhesion was significantly reduced for cells seeded on these treated tissues after 24 h of direct contact. A significant cytotoxicity was found for frozen treated bones in comparison to freeze-dried treated bones. Tissue remodeling with graft stability, no harmful inflammation, and neo-vascularization was observed for freeze-dried chemically treated bones and tendons. Frozen-treated bones were characterized by a lack of matrix recolonization at 4 weeks postimplantation. In conclusion, chemical processing with freeze-drying of human tissues maintains in vitro biocompatibility and in vivo tissue remodeling for clinical application.     

Chemical hydrogels based on a hyaluronic acid-graft-α-elastin derivative as potential scaffolds for tissue engineering

In this work hyaluronic acid (HA) functionalized with ethylenediamine (EDA) has been employed to graft α-elastin. In particular a HA-EDA derivative bearing 50 mol% of pendant amino groups has been successfully employed to produce the copolymer HA-EDA-g-α-elastin containing 32% w/w of protein. After grafting with α-elastin, remaining free amino groups reacted with ethylene glycol diglycidyl ether (EGDGE) for producing chemical hydrogels, proposed as scaffolds for tissue engineering. Swelling degree, resistance to chemical and enzymatic hydrolysis, as well as preliminary biological properties of HA-EDA-g-α-elastin/EGDGE scaffold have been evaluated and compared with a HA-EDA/EGDGE scaffold. The presence of α-elastin grafted to HA-EDA improves attachment, viability and proliferation of primary rat dermal fibroblasts and human umbilical artery smooth muscle cells. Biological performance of HA-EDA-g-α-elastin/EGDGE scaffold resulted comparable to that of a commercial collagen type I sponge (Antema®), chosen as a positive control.     

Reduction of hygrothermal transverse stresses in unidirectional hybrid composites under cyclic environmental conditions

When fiber-reinforced polymer plates are exposed to cyclic environmental conditions, polymer matrix absorbs or desorbs continuously the moisture due to the variation in service temperature and relative humidity. Both temperature and moisture concentration produce an important hygrothermal transverse stresses, which are maximum on both edges of the composite plates. These transverse stresses which are more important at first times of moisture diffusion, can produce a probable damage of composite plates. To extend the durability of our composite plate, interplay hybrid composites are adopted to reduce the transverse stresses on edges. Therefore, a variation of the relationship between thicknesses of unidirectional hybrid composites constituents AS/3501-5 and T300/5208 is carried out in order to find minimal transverse stresses. This thicknesses variation enables us to find the best configuration which gives favourable service conditions of our hybrid composite, i.e., to predict firstly a considerable reduction of hygrothermal transverse stresses at both edges of our hybrid plate, secondly to reduce or to attenuate the edge effect developed in 6 days and 6 weeks periods.     

Crosslinked collagen–gelatin–hyaluronic acid biomimetic film for cornea tissue engineering applications

Cornea disease may lead to blindness and keratoplasty is considered as an effective treatment method. However, there is a severe shortage of donor corneas worldwide. This paper presents the crosslinked collagen (Col)–gelatin (Gel)–hyaluronic acid (HA) films developed by making use of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as the crosslinker. The test results on the physical and biological properties indicate that the CGH631 film (the mass ratio of Col:Gel:HA = 6:3:1) has appropriate optical performance, hydrophilicity and mechanical properties. The diffusion properties of the CGH631 film to NaCl and tryptophan are also satisfactory and the measured data are 2.43 × 10^? 6 cm²/s and 7.97 × 10^? 7 cm²/s, respectively. In addition, cell viability studies demonstrate that the CGH631 film has good biocompatibility, on which human corneal epithelial cells attached and proliferated well. This biocompatible film may have potential use in cornea tissue engineering.     

Preparation and comparative characterization of keratin–chitosan and keratin–gelatin composite scaffolds for tissue engineering applications

We report fabrication of three dimensional scaffolds with well interconnected matrix of high porosity using keratin, chitosan and gelatin for tissue engineering and other biomedical applications. Scaffolds were fabricated using porous Keratin–Gelatin (KG), Keratin–Chitosan (KC) composites. The morphology of both KG and KC was investigated using SEM. The scaffolds showed high porosity with interconnected pores in the range of 20–100 μm. They were further tested by FTIR, DSC, CD, tensile strength measurement, water uptake and swelling behavior. In vitro cell adhesion and cell proliferation tests were carried out to study the biocompatibility behavior and their application as an artificial skin substitute. Both KG and KC composite scaffolds showed similar properties and patterns for cell proliferation. Due to rapid degradation of gelatin in KG, we found that it has limited application as compared to KC scaffold. We conclude that KC scaffold owing to its slow degradation and antibacterial properties would be a better substrate for tissue engineering and other biomedical application.