Injectable gels for tissue engineering

Recently, tissue engineering approaches using injectable, in situ gel forming systems have been reported. In this review, the gelation processes and several injectable systems that exhibit in situ gel formation at physiological conditions are discussed. Applications of selected injectable systems (alginate, chitosan, hyaluronan, polyethylene oxide/polypropylene oxide) in tissue engineering are also described. Injectable polymer formulation can gel in vivo in response to temperature change (thermal gelation), pH change, ionic cross-linking, or solvent exchange. Kinetics of gelation is directly affected by its mechanism. Injectable formulations offer specific advantages over preformed scaffolds such as: possibility of a minimally invasive implantation, an ability to fill a desired shape, and easy incorporation of various therapeutic agents. Several factors need to be considered before an injectable gel can be selected as a candidate for tissue engineering applications. Apart from tissue-specific cell-matrix interactions, the following gel properties need to be considered: gelation kinetics, matrix resorption rate, possible toxicity of degradation products and their elimination routes, and finally possible interference of the gel matrix with histogenesis.     

ECM-based triple layered scaffolds for vascular tissue engineering

The present study focused on the development of three layered small-diameter (<6 mm) extracellular matrix (ECM)-based vessels. These were engineered artificially through the freeze-drying technique. A layer of decellularized bovine aorta (DAM) was deposited on a mandrel and, after lyophilization, it was dipped into a poly-L-lactide acid (PLLA)/polyethylene glycol (PEG) 2000 dichloromethane solution then quickly wrapped with a pre-prepared thin DAM sheet. Mechanical properties of three-layered scaffolds were evaluated by means of uniaxial tensile measurement. Furthermore, human endothelial and smooth muscle cells were seeded on internal and external scaffold surfaces, respectively, and co-cultured for 7 days. Our results demonstrate that i) ECM components provide suitable stimuli for cell adhesion and proliferation, ii) the microporous intermediate PLLA/PEG2000 layer is responsible for the scaffold resistance and iii) the layered deposition technique can be considered a valuable method to obtain layered vascular scaffolds of different sizes and with a good compromise between stiffness and elasticity for optimal cell organization.     

Injectable alginate hydrogels for cell delivery in tissue engineering

Alginate hydrogels are extremely versatile and adaptable biomaterials, with great potential for use in biomedical applications. Their extracellular matrix-like features have been key factors for their choice as vehicles for cell delivery strategies aimed at tissue regeneration. A variety of strategies to decorate them with biofunctional moieties and to modulate their biophysical properties have been developed recently, which further allow their tailoring to the desired application. Additionally, their potential use as injectable materials offers several advantages over preformed scaffold-based approaches, namely: easy incorporation of therapeutic agents, such as cells, under mild conditions; minimally invasive local delivery; and high contourability, which is essential for filling in irregular defects. Alginate hydrogels have already been explored as cell delivery systems to enhance regeneration in different tissues and organs. Here, the in vitro and in vivo potential of injectable alginate hydrogels to deliver cells in a targeted fashion is reviewed. In each example, the selected crosslinking approach, the cell type, the target tissue and the main findings of the study are highlighted.     

Injectable biodegradable polycaprolactone-sebacic acid gels for bone tissue engineering

Tissue engineering constitutes a promising alternative technology to transplantation medicine by creating viable substitutes for failing tissues or organs. The ability to manipulate and reconstitute tissue function has tremendous clinical implications and will most likely play a key role in cell and gene therapies in the coming years. In the present work, a novel injectable and biodegradable biomaterial is reported that could be injected on the human body with a surgical syringe. The material prepared is a blend of polycaprolactone (PCL), a biodegradable and elastic biomedical polymer, and sebacic acid, a natural polymer part of castor oil with low molecular weight to accelerate the slow degradation rate of PCL. The biocompatibility of the blend was evaluated in vitro and its in vivo behavior was also assessed through subcutaneous and bone implantation in rats to evaluate its tissue-forming ability and degradation rate. The results allowed the conclusion that the gel is biocompatible, promotes the differentiation of mesenchymal stem cells, and presents an adequate degradation rate for use in bone tissue engineering. In vivo the gel blends promoted tissue regeneration and adverse reactions were not observed on subcutaneous and bone implants.     

Evaluation of sodium alginate for bone marrow cell tissue engineering

Sodium alginate has applications as a material for the encapsulation and immobilisation of a variety of cell types for immunoisolatory and biochemical processing applications. It forms a biodegradable gel when crosslinked with calcium ions and it has been exploited in cartilage tissue engineering since chondrocytes do not dedifferentiate when immobilised in it. Despite its attractive properties of degradability, ease of processing and cell immobilisation, there is little work demonstrating the efficacy of alginate gel as a substrate for cell proliferation, except when RGD is modified. In this study we investigated the ability of rat bone marrow cells to proliferate and differentiate on alginates of differing composition and purity. The mechanical properties of the gels were investigated. It was found that high purity and high G-type alginate retained 27% of its initial strength after 12 days in culture and that comparable levels of proliferation were observed on this material and tissue culture plastic. Depending on composition, calcium crosslinked alginate can act as a substrate for rat marrow cell proliferation and has potential for use as 3D degradable scaffold.     

Photochemically cross-linked collagen gels as three-dimensional scaffolds for tissue engineering

Collagen gels have many favorable attributes for tissue engineering, but the gels undergo dramatic contraction when cells are added because of the weak noncovalent bonds that form during spontaneous gelation. We hypothesized that photochemically cross-linking collagen gels would make suitable scaffolds for tissue engineering with favorable cell viability and minimal gel contraction. Rose Bengal and riboflavin were chosen as candidate photo-initiators for gel cross-linking using 532- and 458-nm-light wavelengths, respectively. Chondrocyte viability was measured after initial gelation for several concentrations of initiators. Cell viability and gel contraction were then measured using chondrocytes and fibroblasts over 7 days of culture. Rose Bengal used at concentrations necessary for gelation resulted in little or no cell viability. Short-term viability results showed that 0.25- or 0.5-mM concentrations of riboflavin, and 40 s of illumination permitted more than 90% cell viability. Using riboflavin concentrations of 0.25 or 0.5 mM, long-term chondrocyte viability was 113.1 +/- 11.6% and 25.4 +/- 2.7%, respectively, at day 7. Although non-cross-linked chondrocyte constructs contracted to 59.9 +/- 11.8% of their original diameter and fibroblasts contracted to 24.9 +/- 5.0% of their original diameter by day 7, the cross-linked constructs retained 88.8 +/- 7.4% and 85.5 +/- 5.0% of the original diameter, respectively. In conclusion, by photochemically cross-linking collagen gels using riboflavin and visible light, stable gel scaffolds with favorable cell survival can be produced.     

Current status of tissue engineering for cardiovascular structures

Various vascular and valvlular grafts are commonly used in the treatment of cardiovascular disease. Current prosthetic or bioprosthetic materials lack growth potential, and therefore, subsequent replacement further defeats the concept of primary repair early in pediatric cardiac patients. Tissue engineering is a new discipline that offers the potential to create replacement structures from autologous cells and biodegradable polymer scaffolds. Because tissue-engineering constructs contain living cells, they may have the potential for growth, self-repair, and self-remodeling. Cardiac valve leaflets and large conduits in the pulmonary ciruulation have been made with this tissue-engineering approach in lambs. Venous conduits were also created in dogs. Mixed cell populations of endothelial cells and fibroblasts were isolated from explanted peripheral arteries or vein. A synthetic biodegradable scaffold con-sisting of polyglactin and polyglycolic acid fibers was seeded in vitro with mixed cultured cells. After one week, these autologous cell/polymer constructs were reimplanted in animals. Each animal was then followed periodically by echocardiography and angiography. The animals were sacrificed, and the implanted tissues were examined histologically, biochemically, and biomechanically. A 4-hydroxyproline assay was performed to evaluate the collagen content. The implanted conduit diameters increased as the animals grew during the study period. Histologically, the biodegradable polymer scaffold was completely degraded. Collagen analysis of the constructs showed the development of an extracellular matrix. Immunohistochemical staining demonstrated elastin fiber in the matrix and factor VIII on the inner surface of the conduits. In conclusion, a tissue-engineering approach to constructing cardiovascular structures is feasible using cells of either arterial or venous origin. In these tissue-engineered autografts, transplanted autologous cells generated the proper matrix over the polymer scaffold under physiologic conditions.     

Adipose tissue engineering using injectable, oxidized alginate hydrogels

Current treatment modalities for soft tissue augmentation which use autologous grafting and commercially available fillers present a number of challenges and limitations, such as donor site morbidity and volume loss over time. Adipose tissue engineering technology may provide an attractive alternative. This study investigated the feasibility of a degradable alginate hydrogel system with commercially available cryopreserved human adipose stem cells (hADSCs) to engineer adipose tissue. hADSCs were differentiated into adipogenic cells, and encapsulated in alginate hydrogels made susceptible to hydrolysis by partial periodate oxidation of the polymer chains. Cell laden gels were subcutaneously injected into the chest wall of male nude mice, and a cell suspension without alginate served as control. After 10 weeks, specimens were harvested and analyzed morphologically, histologically, and with immunoblotting of tissue extractions. Newly generated tissues were semitransparent and soft in all experimental mice, grossly resembling adipose tissue. Analysis using confocal live imaging, immunohistochemisty and western blot analysis revealed that the newly generated tissue was adipose tissue. This study demonstrates that degradable, injectable alginate hydrogels provide a suitable delivery vehicle for preconditioned cryopreserved hADSCs to engineer adipose tissue.     

High density type I collagen gels for tissue engineering of whole menisci

This study investigates the potential of high density type I collagen gels as an injectable scaffold for tissue engineering of whole menisci, and compares these results with previous strategies using alginate as an injectable scaffold. Bovine meniscal fibrochondrocytes were mixed with collagen and injected into micro-computed tomography-based molds to create 10 and 20 mg ml^?1 menisci that were cultured for up to 4 weeks and compared with cultured alginate menisci. Contraction, histological, confocal microscopy, biochemical and mechanical analysis were performed to determine tissue development. After 4 weeks culture, collagen menisci had preserved their shape and significantly improved their biochemical and mechanical properties. Both 10 and 20 mg ml^?1 menisci maintained their DNA content while significantly improving the glycosaminoglycan and collagen content, at values significantly higher than the alginate controls. Collagen menisci matched the alginate control in terms of the equilibrium modulus, and developed a 3- to 6-fold higher tensile modulus than alginate by 4 weeks. Further fibrochondrocytes were able to reorganize the collagen gels into a more fibrous appearance similar to native menisci.     

Fabrication of a layered microstructured polycaprolactone construct for 3-D tissue engineering

Successful artificial tissue scaffolds support regeneration by promoting cellular organization as well as appropriate mechanical and biological functionality. We have previously shown in vitro that 2-D substrates with micrometer-scale grooves (5 microm deep, 18 microm wide, with 12 microm spacing) can induce cell orientation and ECM alignment. Here, we have transferred this microtopography onto biodegradable polycaprolactone (PCL) thin films. We further developed a technique to layer these cellularized microtextured scaffolds into a 3-D tissue construct. A surface modification technique was used to attach photoreactive acrylate groups on the PCL scaffold surface onto which poly(ethylene glycol)-diacrylate (PEG-DA) gel could be photopolymerized. PEG-DA serves as an adhesive layer between PCL scaffolds, resulting in a VSMC-seeded layered 3-D composite structure that is highly organized and structurally stable. The PCL surface modification chemistry was confirmed via XPS, and the maintenance of cell number and orientation on the modified PCL scaffolds was demonstrated using colorimetric and imaging techniques. Cell number and orientation were also investigated after cells were cultured in the layered 3-D configuration. Such 3-D tissue mimics fabricated with precise cellular organization will enable systematic testing of the effects of cellular orientation on the functional and mechanical properties of tissue-engineered blood vessels.     

Preparation and characterization of alginate/gelatin blend films for cardiac tissue engineering

The aim of this work was the preparation of blends based on alginate and gelatin, with different weight ratio, to combine the advantages of these two natural polymers for application in cardiac tissue engineering. The physicochemical characterization, performed by Fourier transform infrared spectroscopy, differential scanning calorimetry and thermogravimetric analysis, revealed a good miscibility and the presence of interactions among the functional groups of pure biopolymers. Concerning the swelling and degradation tests, performed in different solutions simulating body fluids, both swelling degree and weight losses were higher in phosphate buffer saline (PBS) and for the blends with a higher content of gelatin. These results indicated a better stability of the blends in cell culture medium than in PBS and suggested a mainly hydrolytic degradation process. Cell culture tests, carried out using C2C12 myoblasts, showed a good cell proliferation for all the blends containing more than 60% of gelatin, with the alginate/gelatin 20:80 showing the best response. The same blend was the only one on which cell differentiation was observed. The results obtained in the biological characterization allow to select the alginate/gelatin 20:80 blend as a suitable material to prepare scaffolds for myocardial tissue engineering.     

Copper-releasing, boron-containing bioactive glass-based scaffolds coated with alginate for bone tissue engineering

The aim of this study was to synthesize and characterize new boron-containing bioactive glass-based scaffolds coated with alginate cross-linked with copper ions. A recently developed bioactive glass powder with nominal composition (wt.%) 65 SiO2, 15 CaO, 18.4 Na2O, 0.1 MgO and 1.5 B2O3 was fabricated as porous scaffolds by the foam replica method. Scaffolds were alginate coated by dipping them in alginate solution. Scanning electron microscopy investigations indicated that the alginate effectively attached on the surface of the three-dimensional scaffolds leading to a homogeneous coating. It was confirmed that the scaffold structure remained amorphous after the sintering process and that the alginate coating improved the scaffold bioactivity and mechanical properties. Copper release studies showed that the alginate-coated scaffolds allowed controlled release of copper ions. The novel copper-releasing composite scaffolds represent promising candidates for bone regeneration.     

Optimized growth conditions for tissue engineering of human cardiovascular structures

Optimized in vitro formation of strong tissue is a prerequisite for tissue engineering of cardiovascular structures, such as heart valves and blood vessels. This study evaluates different growth media additives as to cell proliferation, extracellular matrix formation, and mechanical characteristics. Biodegradable polymers were seeded with human vascular myofibroblasts. Group A was cultured with standard medium, groups B, C, and D were in addition supplemented with ascorbate, fibroblast growth factor (bFGF), and both respectively. Analysis included histology, electron microsocopy, mechanical testing, and biochemical assays for cell proliferation (DNA) and extracellular matrix (collagen). DNA content increased in all groups, showing significantly more cells in group C and D after 14d. Collagen increased in all groups, except for C. Morphology showed viable, layered cellular tissue, with collagen fibrils after 2w, most pronounced in B and D. Mechanical properties decreased initially, stabilizing after 2w. In conclusion, standard nutrient media were efficient for seeded human vascular cells cultured on biodegradable meshes. Supplementation with bFGF+ascorbate resulted in enhanced early cell proliferation and structurally more mature tissue formation.     

Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. Structure, gelation rate and mechanical properties

Alginate gels have been used in both drug delivery and cell encapsulation applications in the bead form usually produced by dripping alginate solution into a CaCl2 bath. The major disadvantages to these systems are that the gelation rate is hard to control; the resulting structure is not uniform; and mechanically strong and complex-shaped 3-D structures are difficult to achieve. In this work controlled gelation rate was achieved with CaCO3-GDL and CaSO4-CaCO3-GDL systems, and homogeneous alginate gels were formulated as scaffolds with defined dimensions for tissue engineering applications. Gelation rate increased with increasing total calcium content, increasing proportion of CaSO4, increasing temperature and decreasing alginate concentration. Mechanical properties of the alginate gels were controlled by the compositional variables. Slower gelation systems generate more uniform and mechanically stronger gels than faster gelation systems. The compressive modulus and strength increased with alginate concentration, total calcium content, molecular weight and guluronic acid (G) content of the alginate. MC3T3-E1 osteoblastic cells were uniformly incorporated in the alginate gels and cultured in vitro. These results demonstrated how alginate gel and gel/cell systems could be formulated with controlled structure, gelation rate, and mechanical properties for tissue engineering and other biomedical applications.     

Automatic algorithm for generating complex polyhedral scaffold structures for tissue engineering

In this article, an approach for tissue-engineering (TE) scaffold fabrication by way of integrating computer-based medical imaging, computer graphics, data manipulation techniques, computer-aided design (CAD), and rapid prototyping (RP) technologies is introduced. The aim is to provide a generic solution for the production of scaffolds that can potentially meet the diverse requirements of TE applications. In the work presented, a novel parametric library of open polyhedral unit cells is developed to assist the user in designing the microarchitecture of the scaffold according to the requirements of its final TE application. Once an open polyhedral unit cell design is selected and sized, a specially developed algorithm is employed to assemble the microarchitecture of the scaffold while adhering to the external geometry of the patient's anatomy generated from medical imaging data. RP fabrication techniques are then employed to build the scaffolds according to the CAD-generated designs. The combined application of such technologies promises unprecedented scaffold qualities with spatially and anatomically accurate three-dimensional forms as well as highly consistent and reproducible microarchitectures. The integrated system also has great potential in providing new cost-effective and rapid solutions to customized made-to-order TE scaffold production.     

Injectable dendritic cell-carrying alginate gels for immunization and immunotherapy

Dendritic cell vaccines, in which antigen-loaded dendritic cells (DCs) are injected directly into patients to trigger immune responses, are in development as a treatment for cancer and some infectious diseases. In this study, we tested the concept of delivering DCs in an injectable hydrogel matrix, with the aim of harboring dendritic cells for prolonged time periods at a defined site and trapping/concentrating factors secreted by DCs to establish an inflammatory milieu in situ. To achieve these goals, a self-gelling formulation of alginate was developed, obtained by mixing calcium-loaded alginate microspheres with soluble alginate solution and dendritic cells, a formulation that rapidly gelled in vivo. When injected subcutaneously in mice, these alginate 'vaccination nodes' containing activated DCs attracted both host dendritic cells and a large number of T cells to the injection sites over a week in vivo, while some of the inoculated DCs trafficked to the draining lymph nodes. Using an adoptive transfer model to track a defined population of T cells responding to immunization with antigen-loaded DCs, we show that DC/alginate immunization led to recruitment of activated antigen-specific T cells to the alginate matrix, in a manner dependent on the presence of the DCs. This gel/DC immunization system may thus be of interest for immunotherapy to direct the accumulation of immune cells at solid tumors or infection sites in the presence of supporting factors co-delivered by the hydrogel matrix.     

A layered ultra-porous scaffold for tissue engineering, created via a hydrospinning method

In this work we propose a new method titled "layered hydrospinning." In this method, nanofibers are being collected on top of a liquid reservoir and assembled layer-by-layer to form a 3D scaffold. The geometrical features of fabricated hydrospun scaffolds show a porosity of 99% and pores of a diameter over 100 microm. Cells were seeded during the hydrospinning process, thus achieving an initial even density of cells in the scaffold. We show that human embryonic stem cells and mouse myoblasts cultured on the scaffolds were able to infiltrate further into the scaffolds and proliferate to a greater extent, compared to conventional electrospun scaffolds collected on a plate.     

A highly organized three-dimensional alginate scaffold for cartilage tissue engineering prepared by microfluidic technology

Osteoarthritis is a degenerative disease and frequently involves the knee, hip and phalangeal joints. Current treatments used in small cartilage defects including multiple drilling, abrasion arthroplasty, mosaicplasty, and autogenous chondrocyte transplantation, however, there are problems needed to be solved. The standard treatment for severe osteoarthritis is total joint arthroplasty. The disadvantages of this surgery are the possibility of implant loosening. Therefore, tissue engineering for cartilage regeneration has become a promising topic. We have developed a new method to produce a highly organized single polymer (alginate) scaffold using microfluidic device. Scanning electron microscope and confocal fluoroscope examinations showed that the scaffold has a regular interconnected porous structure in the scale of 250 μm and high porosity. The scaffold is effective in chondrocyte culture; the cell viability test (WST-1 assay), cell toxicity (lactate dehydrogenase assay), cell survival rate, extracellular matrix production (glycosaminoglycans contents), cell proliferation (DNA quantification), and gene expression (real-time PCR) all revealed good results for chondrocyte culture. The chondrocytes can maintain normal phenotypes, highly express aggrecan and type II collagen, and secrete a great deal of extracellular matrix when seeded in the alginate scaffold. This study demonstrated that a highly organized alginate scaffold can be prepared with an economical microfluidic device, and this scaffold is effective in cartilage tissue engineering.     

Mechanical and diffusive properties of homogeneous alginate gels in form of particles and cylinders

In this study, alginate polymers are used to get homogeneous cylindrical or spherical gels. MRI techniques are employed to study homogeneity of these gels. Four different alginates are used and, for each one, five different concentrations for mechanical tests and three different concentrations for release tests are studied. Mechanical tests are performed to get gels' linear viscoelasticity region and then to evaluate their crosslink density in relation to polymer concentration. Afterwards, three model molecules (theophylline, vitamin B(12), and myoglobin) are loaded within gels to study the release kinetics in water from both cylindrical and spherical gels. Diffusion coefficients calculated from these experiments are then used to estimate the polymeric network mesh wideness. This work shows how crosslink density increases with polymer concentration regardless of the alginate type considered. In addition, while vitamin B(12) diffusion coefficient is inversely proportional to crosslink density, myoglobin is too large to diffuse through the polymeric network, whatever the alginate type and polymer concentration. At the same time, theophylline is too small to be sensibly affected by increasing the polymeric network crosslink density. Finally, MRI analysis and vitamin B(12) diffusion coefficient values prove that, structurally speaking, cylinders and spheres are similar and homogeneous.