Collagen Scaffolds For Tissue Engineering Research Articles

Preparation of mesh-like collagen scaffolds for tissue engineering

Collagen meshes with large or small openings were prepared after selective removal of PLGA mesh from PLGA–collagen composite mesh or collagen-coated PLGA mesh. The collagen meshes support human skin fibroblast adhesion and promote cell proliferation.

Antimicrobial quaternary ammonium organosilane cross-linked nanofibrous collagen scaffolds for tissue engineering.

IntroductionIn search for cross-linkers with multifunctional characteristics, the present work investigated the utility of quaternary ammonium organosilane (QOS) as a potential cross-linker for electrospun collagen nanofibers. We hypothesized that the quaternary ammonium ions improve the electrospinnability by reducing the surface tension and confer antimicrobial properties, while the formation of siloxane after alkaline hydrolysis could cross-link collagen and stimulate cell proliferation.Materials and methodsQOS collagen nanofibers were electrospun by incorporating various concentrations of QOS (0.1%–10% w/w) and were cross-linked in situ after exposure to ammonium carbonate. The QOS cross-linked scaffolds were characterized and their biological properties were evaluated in terms of their biocompatibility, cellular adhesion and metabolic activity for primary human dermal fibroblasts and human fetal osteoblasts.Results and discussionThe study revealed that 1) QOS cross-linking increased the flexibility of otherwise rigid collagen nanofibers and improved the thermal stability; 2) QOS cross-linked mats displayed potent antibacterial activity and 3) the biocompatibility of the composite mats depended on the amount of QOS present in dope solution – at low QOS concentrations (0.1% w/w), the mats promoted mammalian cell proliferation and growth, whereas at higher QOS concentrations, cytotoxic effect was observed.ConclusionThis study demonstrates that QOS cross-linked mats possess anti-infective properties and confer niches for cellular growth and proliferation, thus offering a useful approach, which is important for hard and soft tissue engineering and regenerative medicine.

Development of 3D printed fibrillar collagen scaffold for tissue engineering.

Collagen is widely used in tissue engineering because it can be extracted in large quantities, and has excellent biocompatibility, good biodegradability, and weak antigenicity. In the present study, we isolated printable collagen from bovine Achilles tendon and examined the purity of the isolated collagen using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The bands obtained corresponded to α1, α2 and β chains with little contamination from other small proteins. Furthermore, rheological measurements of collagen dispersions (60mg per ml of PBS) at pH7 revealed values of viscosity of 35.62 ± 1.42Pas at shear rate of 10s- 1 and a shear thinning behavior. Collagen gels and solutions can be used for building scaffolds by three-dimensional (3D) printing. After designing and fabricating a low-cost 3D printer we assayed the collagen printing and obtaining 3D printed scaffolds of collagen at pH7. The porosity of the scaffold was 90.22% ± 0.88% and the swelling ratio was 1437% ± 146%. The microstructure of the scaffolds was studied using scanning electron microscopy, and a porous mesh of fibrillar collagen was observed. In addition, the 3D printed collagen scaffold was not cytotoxic with cell viability higher than 70% using Vero and NIH 3T3 cells. In vitro evaluation using both cells lines demonstrated that the collagen scaffolds had the ability to support cell attachment and proliferation. Also a fibrillar collagen mesh was observed after two weeks of culture at 37°C. Overall, these results are promising since they show the capability of the presented protocol to obtain printable fibrillar collagen at pH7 and the potential of the printing technique for building low-cost biocompatible 3D plotted structures which maintained the fibrillar collagen structure after incubation in culture media without using additional strategies as crosslinking.

Physico-chemical characterization of collagen scaffolds for tissue engineering

Abstract The objective was to research the physical and chemical properties of collagen scaffolds (CS) obtained from bone matrix Nukbone ® subject to a demineralization process using hydrochloric acid. The CS samples were characterized by optical and scanning electron microscopy, elemental chemical analysis, X-ray diffraction, spectroscopy Infrared, thermal analysis, differential scanning calorimetry and nitrogen adsorption. The microanalysis were used to set the macro- and microstructures of CS. They showed that the CS retained the morphology of Nukbone ® with interconnected pores and their size between 100 and 500 μm, and it is composed of 20% by weight of HA and the rest is collagen type I. By infrared spectroscopy the functional groups of collagen type I (amide A – 3285, B – 2917, I – 1633, II – 1553 and III – 1239 cm −1 ) were identified. By thermal analysis it was determined that the phenomenon of denaturation of the collagen type I began in the range of 75–85 °C and burned above 200 °C.

The biocompatibility of silk fibroin and acellular collagen scaffolds for tissue engineering in the ear

Recent experimental studies have shown the suitability of silk fibroin scaffold (SFS) and porcine-derived acellular collagen I/III scaffold (ACS) as onlay graft materials for tympanic membrane perforation repair. The aims of this study were to further characterize and evaluate the in vivo biocompatibility of SFS and ACS compared with commonly used materials such as Gelfoam and paper in a rat model. The scaffolds were implanted in subcutaneous (SC) tissue and middle ear (ME) cavity followed by histological and otoscopic evaluation for up to 26 weeks. Our results revealed that SFS and ACS were well tolerated and compatible in rat SC and ME tissues throughout the study. The tissue response adjacent to the implants evaluated by histology and otoscopy showed SFS and ACS to have a milder tissue response with minimal inflammation compared to that of paper. Gelfoam gave similar results to SFS and ACS after SC implantation, but it was found to be associated with pronounced fibrosis and osteoneogenesis after ME implantation. It is concluded that SFS and ACS both were biocompatible and could serve as potential alternative scaffolds for tissue engineering in the ear.

Hydraulic permeability of multilayered collagen gel scaffolds under plastic compression-induced unidirectional fluid flow

Under conditions of free fluid flow, highly hydrated fibrillar collagen gels expel fluid and undergo gravity driven consolidation (self-compression; SC). This process can be accelerated by the application of a compressive stress (plastic compression; PC) in order to generate dense collagen scaffolds for tissue engineering. To define the microstructural evolution of collagen gels under PC, this study applied a two-layer micromechanical model that was previously developed to measure hydraulic permeability (k) under SC. Radially confined PC resulted in unidirectional fluid flow through the gel and the formation of a dense lamella at the fluid expulsion boundary which was confirmed by confocal microscopy of collagen immunoreactivity. Gel mass loss due to PC and subsequent SC were measured and applied to Darcy’s law to calculate the thickness of the lamella and hydrated layer, as well as their relative permeabilities. Increasing PC level resulted in a significant increase in mass loss fraction and lamellar thickness, while the thickness of the hydrated layer dramatically decreased. Permeability of lamella also decreased from 1.8×10−15 to 1.0×10−15m2 in response to an increase in PC level. Ongoing SC, following PC, resulted in a uniform decrease in mass loss and k with increasing PC level and as a function SC time. Experimental k data were in close agreement with those estimated by the Happel model. Calculation of average k values for various two-layer microstructures indicated that they each approached 10−15–10−14m2 at equilibrium. In summary, the two-layer micromechanical model can be used to define the microstructure and permeability of multi-layered biomimetic scaffolds generated by PC.

Oriented Collagen Scaffolds for Tissue Engineering.

Oriented collagen scaffolds were developed in the form of sheet, mesh and tube by arraying flow-oriented collagen string gels and dehydrating the arrayed gels. The developed collagen scaffolds can be any practical size with any direction of orientation for tissue engineering applications. The birefringence of the collagen scaffolds was quantitatively analyzed by parallel Nicols method. Since native collagen in the human body has orientations such as bone, cartilage, tendon and cornea, and the orientation has a special role for the function of human organs, the developed various types of three-dimensional oriented collagen scaffolds are expected to be useful biomaterials for tissue engineering and regenerative medicines.

A cryogenic direct-plotting system for fabrication of 3D collagen scaffolds for tissue engineering

The goal of tissue engineering is to repair or regenerate damaged tissue using a combination of cellular biology and materials engineering techniques. One of the challenging problems in tissue engineering is the development of a reproducible three-dimensional (3D) scaffold to support cell migration and infiltration. Although natural polymers, such as dissolved collagen or alginate, are considered ideal for this purpose, their hydrophilic properties have hindered the fabrication of designed 3D scaffold structures. To overcome this problem, we developed a novel system for the cryogenic plotting of 3D scaffolds. Using this technique, we created various 3D collagen scaffolds with designed pore structures that exhibited desired properties. The diameter of the individual collagen strands, which varied from 250 µm to 500 µm, was reproducibly dependent on processing parameters, and the final collagen scaffold showed little shrinkage (less than 12%) relative to the initial design. To evaluate the fabricated scaffold, we adapted the scaffold to regenerate skin tissue. Immunohistochemical analysis demonstrated that co-cultured keratinocytes and fibroblasts completely migrated throughout the 3D collagen scaffold and keratinocytes were well differentiated on the surface of scaffold like a human skin.

Collagen scaffolds for tissue engineering

There are two major approaches to tissue engineering for regeneration of tissues and organs. One involves cell-free materials and/or factors and one involves delivering cells to contribute to the regeneraion process. Of the many scaffold materials being investigated, collagen type I, with selective removal of its telopeptides, has been shown to have many advantageous features for both of these approaches. Highly porous collagen lattice sponges have been used to support in vitro growth of many types of tissues. Use of bioreactors to control in vitro perfusion of medium and to apply hydrostatic fluid pressure has been shown to enhance histogenesis in collagen scaffolds. Collagen sponges have also been developed to contain differentiating-inducing materials like demineralized bone to stimulate differentiation of cartilage tissue both in vitro and in vivo.

The effect of pore size on permeability and cell attachment in collagen scaffolds for tissue engineering

The permeability of scaffolds and other three-dimensional constructs used for tissue engineering applications is important as it controls the diffusion of nutrients in and waste out of the scaffold as well as influencing the pressure fields within the construct. The objective of this study was to characterize the permeability/fluid mobility of collagen-GAG scaffolds as a function of pore size and compressive strain using both experimental and mathematical modeling techniques. Scaffolds containing four distinct mean pore sizes (151, 121, 110, 96 microns) were fabricated using a freeze-drying process. An experimental device was constructed to measure the permeability of the scaffold variants at different levels of compressive strain (0, 14, 29 and 40% while a low-density open-cell foam cellular solids model utilizing a tetrakaidecahedral unit cell was used to accurately model the permeability of each scaffold variant at all level of applied strain. The results of both the experimental and the mathematical analysis revealed that scaffold permeability increases with increasing pore size and decreases with increasing compressive strain. The excellent comparison between experimentally measured and predicted scaffold permeability suggests that cellular solids modelling techniques can be utilized to predict scaffold permeability under a variety of physiological loading conditions as well as to predict the permeability of future scaffolds with a wide variety of pore microstructures.

Development of poly (lactic-co-glycolic acid)-collagen scaffolds for tissue engineering

Collagen as an important extra-cellular matrix (ECM) in many tissues is weakly antigenic and the structure of collagen sponges is highly porous with interconnected pores effective for cell infiltration and mass transfer of oxygen and nutrients. Its application as a scaffold is limited by poor mechanical strength and rapid biodegradation. In this paper, we attempt to graft hydrolyzed PLGA fiber surfaces with collagen by N-(3-dimethylaminopropyl)- N′-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS), and then embed these collagen-grafted PLGA fibers in collagen sponge to form a hybrid PLGA-collagen scaffold. For further stability, we cross-linked the collagen in the scaffold and used it in rat liver cell cultivation. The scaffold was characterized by mechanical micro-tester, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Results showed that (1) the scaffolds exhibited isotropic and interconnected porous structure; (2) the compression modulus of this scaffold was enhanced 50 fold compared to the collagen scaffolds. The cell attachment and cytotoxicity of this scaffold were studied. Cell attachment was improved remarkably and the cytotoxicity of the hybrid PLGA-collagen scaffold was lower than that of the un-grafted PLGA-collagen scaffolds using alamarBlue™ assay normalized to the DNA content in each scaffold. This new hybrid scaffold has potential applications for tissue engineering.

Microsphere-integrated collagen scaffolds for tissue engineering: Effect of microsphere formulation and scaffold properties on protein release kinetics

A promising approach to control the time and space distribution of signalling molecules inside tissue engineering scaffolds consists in entrapping biodegradable microspheres releasing the protein locally for long time frames. However, a rational design of microsphere-integrated scaffolds requires the knowledge of protein release profiles directly within the polymeric template. In this work, PLGA microspheres encapsulating rhodamine-labelled bovine serum albumin (BSA-Rhod) as a model protein were produced in different formulation conditions and tested for their release features in solution and in collagen and collagen/hyaluronic acid (HA) scaffolds. BSA-Rhod release profiles from single microspheres in solution and within the scaffold were assessed by using a confocal laser scanning microscopy (CLSM)-assisted method. Results suggest that the same diffusion-erosion process controls BSA-Rhod release from microspheres in solution and collagen. Nonetheless, two main factors contribute protein release within the scaffold, that is water activity in the release environment and transport properties of the protein in the gel. While microsphere formulation mainly controls the induction time necessary to activate protein release, polymer scaffold composition governs the release rate. Thus, the fine regulation of a tissue engineering construct may be obtained by an appropriate combination of microspheres and scaffolds, providing a spatial and temporal control over signalling molecule delivery.

Novel porous aortic elastin and collagen scaffolds for tissue engineering

Decellularized vascular matrices are used as scaffolds in cardiovascular tissue engineering because they retain their natural biological composition and three-dimensional (3-D) architecture suitable for cell adhesion and proliferation. However, cell infiltration and subsequent repopulation of these scaffolds was shown to be unsatisfactory due to their dense collagen and elastic fiber networks. In an attempt to create more porous structures for cell repopulation, we selectively removed matrix components from decellularized porcine aorta to obtain two types of scaffolds, namely elastin and collagen scaffolds. Histology and scanning electron microscopy examination of the two scaffolds revealed a well-oriented porous decellularized structure that maintained natural architecture of the aorta. Quantitative DNA analysis confirmed that both scaffolds were completely decellularized. Stress–strain analysis demonstrated adequate mechanical properties for both elastin and collagen scaffolds. In vitro enzyme digestion of the scaffolds suggested that they were highly biodegradable. Furthermore, the biodegradability of collagen scaffolds could be controlled by crosslinking with carbodiimides. Cell culture studies showed that fibroblasts adhered to and proliferated on the scaffold surfaces with excellent cell viability. Fibroblasts infiltrated about 120 μm into elastin scaffolds and about 40 μm into collagen scaffolds after 4 weeks of rotary cell culture. These results indicated that our novel aortic elastin and collagen matrices have the potential to serve as scaffolds for cardiovascular tissue engineering.

A novel tubular scaffold for cardiovascular tissue engineering.

We have developed a counter rotating cone extrusion device to produce the next generation of three-dimensional collagen scaffold for tissue engineering. The device can produce a continuously varying fibril angle from the lumen to the outside of a 5-mm-diameter collagen tube, similar to the pattern of heart muscle cells in the intact heart. Our scaffold is a novel, oriented, type I collagen, tubular scaffold. We selected collagen because we believe there are important signals from the collagen both geometrically and biochemically that elicit the in vivo -like phenotypic response from the cardiomyocytes. We have shown that cardiomyocytes can be cultured in these tubes and resemble an in vivo phenotype. This new model system will provide important information leading to the design and construction of a functional, biologically based assist device.