Matrix For Tissue Engineering Research Articles

Development of novel 3D scaffolds using BioExtruder by the incorporation of silica into polycaprolactone matrix for bone tissue engineering

Development of three-dimensional (3D) scaffolds has acquired a great importance for bone repair and tissue reconstruction. Thus, this paper addresses the development of 3D scaffolds by varying the content of silicon dioxide (SiO2) in polycaprolactone (PCL) matrix. The scaffolds were fabricated by employing a novel fused deposition modelling technique (BioExtruder). The physicochemical properties of the developed 3D scaffolds were systematically studied using various techniques. The thermal properties and stability of the PCL and its composites were assessed using differential scanning calorimetry and thermogravimetric analysis. The morphology of the developed scaffolds was evaluated using scanning electron microscopy and optical microscopy, and found that the pore size was increased from 270 to 320 μm with increasing the SiO2 content in the PCL matrix. The wettability of the developed scaffolds was assessed using contact angle meter. The scaffold incorporating 15 wt% of SiO2 exhibited the highest hydrophilic property as well as thermal stability. The Young’s modulus value determined using universal testing machine indicated that the scaffold developed with 15 wt% of SiO2 exhibits 101.59 MPa. To assess the performance of the scaffolds for tissue engineering applications, the in-vitro cytotoxicity and cell proliferation were systematically carried out using L929 Mouse Fibroblasts and MG63 Osteoblasts, respectively. It was found that the scaffolds did not show any toxic effects towards the cell growth, and the cell proliferation was greatly increased >90% during 7 days of cell culture. Based on the results, it is concluded that the scaffold containing 15 wt% of SiO2 is of potential candidate for bone tissue engineering application.

Noninvasive ultrasound imaging for assessment of intact microstructure of extracellular matrix in tissue engineering.

Development of artificial tissues or organs is one of the actual tasks in regenerative medicine that requires observation and evaluation of intact volume microstructure of tissue engineering products at all stages of their formation, from native donor tissues and decellularized scaffolds to recipient cell migration in the matrix. Unfortunately in practice, methods of vital noninvasive imaging of volume microstructure in matrixes are absent. In this work, we propose a new approach based on high-frequency acoustic microscopy for noninvasive evaluation and visualization of volume microstructure in tissue engineering products. The results present the ultrasound characterization of native rat diaphragms and lungs and their decellularized scaffolds. Verification of the method for visualization of tissue formation in the matrix volume was described in the model samples of diaphragm scaffolds with stepwise collagenization. Results demonstrate acoustic microscopic sensitivity to cell content concentration, variation inlocal density, and orientation of protein fibers in the volume, micron air inclusions, and other inhomogeneities of matrixes.

Expanding the Range of Available Isoelectric Points of Highly Methacryloylated Gelatin

AbstractCross‐linkable gelatin methacryloyl (GM) is widely used for the generation of artificial extracellular matrix in tissue engineering. However, so far, isoelectric points (IEPs) of highly methacryloylated GM derivatives are limited to IEPs below 7 due to the consumption of amino groups in the modification reaction. In this contribution, the synthesis of a new GM derivative, gelatin methacryloyl‐aminoethyl (GME), with an IEP above 7 similar to the gelatin type A (GA) starting material is reported, together with a high degree of methacryloylation. GME is obtained by reacting GM with ethylenediamine (EDA). The impact of the EDA functionalization on the properties of GM and GME derivatives is characterized thoroughly via 1H‐NMR, 1H‐13C‐HSQC‐NMR, IEP, solution viscosity, gel point, and physico‐chemical properties of resulting hydrogels. The second functionalization step results in amino group concentrations similar to GA and with that in an IEP of 9.9. The data suggest that amino functionalization with EDA prevents physical cross‐linking in the same way as described before for acetyl functionalization. Therefore, GME hydrogels are less stiff than GM hydrogels from GMs with a comparable amount of methacrylic functions. With GME, a gelatin derivative is available that is positively charged at neutral pH without being limited to low methacryl modifications.

Fabrication of Three-Dimensional Fluffy Nanofibrous Scaffolds for Tissue Engineering via Electrospinning and CO2 Escaping Foaming

Electrospinning is a widely used technique to fabricate a nanofibrous scaffold to resemble the extracellular matrix (ECM) in tissue engineering. However, the intrinsic property (dense structure and...

Purification of alginate improves its biocompatibility and eliminates cytotoxicity in matrix for bone tissue engineering

There is a growing interest about using natural polymers from renewable sources as biomaterials for applications in tissue engineering. In the present work, alginates were extracted from Undaria pinnatifida, a brown sea weed invasive in Argentinian coast. The isolated alginate was structurally characterized by IR and 1H NMR spectroscopies, intrinsic viscosity and TGA. For comparison purposes, commercial sodium alginate was purified and characterized using the same protocol as for raw material. Toxicity and biocompatibility of sodium alginate obtained from algae were studied using a murine macrophage-like cell line (RAW 264.7) and bone marrow stromal cells (BMSC), respectively. The presence of impurities inhibited both RAW 264.7 and bone marrow stromal cell proliferation and increased nitric oxide production from macrophages, while inhibited osteoblastic differentiation of BMSC. All these effects were reverted by the purification of alginate. In conclusion, alginate purification improves biocompatibility and osteo-induction while decreases its toxicity.

Interpenetrating alginate on gelatin–poly(2-hydroxyethyl methacrylate) as a functional polymeric matrix for cartilage tissue engineering

In this work, we report the fabrication of an interpenetrating alginate network on a gelatin and (2-hydroxyethyl methacrylate) (GAH). The anisotropic architecture of scaffold promotes the proliferation of chondrocytes. The anisotropy provided by the alginate influences the cellular response because high collagen and glycosaminoglycan (GAG) contents were found in the cells cultured on the GAH scaffold compared to the scaffold (GH) without alginate. The in vivo experiments demonstrate that the scaffold and the disposition of matrix components, particularly collagen, could promote neovascularization with the ability to recruit cells from the surrounding tissue.

Development of cartilage extracellular matrix in cartilage tissue engineering

Cartilage tissue engineering, an effective way to repair cartilage defects, requires an ideal scaffold to promote the regeneration performance of stem cells. Cartilage extracellular matrix (CECM) can imitate the living environment of cartilage cells to the greatest extent. CECM not only exhibits good biocompatibility with chondrocytes and stem cells, which can meet the basic requirements of scaffolds, but also promotes chondrocytes to secrete matrix and induce stem cells to differentiate into chondrocytes; as such, this matrix is a better scaffold and has more advantages than existing ones. The promotion and induction effects could be related to various cartilage-related proteins inside. However, the practical application of this technique is hindered by problems, such as poor mechanical properties and insufficient cell penetration of CECM. Association with other materials can compensate for these inadequacies to a certain degree, and finding a combination mode with optimized performance is the application trend of CECM. This review focuses on research of CECM materials in cartilage tissue engineering.

Preparation and Characterization of Nanocomposite Scaffolds (Collagen/β-TCP/SrO) for Bone Tissue Engineering.

Nowadays, production of nanocomposite scaffolds based on natural biopolymer, bioceramic, and metal ions is a growing field of research due to the potential for bone tissue engineering applications. In this study, a nanocomposite scaffold for bone tissue engineering was successfully prepared using collagen (COL), beta-tricalcium phosphate (β-TCP) and strontium oxide (SrO). A composition of β-TCP (4.9g) was prepared by doping with SrO (0.05g). Biocompatible porous nanocomposite scaffolds were prepared by freeze-drying in different formulations [COL, COL/β-TCP (1:2 w/w), and COL/β-TCP-Sr (1:2 w/w)] to be used as a provisional matrix or scaffold for bone tissue engineering. The nanoparticles were characterized by X-ray diffraction, Fourier transforms infrared spectroscopy and energy dispersive spectroscopy. Moreover, the prepared scaffolds were characterized by physicochemical properties, such as porosity, swelling ratio, biodegradation, mechanical properties, and biomineralization. All the scaffolds had a microporous structure with high porosity (~ 95-99%) and appropriate pore size (100-200μm). COL/β-TCP-Sr scaffolds had the compressive modulus (213.44 ± 0.47kPa) higher than that of COL/β-TCP (33.14 ± 1.77kPa). In vitro cytocompatibility, cell attachment and alkaline phosphatase (ALP) activity studies performed using rat bone marrow mesenchymal stem cells. Addition of β-TCP-Sr to collagen scaffolds increased ALP activity by 1.33-1.79 and 2.92-4.57 folds after 7 and 14days of culture, respectively. In summary, it was found that the incorporation of Sr into the collagen-β-TCP scaffolds has a great potential for bone tissue engineering applications.

Influence of Antimycin A, a bacterial toxin, on human dermal fibroblast cell adhesion to tungsten-silicon oxide nanocomposites

Nano- and micro-scale engineered surface structures are often used to control cell morphology and mimic the extracellular matrix in tissue engineering. However, there is little understanding of how toxins produced by common bacteria might affect cell adhesion to these structures. In this study, human dermal fibroblast (GM5565) cells were incubated on patterned tungsten/silicon oxide nanocomposite in media in the presence or absence of Antimycin A. This composite consists of parallel tungsten and silicon oxide lines with identical widths in the range of 0.18 and 50 μm. The morphology of the cells and of their mitochondria was characterized by using high-resolution scanning electron microscopy and fluorescence confocal microscopy. Results show that cells preferentially align along the line axes in a pattern-dependent manner, with a maximum population of cells oriented within 10° of the line axes on the structures containing 10 μm wide lines. Cells treated with Antimycin A, however, show a smaller proportion of cells oriented in this direction as compared to cells cultured in Antimycin A-free media (34.4% vs 53.0%). The majority of mitochondria in cells growing in Antimycin A-free media are tubular in shape and are preferentially positioned on the tungsten lines, whereas these organelles exhibit a circular geometry and are less attracted to the metal lines in the presence of Antimycin A.

SIS/Aligned Fibre Scaffold Designed to Meet Layered Oesophageal Tissue Complexity and Properties

With donor organs not readily available the need for a tissue engineered oesophagus remains high particularly for congenial childhood conditions such as atresia. Previous attempts have not been very successful and challenges remain to be overcome. Small intestine submucosa (SIS) is an a cellular matrix material with good biological properties though, as with these materials, demonstrably poor mechanical properties. In this work electrospinning was used to mechanically reinforce a unique tubular SIS with PLGA nanofibers. It was hypothesised that if attachment could be achieved between the two materials than this would (i) improve the SIS's mechanical properties, (ii) facilitate smooth muscle cell alignment to support directional growth of muscle cells and potentially restore contractility and (iii) allow for the delivery of bioactive molecules (VEGF in this instance). Through a multistage process adhesion between the layers was achieved without chemically altering the SIS. It was also found that altering mandrel rotational speed affected the alignment of the PLGA nanofibers.SIS-PLGA scaffolds performed mechanically better than SIS alone; yield stress improvement was 200% and 400% along the longitudinal and circumferential direction, respectively (greatest with highly aligned fibres produced at 5000 rpm). Smooth muscle cells cultured on the aligned fibres showed resultant unidirectional alignment. In vivo the SIS-PLGA scaffolds demonstrated relatively limited foreign body reaction judged by the type and proportion of immune cells present and lack of fibrous encapsulation. The scaffolds remained intact at 4 weeks which is required for a structural tissue engineering matrix and obtained good cellular infiltration. The incorporation of VEGF within SIS-PLGA scaffolds increased the blood vessel density of the surrounding tissues, highlighting the possible stimulation of endothelialisation by angiogenic factor delivery. Overall, the designed SIS-PLGA-VEGF hybrid scaffolds might be used as a potential matrix platform for oesophageal tissue engineering. In addition to this, achieving improved attachment between layers of a cellular matrix materials and electrospun fibres layers does possess the potential to be applied to other applications.

Fabrication and evaluation of non-mulberry silk fibroin fiber reinforced chitosan based porous composite scaffold for cartilage tissue engineering.

Lack of potential regenerative medicine to reconstruct damaged cartilage tissue has accelerated investigation and development of potential biomaterial for cartilage tissue engineering. In this study, we fabricated micron-sized non-mulberry silk fibroin fiber (SFF) using N,N-Dimethylacetamide (DMAC)/10% LiBr solution and further used to develop SFF reinforced chitosan(CH) based porous scaffold with desired pore size, porosity, swelling and structural stability. The developed scaffold was characterized for its various physico-chemical, mechanical and biological properties. The developed CH/SFF composite scaffold facilitates human mesenchymal stem cell (hMSCs) attachment, colonization and extracellular matrix deposition. Furthermore, hMSCs shows significantly higher sulfated glycosaminoglycan deposition over CH/SFF in comparison to pure chitosan scaffold (control). Immunocytochemistry studies have shown enhanced expression of collagen type II and aggrecan by hMSCs over composite scaffold than chitosan scaffold. Thus, non-mulberry silk fibroin fiber reinforced chitosan based scaffold might be suitable scaffold that can act as a potential artificial matrix for cartilage tissue engineering.

Applications of decellularized extracellular matrix in bone and cartilage tissue engineering.

Regenerative therapies for bone and cartilage injuries are currently unable to replicate the complex microenvironment of native tissue. There are many tissue engineering approaches attempting to address this issue through the use of synthetic materials. Although synthetic materials can be modified to simulate the mechanical and biochemical properties of the cell microenvironment, they do not mimic in full the multitude of interactions that take place within tissue. Decellularized extracellular matrix (dECM) has been established as a biomaterial that preserves a tissue's native environment, promotes cell proliferation, and provides cues for cell differentiation. The potential of dECM as a therapeutic agent is rising, but there are many limitations of dECM restricting its use. This review discusses the recent progress in the utilization of bone and cartilage dECM through applications as scaffolds, particles, and supplementary factors in bone and cartilage tissue engineering.

Bioinspired Pseudozwitterionic Hydrogels with Bioactive Enzyme Immobilization via pH-Responsive Regulation.

Hydrogels are hydrated networks of flexible polymers with versatile biomedical applications, and their resistance to nonspecific protein adsorption is critical. On the other hand, functionalization with other biomacromolecules would greatly enhance their biotechnological potential. The aim of this research is to prepare low fouling hydrogel polymers for selective protein immobilization. Initially, hydrogels were prepared by controlling the composition ratios of 2-carboxyethyl acrylate (CA) and 2-dimethylaminoethyl methacrylate (DMAEMA) monomers in an N, N-methylene-bis-acrylamide (NMBA) cross-linked free radical polymerization reaction. This series of hydrogels (C1D9 to C9D1) were then analyzed by X-ray photoelectron spectroscopy (XPS) and dynamic laser scattering to confirm the actual polymer ratios and surface charge. When the composition ratio was set at CA:6 vs DMEAMA:4 (C6D4), the hydrogel showed nearly neutral surface charge and an equivalent reaction ratio of CA vs DMAEMA in the hydrogel. Subsequent analysis showed excellent antifouling properties, low blood cell adhesion, hemocompatibility, and platelet deactivation. Moreover, this hydrogel exhibited pH responsiveness to protein adsorption and was then used to facilitate the immobilization of lipase as an indication of active protein functionalization while still maintaining a low fouling status. In summary, a mixed-charge nonfouling pseudozwitterionic hydrogel could be prepared, and its pH-responsive adsorption holds potential for designing a biocompatible tissue engineering matrix or membrane enzyme reactors.

From scaffold to structure: the synthetic production of cell derived extracellular matrix for liver tissue engineering

Liver transplant is the only curative treatment option for patients with end-stage liver failure, however there are few donor livers available for transplant. Tissue engineering of a human liver would potentially solve the problem of escalating donor shortages. A major challenge presents itself in the form of the hepatic extracellular matrix (ECM); a finely controlled in vivo niche which supports hepatocytes and plays a critical role in the development of liver disease. Polymers and decellularized tissues each provide some of the necessary biological cues for the hepatocytes, however, neither alone has proved sufficient. Equally, the ability to fine tune the microenvironment using bioactive molecules presents researchers with the opportunity to create personalised niches for hepatocytes, representing both normal and diseased phenotypes. This study combines cell derived ECM with a fibronectin vector and electrospun scaffolding techniques to produce a platform for creating customisable ECM microenvironments for hepatocytes ( image). The resulting poly-L-lactic acid-extracellular matrix (PLA-ECM) scaffolds were validated using HepG2 hepatocytes. As expected, statistically significant mechanical differences were observed between the synthetically derived ECM (SD-ECM) scaffolds and normal ECM (N-ECM) scaffolds, confirming that the ECM has been altered by the fibronectin producing vector. The PLA-ECM scaffolds maintained hepatocyte growth and function and influence the gene expression of key hepatic genes. Furthermore, immunohistochemistry showed SD and N-ECMs differed in ratios of Collagen I, Laminin and Fibronectin. Our results demonstrate that hybrid PLA-ECM scaffolds and the synthetic production of ECM provide a viable, translatable platform for customising microenvironments for hepatocytes. This technology offers a potential solution to current obstacles in regenerative medicine, disease modelling and whole organ tissue engineering.

Future Research Directions in the Design of Versatile Extracellular Matrix in Tissue Engineering.

Native and artificial extracellular matrices (ECMs) have been widely applied in biomedical fields as one of the most effective components in tissue regeneration. In particular, ECM-based drugs are expected to be applied to treat diseases in organs relevant to urology, because tissue regeneration is particularly important for preventing the recurrence of these diseases. Native ECMs provide a complex in vivo architecture and native physical and mechanical properties that support high biocompatibility. However, the applications of native ECMs are limited due to their tissue-specificity and chemical complexity. Artificial ECMs have been fabricated in an attempt to create a broadly applicable scaffold by using controllable components and a uniform formulation. On the other hands, artificial ECMs fail to mimic the properties of a native ECM; consequently, their applications in tissues are also limited. For that reason, the design of a versatile, hybrid ECM that can be universally applied to various tissues is an emerging area of interest in the biomedical field.

Nanostructured chitosan/gelatin/bioactive glass in situ forming hydrogel composites as a potential injectable matrix for bone tissue engineering

Tissue engineering using soft materials has become a very promising approach for repairing damaged cartilage and bone tissue. In this study, novel in situ forming composite hydrogels were synthesized based on chitosan and gelatin biopolymers associated with bioactive glass nanoparticles. The zeta potential at 37 °C ranged from +3.1 ± 1.4 mV to +6.9 ± 3.2 mV and showed the cationic nature of these hydrogels, which are capable of interacting with negatively charged molecules from native extracellular matrix (ECM). Moreover, the zeta potential analysis and FTIR spectra confirmed that network formation of the hydrogels occurs predominantly by molecular interactions. Rheological results revealed that the elastic modulus (G') increased from 5.4 Pa for pure chitosan hydrogels to 12.4 Pa for hydrogel composites with higher contents of gelatin and bioactive glass. Syringeability and injectability tests showed that all formulations can be smoothly expelled from a syringe and needle with the use of moderate force with a maximum value of 4.0 N. These hydrogel nanocomposites were cytocompatible based on the live cell viability responses of human osteosarcoma cell line (SAOS). These results suggest that these nanocomposites are a promising temporary injectable matrix for bone tissue engineering.

Construction of scaffolds composed of acellular cardiac extracellular matrix for myocardial tissue engineering

High rates of mortality and morbidity stemming from cardiovascular diseases unveil extreme limitations in current therapies despite enormous advances in medical and pharmaceutical sciences. Following myocardial infarction (MI), parts of myocardium undergo irreversible remodeling and is substituted by a scar tissue which eventually leads to heart failure (HF). To address this issue, cardiac patches have been utilized to initiate myocardial regeneration. In this study, a porous cardiac patch is fabricated using a mixture of decellularized myocardium extracellular matrix (ECM) and chitosan (CS). Results of rheological measurements, SEM, biodegradation test, and MTT assay showed that the scaffold composed of 3.5% (w/w) CS and 0.5% ECM has the best potential in providing cardiac progenitor cells (CPCs) with a suitable microenvironmental condition for both attachment and growth. This study demonstrates that the fabricated scaffold is capable of transmitting both mechanical and chemical cues that is native to myocardial tissue and supports efficient growth of the CPCs.

Cooperative Assembly of a Peptide Gelator and Silk Fibroin Afford an Injectable Hydrogel for Tissue Engineering.

Silk fibroin (SF) from Bombyx mori has received increasing interest in biomedical fields, because of its slow biodegradability, good biocompatibility, and low immunogenicity. Although SF-based hydrogels have been studied intensively as a potential matrix for tissue engineering, weak gelation performance and low mechanical strength are major limitations that hamper their widespread applicability. Therefore, searching for new strategies to improve the SF gelation property is highly desirable in tissue engineering research. Herein, we report a facile approach to induce rapid gelation of SF by a small peptide gelator (e.g., NapFF). Following the simple mixing of SF and NapFF in water, a stable hydrogel of SF was obtained in a short time period at physiological pH, and the minimum gelation concentration of SF can reach as low as 0.1%. In this process of gelation, NapFF not only can behave itself as a gelator for supramolecular self-assembly, but also can trigger the conformational transition of the SF molecule from random coil to β-sheet structure via hydrophobic and hydrogen-bonding interactions. More importantly, for the generation of a scaffold with favorable cell-surface interactions, a new peptide gelator (NapFFRGD) with Arg-Gly-Asp (RGD) domain was applied to functionalize SF hydrogel with improved bioactivity for cell adhesion and growth. Following encapsulating the vascular endothelial growth factor (VEGF), the SF gel was subcutaneously injected in mice, and served as an effective matrix to trigger the generation of new blood capillaries in vivo.

Conventional, pulsed and high-field electron paramagnetic resonance for studying metal impurities in calcium phosphates of biogenic and synthetic origins

Calcium phosphates (CaP) based materials are widely recognized as the most suitable matrix for bone tissue engineering. The cationic and anionic substitutions of CaP structure by the elements and groups of biological importance seem to be the effective ways to improve the properties of CaP-based substances to achieve the material’s desired parameters. Various analytical and biochemical methods are used for controlling their chemical content, structure, morphology, etc. Unfortunately, magnetic resonance techniques are usually not even considered as necessary tools for CaP inspection.Some aspects of application of the commercially realized electron paramagnetic resonance (EPR) approaches for characterization of CaP powders and ceramics (including the nanosized materials) such as hydroxyapatite and tricalcium phosphates of biogenic and synthetic origins containing intrinsic impurities or intentional dopants are demonstrated. Among them (1) conventional approaches; (2) high-frequency approaches – for measuring small amount of material; (3) pulsed methods – to study size effects, effects of the co-doping by different sorts of ions, to determine the location of paramagnetic centers, etc. The key features and advantages of the EPR techniques for CaP based materials characterization that could complement the data obtained with the recognized analytical methods are stressed.

Applications of Cardiac Extracellular Matrix in Tissue Engineering and Regenerative Medicine.

The role of the cardiac extracellular matrix (cECM) in providing biophysical and biochemical cues to the cells housed within during disease and development has become increasingly apparent. These signals have been shown to influence many fundamental cardiac cell behaviors including contractility, proliferation, migration, and differentiation. Consequently, alterations to cell phenotype result in directed remodeling of the cECM. This bidirectional communication means that the cECM can be envisioned as a medium for information storage. As a result, the reprogramming of the cECM is increasingly being employed in tissue engineering and regenerativemedicine as a method with which to treat disease. In this chapter, an overview of the composition and structure of the cECM as well as its role in cardiac development and disease will be provided. Additionally, therapeutic modulation of cECM for cardiac regeneration as well as bottom-up and top-down approaches to ECM-based cardiac tissue engineering is discussed. Finally, lingering questions regarding the role of cECM in tissue engineering and regenerative medicine are offered as a catalyst for future research.