The Use of a Green Fluorescent Protein Porcine Model to Evaluate Host Tissue Integration into Extracellular Matrix Derived Bionanocomposite Scaffolds

When using heterogeneous extracellular matrix (ECM) derived scaffolds for soft tissue repair, current methods of in vivo evaluation can fail to provide a clear distinction between host collagen and implanted scaffolds making it difficult to assess host tissue integration and remodeling. The purpose of this study is both to evaluate novel scaffolds conjugated with nanoparticles for host tissue integration and biocompatibility and to assess green fluorescent protein (GFP) expressing swine as a new animal model to evaluate soft tissue repair materials. Human-derived graft materials conjugated with nanoparticles were subcutaneously implanted into GFP expressing swine to be evaluated for biocompatibility and tissue integration through histological scoring and confocal imaging. Histological scoring indicates biocompatibility and remodeling of the scaffolds with and without nanoparticles at 1, 3, and 6 months. Confocal microscope images display host tissue integration into scaffolds although nonspecificity of GFP does not allow for quantification of integration. However, the confocal images do allow for spatial observation of host tissue migration into the scaffolds at different depths of penetration. The study concludes that the nanoparticle scaffolds are biocompatible and promote integration and that the use of GFP expressing swine can aid in visualizing the scaffold/host interface and host cell/tissue migration.

90° Peel off Tests of Tissue Engineered Osteochondral Constructs: A New Method to Determine the Osteochondral Integration

One promising treatment of localized osteochondral defects in the knee joint may be the implantation of in vitro tissue-engineered osteochondral constructs. A crucial aspect of this kind of osteochondral construct is the bonding between the bone-scaffold and scaffold-based chondral layer. Here, a 90° peel off test is proposed as an appropriate method to measure the integration of cartilage to bone in osteochondral constructs for different primary methods of bonding the cartilage scaffold to the bone scaffold, with and without seeded chondroblasts. The method was developed and then tested on tissue-engineered constructs. The force/displacement data obtained allow determination of both the maximum force and the total energy required to separate the two layers. The tests showed good reproducibility and good discrimination between measurements as a function of seeding times. Average maximum peel-off forces varied between 10 mN for fibrin glue only to 575 mN for constructs with cells after four weeks of incubation. Linear regression of the area under the curve (AUC) as a function of maximum force shows a high correlation between the two parameters with R2 = 0.97. The main limitation of the test is that the data provide only a modest ability to decide how uniform the bond is over the area between the two layers.

Alignment of Skeletal Muscle Cells Cultured in Collagen Gel by Mechanical and Electrical Stimulation

For in vitro tissue engineering of skeletal muscle, alignment and fusion of the cultured skeletal muscle cells are required. Although the successful alignment of skeletal muscle cells cultured in collagen gel has been reported using a mechanical force, other means of aligning cultured skeletal muscle cells have not been described. However, skeletal muscle cells cultured in a two-dimensional dish have been reported to align in a uniform direction when electrically stimulated. The purpose of this study is to determine if skeletal muscle cells cultured three-dimensionally in collagen gels can be aligned by an electrical load. By adding direct current to cells of the C2C12 skeletal muscle cell line cultured in collagen gel, it was possible to align C2C12 cells in a similar direction. However, the ratio of alignment was better when mechanical force was used as the means of alignment. Thus for tissue engineering of skeletal muscle cells, electrical stimulation may be useful as a supplementary method.

In Vivo Evaluation of a Decellularized Limbal Graft for Limbal Reconstruction

Corneal and limbal epithelial function is highly dependent on its underlying matrix. In this study, we report the in vitro and in vivo effects of a decellularized limbal matrix on corneal and limbal epithelial differentiation and repair. We demonstrate that a limbal matrix helps to maintain epithelial cells in a more proliferative and less differentiated state. We introduce a novel focal injury model to the limbus using an excimer laser and further show that transplanting a decellularized limbal graft after the limbal injury helps to promote epithelialization and reduce corneal haze formation. These results suggest that a decellularized limbal graft may be therapeutically beneficial in clinical cases of focal limbal deficiency.

Engineered Human Muscle Tissue from Skeletal Muscle Derived Stem Cells and Induced Pluripotent Stem Cell Derived Cardiac Cells

During development, cardiac and skeletal muscle share major transcription factors and sarcomere proteins which were generally regarded as specific to either cardiac or skeletal muscle but not both in terminally differentiated adult cardiac or skeletal muscle. Here, we investigated whether artificial muscle constructed from human skeletal muscle derived stem cells (MDSCs) recapitulates developmental similarities between cardiac and skeletal muscle. We constructed 3-dimensional collagen-based engineered muscle tissue (EMT) using MDSCs (MDSC-EMT) and compared the biochemical and contractile properties with EMT using induced pluripotent stem (iPS) cell-derived cardiac cells (iPS-EMT). Both MDSC-EMT and iPS-EMT expressed cardiac specific troponins, fast skeletal muscle myosin heavy chain, and connexin-43 mimicking developing cardiac or skeletal muscle. At the transcriptional level, MDSC-EMT and iPS-EMT upregulated both cardiac and skeletal muscle-specific genes and expressed Nkx2.5 and Myo-D proteins. MDSC-EMT displayed intracellular calcium ion transients and responses to isoproterenol. Contractile force measurements of MDSC-EMT demonstrated functional properties of immature cardiac and skeletal muscle in both tissues. Results suggest that the EMT from MDSCs mimics developing cardiac and skeletal muscle and can serve as a useful in vitro functioning striated muscle model for investigation of stem cell differentiation and therapeutic options of MDSCs for cardiac repair.

A Fibrin-Based Tissue-Engineered Renal Proximal Tubule for Bioartificial Kidney Devices: Development, Characterization and In Vitro Transport Study

A bioartificial renal proximal tubule is successfully engineered as a first step towards a bioartificial kidney for improved renal substitution therapy. To engineer the tubule, a tunable hollow fiber membrane with an exterior skin layer that provides immunoprotection for the cells from extracapillary blood flow and a coarse inner surface that facilitates a hydrogel coating for cell attachment was embedded in a “lab-on-a-chip” model for the small-scale exploratory testing under flow conditions. Fibrin was coated onto the inner surface of the hollow fiber, and human renal proximal tubule epithelial cells were then seeded. Using this model, we successfully cultured a confluent monolayer, as ascertained by immunofluorescence staining for ZO-1 tight junctions and other proximal tubule markers, scanning electron microscopy, and FITC-inulin recovery studies. Furthermore, the inulin studies, combined with the creatinine and glucose transport profiles, suggested that the confluent monolayer exhibits functional transport capabilities. The novel approaches here may eventually improve current renal substitution technology for renal failure patients.

Studies on a Novel Bioreactor Design for Chondrocyte Culture

A bioreactor system plays an important role in tissue engineering and enables reproduction and controlled changes in the environmental factor. The bioreactor provides technical means to perform controlled processes in safe and reduced reproducible generation of time. Cartilage cells were grown in vitro by mimicking the in vivo condition. The basic unit of cartilage, that is, chondrocyte, requires sufficient shear, strain, and hydrodynamic pressure for regular growth as it is nonvascular tissue. An attempt has been made to design a novel airlift reactor for chondrocyte culture, and the reactor has been evaluated for its performance. The design includes internal loop wavy riser airlift reactor for chondrocyte culture with 5% CO2 sparging which gives a good yield of chondrocyte after 28 days. The wavy riser provides more surfaces for collision of fluid flow so to create the turbulence. Also, the horizontal semicircular baffles create an angle of 180° which helps in high shear rate. The optimized L/D ratio of the designed airlift reactor (for chondrocyte culture) is 5.67, and it also exhibits good mixing performance.

Biomanufacturing versus Superficial Cell Seeding: Simulation of Chondrocyte Proliferation in a Cylindrical Cartilage Scaffold

Local volume averaging approach was used for modeling and simulation of cell growth and proliferation, as well as glucose transfer within a cylindrical cartilage scaffold during cell cultivation. The scaffold matrix including the nutrient solution filling spaces among seeded cell colonies was treated as a porous medium. Applying differential mass balance of cells and glucose to a representative elementary volume of the scaffold, two diffusional mass transfer models were developed based on local volume averaged properties. The derived governing equations take into account time-dependent glucose diffusion, glucose consumption by cells, cell migration, apoptosis, and cell reproduction within the scaffold. Since the volumetric fraction of cells in the scaffold relies on cell growth, which strongly depends on glucose concentration in the scaffold, the governing equations were solved simultaneously using implicit finite difference method and Gauss-Seidel technique. Simulation results showed that cell volumetric fraction of the scaffold can reach about 45% after 50 days if a culture medium with a glucose concentration of 45 kgm−3 is used. Also, simulation results indicate that more uniform and higher average cell volume fraction of the scaffold can be obtained if biomanufacturing-based cell seeding is used across the scaffold rather than cell seeding on the scaffold surface.