Christoph Meinert

Effect of preculture and loading on expression of matrix molecules, matrix metalloproteinases, and cytokines by expanded osteoarthritic chondrocytes.

OBJECTIVE One of the pathologic changes that occurs during osteoarthritis (OA) is the degeneration of the pericellular matrix (PCM). Since the PCM is likely to be involved in mechanotransduction, this study was undertaken to investigate the effects of PCM-like matrix accumulation in zonal OA chondrocytes and their influence on chondrocyte response to compression. METHODS Superficial and middle/deep zone chondrocytes from macroscopically normal cartilage of OA knees were expanded and encapsulated in alginate gels. The effects of compression (short-term or long-term) and preculture on chondrocyte expression of various matrix molecules, cytokines, and matrix metalloproteinases (MMPs) were assessed. Additionally, nonexpanded chondrocytes were encapsulated in alginate and cultured in the presence or absence of transforming growth factor β1 (TGFβ1) and dexamethasone and analyzed following short-term compression experiments. RESULTS Expanded OA chondrocytes (superficial and middle/deep zone) that were precultured for 2 weeks under free-swelling conditions prior to dynamic compression responded more sensitively to loading and had increased matrix accumulation, increased interleukin-1β (IL-1β) and IL-4 levels, and decreased levels of MMP-2 (in the middle/deep zone) compared to the nonloaded controls. Compression also decreased MMP-3 and MMP-13 levels even without preculture. Nonexpanded chondrocytes did not respond to compression, but differences in gene expression were found depending on the zone of harvest, time in culture, and medium composition. CONCLUSION Our findings demonstrate that with predeposited PCM-like matrix, compressive stimulation can enhance matrix protein accumulation in expanded OA chondrocytes. Investigations into how PCM or other matrix components differentially affect this balance under mechanical loading may provide invaluable insight into OA pathogenesis and the use of expanded cells in tissue engineering and regenerative medicine-based applications.

A novel bioreactor system for biaxial mechanical loading enhances the properties of tissue-engineered human cartilage

The ex vivo engineering of autologous cartilage tissues has the potential to revolutionize the clinical management of joint disorders. Yet, high manufacturing costs and variable outcomes associated with tissue-engineered implants are still limiting their application. To improve clinical outcomes and facilitate a wider use of engineered tissues, automated bioreactor systems capable of enhancing and monitoring neotissues are required. Here, we developed an innovative system capable of applying precise uni- or biaxial mechanical stimulation to developing cartilage neotissues in a tightly controlled and automated fashion. The bioreactor allows for simple control over the loading parameters with a user-friendly graphical interface and is equipped with a load cell for monitoring tissue maturation. Applying our bioreactor, we demonstrate that human articular chondrocytes encapsulated in hydrogels composed of gelatin methacryloyl (GelMA) and hyaluronic acid methacrylate (HAMA) respond to uni- and biaxial mechanical stimulation by upregulation of hyaline cartilage-specific marker genes. We further demonstrate that intermittent biaxial mechanostimulation enhances accumulation of hyaline cartilage-specific extracellular matrix. Our study underlines the stimulatory effects of mechanical loading on the biosynthetic activity of human chondrocytes in engineered constructs and the need for easy-to-use, automated bioreactor systems in cartilage tissue engineering.

Attenuated kallikrein‐related peptidase activity disrupts desquamation and leads to stratum corneum thickening in human skin equivalent models

Epidermal homeostasis is maintained through the balance between keratinocyte proliferation, differentiation and desquamation; however, human skin equivalent (HSE) models are known to differentiate excessively. In native tissue, proteases such as kallikrein‐related peptidase (KLK) 5 and KLK7 cleave the extracellular components of corneodesmosomes; proteins corneodesmosin, desmocollin 1 and desmoglein 1, loosening the cellular connections and enabling desquamation. The actions of KLK7 are tightly controlled by protease inhibitors, skin‐derived antileucoproteinase (SKALP) and lymphoepithelial Kazal‐type‐related inhibitor (LEKTI), which also inhibits KLK5, localizing protease activity to the stratum corneum.

Tailoring hydrogel surface properties to modulate cellular response to shear loading.

Biological tissues at articulating surfaces, such as articular cartilage, typically have remarkable low-friction properties that limit tissue shear during movement. However, these frictional properties change with trauma, aging, and disease, resulting in an altered mechanical state within the tissues. Yet, it remains unclear how these surface changes affect the behaviour of embedded cells when the tissue is mechanically loaded. Here, we developed a cytocompatible, bilayered hydrogel system that permits control of surface frictional properties without affecting other bulk physicochemical characteristics such as compressive modulus, mass swelling ratio, and water content. This hydrogel system was applied to investigate the effect of variations in surface friction on the biological response of human articular chondrocytes to shear loading. Shear strain in these hydrogels during dynamic shear loading was significantly higher in high-friction hydrogels than in low-friction hydrogels. Chondrogenesis was promoted following dynamic shear stimulation in chondrocyte-encapsulated low-friction hydrogel constructs, whereas matrix synthesis was impaired in high-friction constructs, which instead exhibited increased catabolism. Our findings demonstrate that the surface friction of tissue-engineered cartilage may act as a potent regulator of cellular homeostasis by governing the magnitude of shear deformation during mechanical loading, suggesting a similar relationship may also exist for native articular cartilage. STATEMENT OF SIGNIFICANCE Excessive mechanical loading is believed to be a major risk factor inducing pathogenesis of articular cartilage and other load-bearing tissues. Yet, the mechanisms leading to increased transmission of mechanical stimuli to cells embedded in the tissue remain largely unexplored. Here, we demonstrate that the tribological properties of loadbearing tissues regulate cellular behaviour by governing the magnitude of mechanical deformation arising from physiological tissue function. Based on these findings, we propose that changes to articular surface friction as they occur with trauma, aging, or disease, may initiate tissue pathology by increasing the magnitude of mechanical stress on embedded cells beyond a physiological level.

Immune system augmentation via humanization using stem/progenitor cells and bioengineering in a breast cancer model study: Humanized mouse model for cancer study

Despite significant advances, most current in vivo models fail to fully recapitulate the biological processes that occur in humans. Here we aimed to develop an advanced humanized model with features of an organ bone by providing different bone tissue cellular compartments including preosteoblasts, mesenchymal stem/stromal (MSCs), endothelial and hematopoietic cells in an engineered microenvironment. The bone compartment was generated by culturing the human MSCs, umbilical vein endothelial cells with gelatin methacryloyl hydrogels in the center of a melt‐electrospun polycaprolactone tubular scaffolds, which were seeded with human preosteoblasts. The tissue engineered bone (TEB) was subcutaneously implanted into the NSG mice and formed a morphologically and functionally organ bone. Mice were further humanized through the tail vein injection of human cord blood derived CD34+ cells, which then populated in the mouse bone marrow, spleen and humanized TEB (hTEB). 11 weeks after CD34+ transplantation, metastatic breast cancer cells (MDA‐MB‐231BO) were orthotopically injected. Cancer cell injection resulted in the formation of a primary tumor and metastasis to the hTEB and mouse organs. Less frequent metastasis and lower tumor burden were observed in hematochimeric mice, suggesting an immune‐mediated response against the breast cancer cells. Overall, our results demonstrate the efficacy of tissue engineering approaches to study species‐specific cancer‐bone interactions. Further studies using genetically modified hematopoietic stem cells and bioengineered microenvironments will enable us to address the specific roles of signaling molecules regulating hematopoietic niches and cancer metastasis in vivo.

Engineering Anisotropic Muscle Tissue using Acoustic Cell Patterning

Abstract Tissue engineering has offered unique opportunities for disease modeling and regenerative medicine; however, the success of these strategies is dependent on faithful reproduction of native cellular organization. Here, it is reported that ultrasound standing waves can be used to organize myoblast populations in material systems for the engineering of aligned muscle tissue constructs. Patterned muscle engineered using type I collagen hydrogels exhibits significant anisotropy in tensile strength, and under mechanical constraint, produced microscale alignment on a cell and fiber level. Moreover, acoustic patterning of myoblasts in gelatin methacryloyl hydrogels significantly enhances myofibrillogenesis and promotes the formation of muscle fibers containing aligned bundles of myotubes, with a width of 120–150 µm and a spacing of 180–220 µm. The ability to remotely pattern fibers of aligned myotubes without any material cues or complex fabrication procedures represents a significant advance in the field of muscle tissue engineering. In general, these results are the first instance of engineered cell fibers formed from the differentiation of acoustically patterned cells. It is anticipated that this versatile methodology can be applied to many complex tissue morphologies, with broader relevance for spatially organized cell cultures, organoid development, and bioelectronics.