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A biotechnological platform consisting of two-color 3D super-resolution readout and a microfluidic system was developed to investigate platelet interaction with a layer of perfused endothelial cells under flow conditions. Platelet activation has been confirmed via CD62P clustering on the membrane and mitochondrial morphology of ECs at the single cell level were examined using 3D two-color single-molecule localization microscopy and classified applying machine learning. To compare binding of activated platelets to intact or stressed ECs, a femtosecond laser was used to induced damage to single ECs within the perfused endothelial layer. We observed that activated platelets bound to the perfused ECs layer preferentially in the proximity to single stressed ECs. Platelets activated under flow were ∼6 times larger compared to activated ones under static conditions. The CD62P expression indicated more CD62P proteins on membrane of dynamically activated platelets, with a tendency to higher densities at the platelet/EC interface. Platelets activated under static conditions showed a less pronounced CD62P top/bottom asymmetry. The clustering of CD62P in the platelet membrane differs depending on the activation conditions. Our results confirm that nanoscopic analysis using two-color 3D super-resolution technology can be used to assess platelet interaction with a stressed endothelium under dynamic conditions.

Regeneration of bone defects is often limited due to compromised bone tissue physiology. Previous studies suggest that engineered extracellular matrices enhance the regenerative capacity of mesenchymal stromal cells. In this study, we used human-induced pluripotent stem cells, a scalable source of young mesenchymal progenitors (hiPSC-MPs), to generate extracellular matrix (iECM) and test its effects on the osteogenic capacity of human bone-marrow mesenchymal stromal cells (BMSCs). iECM was deposited as a layer on cell culture dishes and into three-dimensional (3D) silk-based spongy scaffolds. After decellularization, iECM maintained inherent structural proteins including collagens, fibronectin and laminin, and contained minimal residual DNA. Young adult and aged BMSCs cultured on the iECM layer in osteogenic medium exhibited a significant increase in proliferation, osteogenic marker expression, and mineralization as compared to tissue culture plastic. With BMSCs from aged donors, matrix mineralization was only detected when cultured on iECM, but not on tissue culture plastic. When cultured in 3D iECM/silk scaffolds, BMSCs exhibited significantly increased osteogenic gene expression levels and bone matrix deposition. iECM layer showed a similar enhancement of aged BMSC proliferation, osteogenic gene expression, and mineralization compared with extracellular matrix layers derived from young adult or aged BMSCs. However, iECM increased osteogenic differentiation and decreased adipocyte formation compared with single protein substrates including collagen and fibronectin. Together, our data suggest that the microenvironment comprised of iECM can enhance the osteogenic activity of BMSCs, providing a bioactive and scalable biomaterial strategy for enhancing bone regeneration in patients with delayed or failed bone healing.

Medication-related osteonecrosis of the jaw (MRONJ) is a complication of certain pharmacological treatments such as bisphosphonates, denosumab, and angiogenesis inhibitors. There are currently no guidelines on its management, particularly in advanced stages. The human amniotic membrane (hAM) has low immunogenicity and exerts anti-inflammatory, antifibrotic, antimicrobial, antiviral, and analgesic effects. It is a source of stem cells and growth factors promoting tissue regeneration. hAM acts as an anatomical barrier with suitable mechanical properties (permeability, stability, elasticity, flexibility, and resorbability) to prevent the proliferation of fibrous tissue and promote early neovascularization at the surgical site. In oral surgery, hAM stimulates healing and facilitates the proliferation and differentiation of epithelial cells in the oral mucosa and therefore its regeneration. We proposed using cryopreserved hAM to eight patients suffering from cancer (11 lesions) with stage 2–3 MRONJ on a compassionate use basis. A collagen sponge was added in some cases to facilitate hAM grafting. One or three hAMs were applied and one patient had a reapplication. Three patients had complete closure of the surgical site with proper epithelialization at 2 weeks, and two of them maintained it until the last follow-up. At 1 week after surgery, three patients had partial wound dehiscence with partial healing 3 months later and two patients had complete wound dehiscence. hAM reapplication led to complete healing. All patients remained asymptomatic with excellent immediate significant pain relief, no infections, and a truly positive impact on the patients’ quality of life. No adverse events occurred. At 6 months of follow-up, 80% of lesions had complete or partial wound healing (30 and 50%, respectively), while 62.5% of patients were in stage 3. Radiological evaluations found that 85.7% of patients had stable bone lesions (n = 5) or new bone formation (n = 1). One patient had a worsening MRONJ but remained asymptomatic. One patient did not attend his follow-up radiological examination. For the first time, this prospective pilot study extensively illustrates both the handling and surgical application of hAM in MRONJ, its possible association with a collagen sponge scaffold, its outcome at the site, the application of multiple hAM patches at the same time, and its reapplication.

Fibrin hydrogels have proven highly suitable scaffold materials for skeletal muscle tissue engineering in the past. Certain parameters of those types of scaffolds, however, greatly affect cellular mechanobiology and therefore the myogenic outcome. The aim of this study was to identify the influence of apparent elastic properties of fibrin scaffolds in 2D and 3D on myoblasts and evaluate if those effects differ between murine and human cells. Therefore, myoblasts were cultured on fibrin-coated multiwell plates (“2D”) or embedded in fibrin hydrogels (“3D”) with different elastic moduli. Firstly, we established an almost linear correlation between hydrogels’ fibrinogen concentrations and apparent elastic moduli in the range of 7.5 mg/ml to 30 mg/ml fibrinogen (corresponds to a range of 7.7–30.9 kPa). The effects of fibrin hydrogel elastic modulus on myoblast proliferation changed depending on culture type (2D vs 3D) with an inhibitory effect at higher fibrinogen concentrations in 3D gels and vice versa in 2D. The opposite effect was evident in differentiating myoblasts as shown by gene expression analysis of myogenesis marker genes and altered myotube morphology. Furthermore, culture in a 3D environment slowed down proliferation compared to 2D, with a significantly more pronounced effect on human myoblasts. Differentiation potential was also substantially impaired upon incorporation into 3D gels in human, but not in murine, myoblasts. With this study, we gained further insight in the influence of apparent elastic modulus and culture type on cellular behavior and myogenic outcome of skeletal muscle tissue engineering approaches. Furthermore, the results highlight the need to adapt parameters of 3D culture setups established for murine cells when applied to human cells.

Biomechanical cues such as shear stress, stretching, compression, and matrix elasticity are vital in the establishment of next generation physiological in vitro tissue models. Matrix elasticity, for instance, is known to guide stem cell differentiation, influence healing processes and modulate extracellular matrix (ECM) deposition needed for tissue development and maintenance. To better understand the biomechanical effect of matrix elasticity on the formation of articular cartilage analogs in vitro, this study aims at assessing the redifferentiation capacity of primary human chondrocytes in three different hydrogel matrices of predefined matrix elasticities. The hydrogel elasticities were chosen to represent a broad spectrum of tissue stiffness ranging from very soft tissues with a Young’s modulus of 1 kPa up to elasticities of 30 kPa, representative of the perichondral-space. In addition, the interplay of matrix elasticity and transforming growth factor beta-3 (TGF-β3) on the redifferentiation of primary human articular chondrocytes was studied by analyzing both qualitative (viability, morphology, histology) and quantitative (RT-qPCR, sGAG, DNA) parameters, crucial to the chondrotypic phenotype. Results show that fibrin hydrogels of 30 kPa Young’s modulus best guide chondrocyte redifferentiation resulting in a native-like morphology as well as induces the synthesis of physiologic ECM constituents such as glycosaminoglycans (sGAG) and collagen type II. This comprehensive study sheds light onto the mechanobiological impact of matrix elasticity on formation and maintenance of articular cartilage and thus represents a major step toward meeting the need for advanced in vitro tissue models to study both re- and degeneration of articular cartilage.

Effective regeneration of bone defects often presents significant challenges, particularly in patients with decreased tissue regeneration capacity due to extensive trauma, disease, and/or advanced age. A number of studies have focused on enhancing bone regeneration by applying mesenchymal stromal cells (MSCs) or MSC-based bone tissue engineering strategies. However, translation of these approaches from basic research findings to clinical use has been hampered by the limited understanding of MSC therapeutic actions and complexities, as well as costs related to the manufacturing, regulatory approval, and clinical use of living cells and engineered tissues. More recently, a shift from the view of MSCs directly contributing to tissue regeneration toward appreciating MSCs as “cell factories” that secrete a variety of bioactive molecules and extracellular vesicles with trophic and immunomodulatory activities has steered research into new MSC-based, “cell-free” therapeutic modalities. The current review recapitulates recent developments, challenges, and future perspectives of these various MSC-based bone tissue engineering and regeneration strategies.

There is continual demand for animal models that allow a quantitative assessment of angiogenic properties of biomaterials, therapies, and pharmaceuticals. In its simplest form, this is done by subcutaneous material implantation and subsequent vessel counting which usually omits spatial data. We have refined an implantation model and paired it with a computational analytic routine which outputs not only vessel count but also vessel density, distribution, and vessel penetration depth, that relies on a centric vessel as a reference point. We have successfully validated our model by characterizing the angiogenic potential of a fibrin matrix in conjunction with recombinant human vascular endothelial growth factor (rhVEGF165). The inferior epigastric vascular pedicles of rats were sheathed with silicone tubes, which were subsequently filled with 0.2 ml of fibrin and different doses of rhVEGF165, centrically embedding the vessels. Over 4 weeks, tissue samples were harvested and subsequently immunohistologically stained and computationally analyzed. The model was able to detect variations over the angiogenic potentials of growth factor spiked fibrin matrices. Adding 20 ng of rhVEGF165 resulted in a significant increase in vasculature while 200 ng of rhVEGF165 did not improve vascular growth. Vascularized tissue volume increased during the first week and vascular density increased during the second week. Total vessel count increased significantly and exhibited a peak after 2 weeks which was followed by a resorption of vasculature by week 4. In summary, a simple implantation model to study in vivo vascularization with only a minimal workload attached was enhanced to include morphologic data of the emerging vascular tree.