Macrophages are key players of the innate immune system that can roughly be divided into the pro-inflammatory M1 type and the anti-inflammatory, pro-healing M2 type. While a transient initial pro-inflammatory state is helpful, a prolonged inflammation deteriorates a proper healing and subsequent regeneration. One promising strategy to drive macrophage polarization by biomaterials is precise control over biomaterial geometry. For regenerative approaches, it is of particular interest to identify geometrical parameters that direct human macrophage polarization. For this purpose, we advanced melt electrowriting (MEW) towards the fabrication of fibrous scaffolds with box-shaped pores and precise inter-fiber spacing from 100 μm down to only 40 μm. These scaffolds facilitate primary human macrophage elongation accompanied by differentiation towards the M2 type, which was most pronounced for the smallest pore size of 40 μm. These new findings can be important in helping to design new biomaterials with an enhanced positive impact on tissue regeneration.

In vitro co-cultures of different primary human cell types are pivotal for the testing and evaluation of biomaterials under conditions that are closer to the human in vivo situation. Especially co-cultures of macrophages and mesenchymal stem cells (MSCs) are of interest, as they are both present and involved in tissue regeneration and inflammatory reactions and play crucial roles in the immediate inflammatory reactions and the onset of regenerative processes, thus reflecting the decisive early phase of biomaterial contact with the host. A co-culture system of these cell types might thus allow for the assessment of the biocompatibility of biomaterials. The establishment of such a co-culture is challenging due to the different in vitro cell culture conditions. For human macrophages, medium is usually supplemented with human serum (hS), whereas hMSC culture is mostly performed using fetal calf serum (FCS), and these conditions are disadvantageous for the respective other cell type. We demonstrate that human platelet lysate (hPL) can replace hS in macrophage cultivation and appears to be the best option for co-cultivation of human macrophages with hMSCs. In contrast to FCS and hS, hPL maintained the phenotype of both cell types, comparable to that of their respective standard culture serum, as well as the percentage of each cell population. Moreover, the expression profile and phagocytosis activity of macrophages was similar to hS.

Melt electrowriting (MEW) is an additive manufacturing technology that produces readily handleable fibrous scaffolds with controlled geometry to support cell infiltration. Although MEW scaffolds have excellent potential for cell delivery in regenerative medicine applications, studies to date have primarily focused on polymers such as poly­(ε-caprolactone) (PCL) that lack bioactive cues to affect cell function. To address this aspect, MEW scaffolds with extracellular matrix (ECM) coatings were developed as a proadipogenic platform for human mesenchymal stromal cells (hMSCs). More specifically, highly flexible PCL scaffolds fabricated through MEW were coated with a complex ECM suspension prepared from human decellularized adipose tissue (DAT), purified fibronectin, or laminin to determine the effects of two key bioactive proteins present within adipose-derived ECM. In vitro studies exploring the response of human bone marrow-derived mesenchymal stromal cells cultured under adipogenic differentiation conditions indicated a high level of differentiation on all substrates studied, including unmodified PCL scaffolds and two-dimensional controls. To more fully assess the intrinsic proadipogenic capacity of the composite biomaterials, a modified culture regime was established that involved a short-term adipogenic induction in differentiation medium, followed by continued culture in maintenance medium supplemented with insulin for up to 3 weeks. Under these conditions, adipogenic differentiation was enhanced on all fiber scaffolds as compared to the tissue culture controls. Notably, the highest adipogenic response was consistently observed on the PCL + DAT scaffolds, based on the analysis of multiple markers including adipogenic gene [lipoprotein lipase, fatty acid binding protein 4 (FABP4), adiponectin, perilipin 1] and protein (FABP4, leptin) expression and intracellular triglyceride accumulation. Taken together, the PCL scaffolds incorporating DAT provide an adipoinductive microenvironment for the hMSCs, with particular applicability of this cell-instructive delivery platform for applications in plastic and reconstructive surgery.

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Physical interaction of skeletal precursors with multiple myeloma cells has been shown to suppress their osteogenic potential while favoring their tumor-promoting features. Although several transcriptome analyses of myeloma patient-derived mesenchymal stem cells have displayed differences compared to their healthy counterparts, these analyses insufficiently reflect the signatures mediated by tumor cell contact, vary due to different methodologies, and lack results in lineage-committed precursors. To determine tumor cell contact-mediated changes on skeletal precursors, we performed transcriptome analyses of mesenchymal stem cells and osteogenic precursor cells cultured in contact with the myeloma cell line INA-6. Comparative analyses confirmed dysregulation of genes which code for known disease-relevant factors and additionally revealed upregulation of genes that are associated with plasma cell homing, adhesion, osteoclastogenesis, and angiogenesis. Osteoclast-derived coupling factors, a dysregulated adipogenic potential, and an imbalance in favor of anti-anabolic factors may play a role in the hampered osteoblast differentiation potential of mesenchymal stem cells. Angiopoietin-Like 4 (ANGPTL4) was selected from a list of differentially expressed genes as a myeloma cell contact-dependent target in skeletal precursor cells which warranted further functional analyses. Adhesion assays with full-length ANGPTL4-coated plates revealed a potential role of this protein in INA-6 cell attachment. This study expands knowledge of the myeloma cell contact-induced signature in the stromal compartment of myelomatous bones and thus offers potential targets that may allow detection and treatment of myeloma bone disease at an early stage.

Biointerface engineering is a wide‐spread strategy to improve the healing process and subsequent tissue integration of biomaterials. Especially the integration of specific peptides is one promising strategy to promote the regenerative capacity of implants and 3D scaffolds. In vivo, these tailored interfaces are, however, first confronted with the innate immune response. Neutrophils are cells with pronounced proteolytic potential and the first recruited immune cells at the implant site; nonetheless, they have so far been underappreciated in the design of biomaterial interfaces. Herein, an in vitro approach is introduced to model and analyze the neutrophil interaction with bioactivated materials at the example of nano‐bioinspired electrospun surfaces that reveals the vulnerability of a given biointerface design to the contact with neutrophils. A sacrificial, transient hydrogel coating that demonstrates optimal protection for peptide‐modified surfaces and thus alleviates the immediate cleavage by neutrophil elastase is further introduced.

Multipotent adult stem cells/precursor cells, especially of the mesenchymal and endothelial lineage, may have great potential for bone tissue engineering. Although their potential is highly recognized, not much is known about the underlying molecular mechanisms that initiate the regeneration process, connect osteogenesis, and angiogenesis and, finally, orchestrate renewal of bone tissue. Our study addressed these questions by generating two in vitro cell culture models to examine the changes in the global gene expression patterns of endothelial precursor cells and mesenchymal stem cells after 24 hours of either humoral (conditioned medium) or direct cell-cell interaction (co-culture). Endothelial precursor cells were isolated from human buffy coat and mesenchymal stem cells from the bone marrow of the femoral head. The comparison of the treated and control cells by microarray analyses revealed in total more than 1500 regulated genes, which were analyzed for their affiliation to angiogenesis and osteogenesis. Expression array analyses at the RNA and protein level revealed data with respect to regulated genes, pathways and targets that may represent a valid basis for further dissection of the systems biology of regeneration processes. It may also be helpful for the reconstitution of the natural composition of a regenerative microenvironment when targeting tissue regeneration both in vitro and in situ.

Abstract:Macrophages are important cells of the innate immune system. They exhibit a high plasticity in phenotypes and play a major role in healing by initiating the early inflammatory reactions via the pro-inflammatory M1 phenotype. The anti-inflammatory M2 phenotype is assumed to induce regenerative processes and vascularization in subsequent tissue repair. Especially for regenerative processes, their interplay with multipotent human mesenchymal stromal cells (hMSCs) is decisive. Accordingly, in vitro co-culture models of these cell types are an important starting point for unraveling regenerative mechanisms. In our study, we compared direct co-culture, transwell-systems, and the use of conditioned medium to investigate the mitochondria transfer between the two cell types and the influence of hMSCs' presence on the phagocytic activity of macrophages. Using flow cytometry and fluorescence microscopy, we visualized the transfer of mitochondria in both directions: from hMSCs to macrophages and most notably also vice versa. Both cell types release mitochondria and internalize them in direct contact via tunneling nanotubes, as well as in indirect contact due to extracellular vesicles (EVs). Mitochondria were non-directionally released into the medium and could be transferred via conditioned medium. After three hours of direct and indirect co-culture, the majority of the cells showed a mitochondrial uptake. Co-cultivation also led to an increase of phagocytic activity of macrophages, with the highest phagocytic rate after 48 h and most pronounced in direct co-cultivation.