Periprosthetic Joint Infections of the Knee Lastingly Impact the Bone Homeostasis.

After periprosthetic joint infection (PJI)-dependent revision surgery, a significantly elevated number of patients suffer from prosthesis failure due to aseptic loosening and require additional revision surgery despite clearance of the initial infection. The mechanisms underlying this pathology are not well understood, as it has been assumed that the bone stock recovers after revision surgery. Despite clinical evidence suggesting decreased osteogenic potential in PJI, understanding of the underlying biology remains limited. In this study, we investigated the impact of PJI on bone homeostasis in a two-stage exchange approach at explantation and reimplantation. Sixty-four human tibial and femoral specimens (20 control, 20 PJI septic explantation, and 24 PJI prosthesis reimplantation samples) were analyzed for their bone microstructure, cellular composition, and expression of relevant genetic markers. Samples were analyzed using X-ray microtomography, Alcian blue and tartrate-resistant acid phosphatase staining, and RT-qPCR. In patients with PJI, bone volume (BV/TV; 0.173 ± 0.026; p < 0.001), trabecular thickness (164.262 ± 18.841 µm; p < 0.001), and bone mineral density (0.824 ± 0.017 g/cm2 ; p = 0.049) were reduced; trabecular separation (1833.939 ± 178.501 µm; p = 0.005) was increased. While prevalence of osteoclasts was elevated (N.Oc/BS: 0.663 ± 0.102, p < 0.001), osteoblast cell numbers were lower at explantation (N.Ob/BS: 0.149 ± 0.021; p = 0.047). Mean expression of bone homeostasis markers osteocalcin, osteopontin, Runx2, TSG-6, and FGF-2 was significantly reduced at prosthesis explantation. Despite partial recovery, all analyzed parameters were still significantly impacted at reimplantation. In contrast, mean expression of osteoclastogenesis-stimulating cytokine IL-17a was significantly increased at both explantation and reimplantation. In this study, we found a strong and lasting impact of PJI on the bone homeostasis on a molecular, cellular, and microstructural level. These changes may be responsible for the increased risk of prosthesis failure due to aseptic loosening. Our data suggest there is significant potential in modulating bone homeostasis to improve prosthesis fixation and long-term clinical outcome in affected patients. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Collagen Fibrils Mechanically Contribute to Tissue Contraction in an In Vitro Wound Healing Scenario.

Wound contraction is an ancient survival mechanism of vertebrates that results from tensile forces supporting wound closure. So far, tissue tension was attributed to cellular forces produced by tissue-resident (myo-)fibroblasts alone. However, difficulties in explaining pathological deviations from a successful healing path motivate the exploration of additional modulatory factors. Here, it is shown in a biomaterial-based in vitro wound healing model that the storage of tensile forces in the extracellular matrix has a significant, so-far neglected contribution to macroscopic tissue tension. In situ monitoring of tissue forces together with second harmonic imaging reveal that the appearance of collagen fibrils correlates with tissue contraction, indicating a mechanical contribution of tensioned collagen fibrils in the contraction process. As the re-establishment of tissue tension is key to successful wound healing, the findings are expected to advance the understanding of tissue healing but also underlying principles of misregulation and impaired functionality in scars and tissue contractures.

Mesoscale substrate curvature overrules nanoscale contact guidance to direct bone marrow stromal cell migration.

The intrinsic architecture of biological tissues and of implanted biomaterials provides cells with large-scale geometrical cues. To understand how cells are able to sense and respond to complex structural environments, a deeper insight into the cellular response to multi-scale and conflicting geometrical cues is needed. In this study, we subjected human bone marrow stromal cells (hBMSCs) to mesoscale cylindrical surfaces (diameter 250-5000 µm) and nanoscale collagen fibrils (diameter 100-200 nm) that were aligned perpendicular to the cylinder axis. On flat surfaces and at low substrate curvatures (cylinder diameter d > 1000 µm), cell alignment and migration were governed by the nanoscale collagen fibrils, consistent with the contact guidance effect. With increasing surface curvature (decreasing cylinder diameter, d < 1000 µm), cells increasingly aligned and migrated along the cylinder axis, i.e. the direction of zero curvature. An increase in phosphorylated myosin light chain levels was observed with increasing substrate curvature, suggesting a link between substrate-induced cell bending and the F-actin-myosin machinery. Taken together, this work demonstrates that geometrical cues of up to 10× cell size can play a dominant role in directing hBMSC alignment and migration and that the effect of nanoscale contact guidance can even be overruled by mesoscale curvature guidance.

Surface Curvature Differentially Regulates Stem Cell Migration and Differentiation via Altered Attachment Morphology and Nuclear Deformation.

Signals from the microenvironment around a cell are known to influence cell behavior. Material properties, such as biochemical composition and substrate stiffness, are today accepted as significant regulators of stem cell fate. The knowledge of how cell behavior is influenced by 3D geometric cues is, however, strongly limited despite its potential relevance for the understanding of tissue regenerative processes and the design of biomaterials. Here, the role of surface curvature on the migratory and differentiation behavior of human mesenchymal stem cells (hMSCs) has been investigated on 3D surfaces with well-defined geometric features produced by stereolithography. Time lapse microscopy reveals a significant increase of cell migration speed on concave spherical compared to convex spherical structures and flat surfaces resulting from an upward-lift of the cell body due to cytoskeletal forces. On convex surfaces, cytoskeletal forces lead to substantial nuclear deformation, increase lamin-A levels and promote osteogenic differentiation. The findings of this study demonstrate a so far missing link between 3D surface curvature and hMSC behavior. This will not only help to better understand the role of extracellular matrix architecture in health and disease but also give new insights in how 3D geometries can be used as a cell-instructive material parameter in the field of biomaterial-guided tissue regeneration.

Crosstalk between immune cells and mesenchymal stromal cells in a 3D bioreactor system.

INTRODUCTION: Mesenchymal stromal cells (MSC), known for their high immune modulatory capacity are promising tools for several cell-based therapies. To better mimic the in vivo situation of MSC interactions with immune cells, we applied an artificial lymph node (ALN)-bioreactor culture system combining a miniaturized perfusion bioreactor with a 3D matrix-based cell culture of immune competent cells forming micro-organoids. METHODS: Rat lymph node cells and allogeneic bone marrow-derived MSCs were seeded in a 20:1 ratio within the agarose matrix of the ALN-reactor. Lymphocytes were pre-incubated with Concanavalin A (ConA) and then co-cultured with MSC in the matrix with additional ConA in the perfusing medium. Live/dead staining showed survival of the co-cultures during the 8-day ALN-reactor run. Paraffin sections of bioreactor matrices were analyzed by proliferating cell nuclear antigen (PCNA)-specific stai-ning to determine MSC proliferation. Immune modulatory capacity was defined by daily analysis of cytokine secretion profiles (TNFa, IFNy, IL-1a, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-12p40/p70, GM-CSF). RESULTS: Cytokine peak secretion at day 2 was significantly inhibited by MSCs for TNFa (96.8 ± 4.8%) and IFNy (88.7 ± 12.0%) in 3D co-cultures. In contrast, other cytokines (IL-1, IL-6, IL-12) were induced. Furthermore, we detected a significantly higher (58.8%) fraction of proliferating MSCs in the presence of immune cells compared to control bioreactors loaded with MSCs only. CONCLUSIONS: In the future, this system might be an excellent tool to investigate the mechanisms of MSC-mediated immune modulation during simulated in vivo conditions.

The impact of substrate stiffness and mechanical loading on fibroblast-induced scaffold remodeling.

Fibroblasts as many other cells are known to form, contract, and remodel the extracellular matrix (ECM). The presented study aims to gain an insight into how mechanical boundary conditions affect the production of ECM components, their remodeling, and the feedback of the altered mechanical cell environment on these processes. The influence of cyclic mechanical loading (f=1 Hz, 10% axial compression) and scaffold stiffness (E=1.2 and 8.5 kPa) on the mechanical properties of fibroblast-seeded scaffold constructs were investigated in an in vitro approach over 14 days of culture. To do so, a newly developed bioreactor system was employed. While mechanical loading resulted in a clear upregulation of procollagen-I and fibronectin production, scaffold stiffness showed to primarily influence matrix metalloproteinase-1 (MMP-1) secretion and cell-induced scaffold contraction. Higher stiffness of the collagen scaffolds resulted in an up to twofold higher production of collagen-degrading MMP-1. The changes of mechanical parameters like Young's modulus, maximum compression force, and elastic portion of compression force over time suggest that from initially distinct mechanical starting conditions (scaffold stiffness), the construct's mechanical properties converge over time. As a consequence of mechanical loading a shift toward higher construct stiffness was observed. The results suggest that scaffold stiffness has only a temporary effect on cell behavior, while the impact of mechanical loading is preserved over time. Thus, it is concluded that the mechanical environment of the cell after remodeling is depending on mechanical loading rather than on initial scaffold stiffness.