15 research outputs found
Herbal Extracts of Ginseng and Maqui Berry Show Only Minimal Effects on an In Vitro Model of Early Fracture Repair of Smokers
Smoking Impairs Hematoma Formation and Dysregulates Angiogenesis as the First Steps of Fracture Healing
Bone fracture healing is an overly complex process in which inflammation, osteogenesis, and angiogenesis are tightly coupled, and delayed fracture repair is a very common health risk. One of the major causes of delayed healing is the formation of insufficient vasculature. Precise regulation of blood vessels in bone and their interplay with especially osteogenic processes has become an emerging topic within the last years; nevertheless, regulation of angiogenesis in (early) diseased fracture repair is still widely unknown. Here, we aim to develop an in vitro model for the analysis of early fracture healing which also enables the analysis of angiogenesis as a main influencing factor. As smoking is one of the main risk factors for bone fractures and developing a delay in healing, we model smoking and non-smoking conditions in vitro to analyze diverging reactions. Human in vitro fracture hematomas mimicking smokers’ and non-smokers’ hematomas were produced and analyzed regarding cell viability, inflammation, osteogenic and chondrogenic differentiation, and angiogenic potential. We could show that smokers’ blood hematomas were viable and comparable to non-smokers. Smokers’ hematomas showed an increase in inflammation and a decrease in osteogenic and chondrogenic differentiation potential. When analyzing angiogenesis, we could show that the smokers’ hematomas secrete factors that drastically reduced HUVEC proliferation and tube formation. With an angiogenesis array and gene expression analysis, we could identify the main influencing factors: Anpgt1/2, Tie2, and VEGFR2/3. In conclusion, our model is suitable to mimic smoking conditions in vitro showing that smoking negatively impacts early vascularization of newly formed tissue
Use of in vitro bone models to screen for altered bone metabolism, osteopathies, and fracture healing: challenges of complex models
Approx. every third hospitalized patient in Europe suffers from musculoskeletal injuries or diseases. Up to 20% of these patients need costly surgical revisions after delayed or impaired fracture healing. Reasons for this are the severity of the trauma, individual factors, e.g, the patients' age, individual lifestyle, chronic diseases, medication, and, over 70 diseases that negatively affect the bone quality. To investigate the various disease constellations and/or develop new treatment strategies, many in vivo, ex vivo, and in vitro models can be applied. Analyzing these various models more closely, it is obvious that many of them have limits and/or restrictions. Undoubtedly, in vivo models most completely represent the biological situation. Besides possible species-specific differences, ethical concerns may question the use of in vivo models especially for large screening approaches. Challenging whether ex vivo or in vitro bone models can be used as an adequate replacement for such screenings, we here summarize the advantages and challenges of frequently used ex vivo and in vitro bone models to study disturbed bone metabolism and fracture healing. Using own examples, we discuss the common challenge of cell-specific normalization of data obtained from more complex in vitro models as one example of the analytical limits which lower the full potential of these complex model systems
Establishment of an In Vitro Scab Model for Investigating Different Phases of Wound Healing
Chronic wounds are a serious problem in clinical work and a heavy burden for individuals and society. In order to develop novel therapies, adequate model systems for the investigation of wound healing are required. Although in past years different in vitro and in vitro wound healing models have been established, a true human-like model does still not exist. Animal models are limited in their use due to species-specific differences in the skin, a lengthy manufacturing process, experimental costs, and ethical concerns. Both 2D and 3D in vitro models are usually comprised of only one or two skin cell types and fail to capture the reaction between blood cells and skin cells. Thus, our aim was to develop an in vitro scab model to investigate early reactions in the wound healing process. The here established scab model is comprised of HaCaT cells and freshly collected blood from healthy volunteers. The generated scabs were stably cultured for more than 2 weeks. TGF-β signaling is well known to regulate the early phases of wound healing. All three TGF-β isoforms and target genes involved in extracellular matrix composition and degradation were expressed in the in vitro scabs. To validate the in vitro scab model, the effects of either additional stimulation or the inhibition of the TGF-β signaling pathway were investigated. Exogenous application of TGF-β1 stimulated matrix remodeling, which loosened the structure of the in vitro scabs with time, also induced expression of the inhibitory Smad7. Inhibition of the endogenous TGF-β signaling, on the contrary, resulted in a rapid condensation and degranulation of the in vitro scabs. In summary, the here established in vitro scab model can be used to analyze the first phases of wound healing where blood and skin cells interact, as it is viable and responsive for more than 2 weeks
Establishment of an In Vitro Scab Model for Investigating Different Phases of Wound Healing
Chronic wounds are a serious problem in clinical work and a heavy burden for individuals and society. In order to develop novel therapies, adequate model systems for the investigation of wound healing are required. Although in past years different in vitro and in vitro wound healing models have been established, a true human-like model does still not exist. Animal models are limited in their use due to species-specific differences in the skin, a lengthy manufacturing process, experimental costs, and ethical concerns. Both 2D and 3D in vitro models are usually comprised of only one or two skin cell types and fail to capture the reaction between blood cells and skin cells. Thus, our aim was to develop an in vitro scab model to investigate early reactions in the wound healing process. The here established scab model is comprised of HaCaT cells and freshly collected blood from healthy volunteers. The generated scabs were stably cultured for more than 2 weeks. TGF-β signaling is well known to regulate the early phases of wound healing. All three TGF-β isoforms and target genes involved in extracellular matrix composition and degradation were expressed in the in vitro scabs. To validate the in vitro scab model, the effects of either additional stimulation or the inhibition of the TGF-β signaling pathway were investigated. Exogenous application of TGF-β1 stimulated matrix remodeling, which loosened the structure of the in vitro scabs with time, also induced expression of the inhibitory Smad7. Inhibition of the endogenous TGF-β signaling, on the contrary, resulted in a rapid condensation and degranulation of the in vitro scabs. In summary, the here established in vitro scab model can be used to analyze the first phases of wound healing where blood and skin cells interact, as it is viable and responsive for more than 2 weeks
Microcurrent Reverses Cigarette Smoke-Induced Angiogenesis Impairment in Human Keratinocytes In Vitro
Cigarette smoking (CS) leads to several adverse health effects, including diseases, disabilities, and even death. Post-operative and trauma patients who smoke have an increased risk for complications, such as delayed bone or wound healing. In clinical trials, microcurrent (MC) has been shown to be a safe, non-invasive, and effective way to accelerate wound healing. Our study aimed to investigate if MC with the strength of 100 μA may be beneficial in treating CS-related healing impairment, especially in regard to angiogenesis. In this study, we investigated the effect of human keratinocyte cells (HaCaT) on angiogenesis after 72 h of cigarette smoke extract (CSE) exposure in the presence or absence of 100 μA MC. Cell viability and proliferation were evaluated by resazurin conversion, Sulforhodamine B, and Calcein-AM/Hoechst 33342 staining; the pro-angiogenic potential of HaCaT cells was evaluated by tube formation assay and angiogenesis array assay; signaling pathway alterations were investigated using Western blot. Constant exposure for 72 h to a 100 μA MC enhanced the angiogenic ability of HaCaT cells, which was mediated through the PI3K-Akt signaling pathway. In conclusion, the current data indicate that 100 μA MC may support wound healing in smoking patients by enhancing angiogenesis
Impact of four protein additives in cryogels on osteogenic differentiation of adipose-derived mesenchymal stem cells
Human adipose-derived mesenchymal stem/stromal cells (Ad-MSCs) have great potential for bone tissue engineering. Cryogels, mimicking the three-dimensional structure of spongy bone, represent ideal carriers for these cells. We developed poly(2-hydroxyethyl methacrylate) cryogels, containing hydroxyapatite to mimic inorganic bone matrix. Cryogels were additionally supplemented with different types of proteins, namely collagen (Coll), platelet rich plasma (PRP), immune cells-conditioned medium (CM), and RGD peptides (RGD). The different protein components did not affect scaffolds’ porosity or water-uptake capacity, but altered pore size and stiffness. Stiffness was highest in scaffolds with PRP (82.3 kPa), followed by Coll (55.3 kPa), CM (45.6 kPa), and RGD (32.8 kPa). Scaffolds with PRP, CM, and Coll had the largest pore diameters (~60 µm). Ad MSCs were osteogenically differentiated on these scafffolds for 14 days. Cell attachment and survival rates were comparable for all four scaffolds. Runx2 and osteocalcin levels only increased in Ad-MSCs on Coll, PRP and CM cryogels. Osterix levels increased slightly in Ad-MSCs differentiated on Coll and PRP cryogels. With differentiation alkaline phosphatase activity decreased under all four conditions. In summary, besides Coll cryogel our PRP cryogel constitutes as an especially suitable carrier for bone tissue engineering. This is of special interest, as this scaffold can be generated with patients’ PRP
Maqui Berry and Ginseng Extracts Reduce Cigarette Smoke-Induced Cell Injury in a 3D Bone Co-Culture Model
3D Environment Is Required In Vitro to Demonstrate Altered Bone Metabolism Characteristic for Type 2 Diabetics
A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings