Increased Levels of BAMBI Inhibit Canonical TGF-ß Signaling in Chronic Wound Tissues.

Chronic wounds affect more than 2% of the population worldwide, with a significant burden on affected individuals, healthcare systems, and societies. A key regulator of the entire wound healing cascade is transforming growth factor beta (TGF-ß), which regulates not only inflammation and extracellular matrix formation but also revascularization. This present work aimed at characterizing wound tissues obtained from acute and chronic wounds regarding angiogenesis, inflammation, as well as ECM formation and degradation, to identify common disturbances in the healing process. Serum and wound tissues from 38 patients (N = 20 acute and N = 18 chronic wounds) were analyzed. The patients' sera suggested a shift from VEGF/VEGFR to ANGPT/TIE2 signaling in the chronic wounds. However, this shift was not confirmed in the wound tissues. Instead, the chronic wound tissues showed increased levels of MMP9, a known activator of TGF-ß. However, regulation of TGF-ß target genes, such as CTGF, COL1A1, or IL-6, was absent in the chronic wounds. In wound tissues, all three TGF-ß isoforms were expressed with increased levels of TGF-ß1 and TGF-ß3 and a reporter assay confirmed that the expressed TGF-ß was activated. However, Western blots and immunostaining showed decreased canonical TGF-ß signaling in the respective chronic wound tissues, suggesting the presence of a TGF-ß inhibitor. As a potential regulatory mechanism, the TGF-ß proteome profiler array suggested elevated levels of the TGF-ß pseudo-receptor BAMBI. Also, tissue expression of BAMBI was significantly increased not only in chronic wounds (10.6-fold) but also in acute wounds that had become chronic (9.5-fold). In summary, our data indicate a possible regulatory role of BAMBI in the development of chronic wounds. The available few in vivo studies support our findings by postulating a therapeutic potential of BAMBI for controlling scar formation.

Herbal Extracts of Ginseng and Maqui Berry Show Only Minimal Effects on an In Vitro Model of Early Fracture Repair of Smokers.

Smoking is a major risk factor for delayed fracture healing, affecting several aspects of early fracture repair, including inflammation, osteogenesis, and angiogenesis. Panax ginseng (GE) and maqui berry extract (MBE) were shown in our previous studies to reduce smoke-induced cellular damage in late bone-healing in vitro models. We aimed here to analyze their effects on the early fracture repair of smokers in a 3D co-culture model of fracture hematomas and endothelial cells. Both extracts did not alter the cellular viability at concentrations of up to 100 µg/mL. In early fracture repair in vitro, they were unable to reduce smoking-induced inflammation and induce osteo- or chondrogenicity. Regarding angiogenesis, smoking-induced stress in HUVECs could not be counteracted by both extracts. Furthermore, smoking-impaired tube formation was not restored by GE but was harmed by MBE. However, GE promoted angiogenesis initiation under smoking conditions via the Angpt/Tie2 axis. To summarize, cigarette smoking strikingly affected early fracture healing processes in vitro, but herbal extracts at the applied doses had only a limited effect. Since both extracts were shown before to be very effective in later stages of fracture healing, our data suggest that their early use immediately after fracture does not appear to negatively impact later beneficial effects.

Maqui Berry and Ginseng Extracts Reduce Cigarette Smoke-Induced Cell Injury in a 3D Bone Co-Culture Model.

Cigarette smoking-induced oxidative stress has harmful effects on bone metabolism. Maqui berry extract (MBE) and ginseng extract (GE) are two naturally occurring antioxidants that have been shown to reduce oxidative stress. By using an osteoblast and osteoclast three-dimensional co-culture system, we investigated the effects of MBE and GE on bone cells exposed to cigarette smoke extract (CSE). The cell viability and function of the co-culture system were measured on day 14. Markers of bone cell differentiation and oxidative stress were evaluated at gene and protein levels on day 7. The results showed that exposure to CSE induced osteoporotic-like alterations in the co-culture system, while 1.5 µg/mL MBE and 50 µg/mL GE improved CSE-impaired osteoblast function and decreased CSE-induced osteoclast function. The molecular mechanism of MBE and GE in preventing CSE-induced bone cell damage is linked with the inhibition of the NF-κB signaling pathway and the activation of the Nrf2 signaling pathway. Therefore, MBE and GE can reduce CSE-induced detrimental effects on bone cells and, thus, prevent smoking-induced alterations in bone cell homeostasis. These two antioxidants are thus suitable supplements to support bone regeneration in smokers.

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.

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.

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-ß signaling is well known to regulate the early phases of wound healing. All three TGF-ß 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-ß signaling pathway were investigated. Exogenous application of TGF-ß1 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-ß 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.

Effects of immune cells on mesenchymal stem cells during fracture healing.

In vertebrates, bone is considered an osteoimmune system which encompasses functions of a locomotive organ, a mineral reservoir, a hormonal organ, a stem cell pool and a cradle for immune cells. This osteoimmune system is based on cooperatively acting bone and immune cells, cohabitating within the bone marrow. They are highly interdependent, a fact that is confounded by shared progenitors, mediators, and signaling pathways. Successful fracture healing requires the participation of all the precursors, immune and bone cells found in the osteoimmune system. Recent evidence demonstrated that changes of the immune cell composition and function may negatively influence bone healing. In this review, first the interplay between different immune cell types and osteoprogenitor cells will be elaborated more closely. The separate paragraphs focus on the specific cell types, starting with the cells of the innate immune response followed by cells of the adaptive immune response, and the complement system as mediator between them. Finally, a brief overview on the challenges of preclinical testing of immune-based therapeutic strategies to support fracture healing will be given.

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.

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.

Material-Dependent Formation and Degradation of Bone Matrix-Comparison of Two Cryogels.

Cryogels represent ideal carriers for bone tissue engineering. We recently described the osteogenic potential of cryogels with different protein additives, e.g., platelet-rich plasma (PRP). However, these scaffolds raised concerns as different toxic substances are required for their preparation. Therefore, we developed another gelatin (GEL)-based cryogel. This study aimed to compare the two scaffolds regarding their physical characteristics and their influence on osteogenic and osteoclastic cells. Compared to the PRP scaffolds, GEL scaffolds had both larger pores and thicker walls, resulting in a lower connective density. PRP scaffolds, with crystalized calcium phosphates on the surface, were significantly stiffer but less mineralized than GEL scaffolds with hydroxyapatite incorporated within the matrix. The GEL scaffolds favored adherence and proliferation of the osteogenic SCP-1 and SaOS-2 cells. Macrophage colony-stimulating factor (M-CSF) and osteoprotegerin (OPG) levels seemed to be induced by GEL scaffolds. Levels of other osteoblast and osteoclast markers were comparable between the two scaffolds. After 14 days, mineral content and stiffness of the cryogels were increased by SCP-1 and SaOS-2 cells, especially of PRP scaffolds. THP-1 cell-derived osteoclastic cells only reduced mineral content and stiffness of PRP cryogels. In summary, both scaffolds present powerful advantages; however, the possibility to altered mineral content and stiffness may be decisive when it comes to using PRP or GEL scaffolds for bone tissue engineering.

Cell-Type-Specific Quantification of a Scaffold-Based 3D Liver Co-Culture.

In order to increase the metabolic activity of human hepatocytes and liver cancer cell lines, many approaches have been reported in recent years. The metabolic activity could be increased mainly by cultivating the cells in 3D systems or co-cultures (with other cell lines). However, if the system becomes more complex, it gets more difficult to quantify the number of cells (e.g., on a 3D matrix). Until now, it has been impossible to quantify different cell types individually in 3D co-culture systems. Therefore, we developed a PCR-based method that allows the quantification of HepG2 cells and 3T3-J2 cells separately in a 3D scaffold culture. Moreover, our results show that this method allows better comparability between 2D and 3D cultures in comparison to the often-used approaches based on metabolic activity measurements, such as the conversion of resazurin.

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 scaffolds 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.