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.

Many cells in the human body strongly react on decreased oxygen concentrations, generally defined as hypoxia. Therefore, inducing hypoxia in vitro is essential for research. Classically, hypoxia is induced using a hypoxia chamber, but alternative methods exist that do not require special equipment. Here, we compared three different methods to induce hypoxia without a hypoxia chamber: the chemical stabilization of HIF-1α by CoCl₂, the decrease in pericellular oxygen concentrations by increased media height, and the consumption of oxygen by an enzymatic system. Hypoxia induction was further analyzed within three different cell culture systems: 2D (adherent) osteoprogenitor cells, monocytic (suspension) cells, and in a 3D in vitro fracture hematoma model. The different methods were analyzed within the scope of fracture healing regarding inflammation and differentiation. We could show that all three induction methods were feasible for hypoxia induction within adherent cells. Increased media heights did not stimulate a hypoxic response within suspension cells and in the 3D system. Chemical stabilization of HIF-1α showed limitations when looking at the expression of cytokines in osteoprogenitors and monocytes. Enzymatic reduction of oxygen proofed to be most effective within all three systems inducing inflammation and differentiation.

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.

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.

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.

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.