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Ectopic bone formation is a unique biologic entity--distinct from other areas of skeletal biology. Animal research models of ectopic bone formation most often employ rodent models and have unique advantages over orthotopic (bone) environments, including a relative lack of bone cytokine stimulation and cell-to-cell interaction with endogenous (host)...
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... Absorbances were measured with a spectrophotometer at 610 nm, and the Ca 2+ concentration was calculated according to the manufacturer's protocol. An ectopic bone formation model was further established using a muscle pouch implantation method as reported before 41 . Briefly, a 2-mm length longitudinal incision was made along the bilateral hind limb after mice anesthetization using isoflurane gas, and a 5-mm in-depth pocket was created by separating muscle fibers within hamstrings. ...
... This could be coupled to systematic analyses of changes in the secreted factors and matrix composition of CTs over time to elucidate the lack of ECO resulting from the prolonged in vitro maturation. An ectopic mouse model was chosen in this study to assess the intrinsic osteogenicity of CTs, but it is important to address how such engineered grafts would perform in an orthotopic bone defect environment, with the involvement of more factors that might have a pivotal role in the remodelling process such as cytokines from the bone niche, bone-forming cells, endogenous progenitor cells, biomechanical forces and inflammatory cues [37]. It is known that inflammation influences both graft survival and the process of regeneration [38]. ...
Endochondral ossification (ECO), the major ossification process during embryogenesis and bone repair, involves the formation of a cartilaginous template remodelled into a functional bone organ. Adipose-derived stromal cells (ASC), non-skeletal multipotent progenitors from the stromal vascular fraction (SVF) of human adipose tissue, were shown to recapitulate ECO and generate bone organs in vivo when primed into a hypertrophic cartilage tissue (HCT) in vitro. However, the reproducibility of ECO was limited and the major triggers remain unknown. We studied the effect of the expansion of cells and maturation of HCT on the induction of the ECO process. SVF cells or expanded ASC were seeded onto collagen sponges, cultured in chondrogenic medium for 3–6 weeks and implanted ectopically in nude mice to evaluate their bone-forming capacities. SVF cells from all tested donors formed mature HCT in 3 weeks whereas ASC needed 4–5 weeks. A longer induction increased the degree of maturation of the HCT, with a gradually denser cartilaginous matrix and increased mineralization. This degree of maturation was highly predictive of their bone-forming capacity in vivo, with ECO achieved only for an intermediate maturation degree. In parallel, expanding ASC also resulted in an enrichment of the stromal fraction characterized by a rapid change of their proteomic profile from a quiescent to a proliferative state. Inducing quiescence rescued their chondrogenic potential. Our findings emphasize the role of monolayer expansion and chondrogenic maturation degree of ASC on ECO and provides a simple, yet reproducible and effective approach for bone formation to be tested in specific clinical models.
... The renal subcapsular space is considered a preferred site for xenotransplantation of pancreatic β-cells, stem cells, and cancer cells because it provides an easily accessible, nutrition-rich pocket that supports engraftment. In particular, renal subcapsular xenotransplantation is a suitable model for ectopic bone formation because it lacks bone cytokine stimulation and interaction with endogenous (host) bone-forming cells, allowing for more controlled osteogenesis [19]. ...
... Choice of the implantation model is extremely important. Currently, the most commonly used models are subcutaneous implantation, implantation into a muscle pocket (intramuscular implantation), and implantation into a renal capsule [14]. Intramuscular implantation model has several advantages over the other two models. ...
... In particular, it is almost as convenient in terms of surgical technique as subcutaneous implantation and much simpler than renal capsule implantation, while being fully adoptable to large laboratory animals and humans. Compared to the subcutaneous implantation, intramuscular implantation usually provides a more pronounced and earlier formation of bone tissue [15], which may be due to the better blood supply to muscle tissue, as well as presence of the satellite progenitor cells in the skeletal muscles capable of osteogenic differentiation under the action of appropriate inducers, such as bone morphogenetic proteins (BMPs) [14]. For example, in the study conducted on dogs and pigs, intramuscular implantation showed bone formation after 45 days, while bone formation through subcutaneous implantation took 60 days [16]. ...
... At the same time, in the case of introduction of a similar implant into the critical-sized cranial defects in mice, after 9 weeks from the beginning of the experiment and with half the amount of BMP-2, almost complete remodeling of DBM with formation of the newly formed bone tissue with osteocytes included in it was observed [23]. Different rates of remodeling may be due to various reasons, in particular, variations in the intensity and pathways of osteogenesis due to different origins of osteoprogenitor cells: in the case of orthotopic osteogenesis they are mesenchymal stromal stem cells from the bone marrow of the maternal bone and periosteum, while in the case of ectopic osteogenesis in the muscle pocket they are satellite cells of skeletal muscle progenitors [14]. It is of interest to investigate the fate of such implants at later terms. ...
High efficiency of hybrid implants based on calcium-magnesium silicate ceramic, diopside, as a carrier of recombinant BMP-2 and xenogenic demineralized bone matrix (DBM) as a scaffold for bone tissue regeneration was demonstrated previously using the model of critical size cranial defects in mice. In order to investigate the possibility of using these implants for growing autologous bone tissue using in vivo bioreactor principle in the patient’s own body, effectiveness of ectopic osteogenesis induced by them in intramuscular implantation in mice was studied. At the dose of 7 μg of BMP-2 per implant, dense agglomeration of cells, probably skeletal muscle satellite precursor cells, was observed one week after implantation with areas of intense chondrogenesis, initial stage of indirect osteogenesis, around the implants. After 12 weeks, a dense bone capsule of trabecular structure was formed covered with periosteum and mature bone marrow located in the spaces between the trabeculae. The capsule volume was about 8-10 times the volume of the original implant. There were practically no signs of inflammation and foreign body reaction. Microcomputed tomography data showed significant increase of the relative bone volume, number of trabeculae, and bone tissue density in the group of mice with BMP-2-containing implant in comparison with the group without BMP-2. Considering that DBM can be obtained in practically unlimited quantities with required size and shape, and that BMP-2 is obtained by synthesis in E. coli cells and is relatively inexpensive, further development of the in vivo bioreactor model based on the hybrid implants constructed from BMP-2, diopside, and xenogenic DBM seems promising.
... In situ studies are based on the three valid models of ectopic (heterotopic) assay, including subcutaneous, intramuscular or renal capsule implantation of scaffolds [70]. Design, geometry, and size of the pores combined with appropriate porosity of the scaffolds induce ectopic (outside the bone) osteoinduction of MSCs [71] to form the bone cells de novo. ...
... The orthotopic test examines bone regeneration in vivo at the correct anatomical sites [70]. In this case, in addition to osteoinduction, significant ingrowth of bone tissue into the pores of the scaffold Table 2 Some examples of spheroids to promote/generate a growth of mesenchymal cells. ...
... No ectopic bone was found in the vicinity of the implants, which is rare following subcutaneous implantation given the absence of mesenchymal stem cells in the intradermal space. [41] However, it is known that progenitor cells found in the blood are phenotypically similar to mesenchymal stem cells in bone marrow and can accu-mulate at the site of injury in order to regenerate damaged tissue. [42] The Runx2 mRNA expressed in this study (from soft tissues) would have mainly originated from circulating mesenchymal stem cells. ...
Abstract Median sternotomy provides access to the heart and surrounding valves for cardiothoracic surgery. However, complications arise such as micro‐motion and separation between the two sternal halves. To prevent these complications, the use of a bone adhesive has been proposed to augment the mechanical union of the two sternal halves when combined with wire cerclage. Our group has developed a bone adhesive for sternal fixation, utilizing a glass polyalkenoate cement (GPC) based on a zinc silicate ionomeric glass (mole fraction: SiO2:0.48, ZnO:0.36, CaO:0.12, SrO:0.04). Here we present the first soft‐tissue biological safety assessment of this novel Sr/Zn‐GPC, where we implanted the material subcutaneously in rats for 6 and 12 weeks. Polymethyl‐methacrylate (PMMA) bone cement was used as a baseline control with respect to inflammation and biocompatibility. Histological assessment revealed no adverse tissue reaction nor ectopic bone formation in response to the novel material. Additionally, relative expression of pro‐inflammatory and osteogenic genes (Col1a1, SOX9, Runx2, IL‐1, IL‐6, and TNF‐α) was assessed in the tissue surrounding implants and revealed no significant differences in expression between the Sr/Zn‐GPC and PMMA implants.
... Animal models of ectopic bone formation have clear advantages over orthotopic transplantation because of the environments lacking cytokine interference and interactions with endogenous cell types, e.g., bone-forming cells. Three ectopic locationssubcutaneous, intramuscular, and kidney capsule-have been mainly used for transplantation studies (Scott et al., 2012). Subcutaneous implantation is the simplest method, but pertinent concerns arise because of several caveats related to the paucity of bone formation (Yang et al., 1996). ...
Adult stem cells not only maintain tissue homeostasis but are also critical for tissue regeneration during injury. Skeletal stem cells are multipotent stem cells that can even generate bones and cartilage upon transplantation to an ectopic site. This tissue generation process requires essential stem cell characteristics including self-renewal, engraftment, proliferation, and differentiation in the microenvironment. Our research team has successfully characterized and isolated skeletal stem cells (SSCs) from the cranial suture called suture stem cells (SuSCs), which are responsible for craniofacial bone development, homeostasis, and injury-induced repair. To assess their stemness features, we have demonstrated the use of kidney capsule transplantation for an in vivo clonal expansion study. The results show bone formation at a single-cell level, thus permitting a faithful assessment of stem cell numbers at the ectopic site. The sensitivity in assessing stem cell presence permits using kidney capsule transplantation to determine stem cell frequency by limiting dilution assay. Here, we described detailed protocols for kidney capsule transplantation and limiting dilution assay. These methods are extremely valuable both for the evaluation of skeletogenic ability and the determination of stem cell frequency.
... Subcutaneous implantation is a common and practical strategy in bone tissue engineering with a unique biologic entity that distinct from other areas of skeletal biology [61,62]. This strategy often was applied rodent models and have unique advantages over orthotopic (bone) environments, including a relative lack of bone cytokine stimulation and cell-to-cell interaction with endogenous (host) bone-forming cells [61]. ...
... Subcutaneous implantation is a common and practical strategy in bone tissue engineering with a unique biologic entity that distinct from other areas of skeletal biology [61,62]. This strategy often was applied rodent models and have unique advantages over orthotopic (bone) environments, including a relative lack of bone cytokine stimulation and cell-to-cell interaction with endogenous (host) bone-forming cells [61]. This allows for relatively controlled in vivo experimental bone formation. ...
Bone morphogenetic protein (BMP-2) has been approved by the FDA to promote bone regeneration, but uncertain osteogenic effect and dose-dependent side effects may occur. Osteoimmunomodulation plays an important role in growth factor-based osteogenesis. Here, we explored how proinflammatory signals affect the dose-dependent osteogenic potential of BMP-2. We observed that the expression level of local IL-1β did not increase with the dose of BMP-2 in the mouse osteogenesis model. A low dose of BMP-2 could not promote new bone formation, but trigger the release of IL-1β from M1 macrophages. As the dose of BMP-2 increased, the IL-1β expression and M1 infiltration in local microenvironment were inhibited by IL-1Ra from MSCs under osteogenic differentiation induced by BMP-2, and new bone tissues formed, even excessively. Anti-inflammatory drugs (Dexamethasone, Dex) promoted osteogenesis via inhibiting M1 polarization and enhancing BMP-2-induced MSC osteo-differentiation. Thus, we suggest that the osteogenic effect of BMP-2 involves macrophage-MSC interaction that is dependent on BMP-2 dose and based on IL-1R1 ligands, including IL-1β and IL-1Ra. The dose of BMP-2 could be reduced by introducing immunoregulatory strategies.
... Some studies have analyzed the differentiation of MSCs using their transplantation into experimental animals intraperitoneally (Kramann et al., 2013) or intramuscularly (Qu et al., 2011;Tuček et al., 2017); however, these methods have disadvantages associated with the difficulty of tracking the fate of cells in the abdominal cavity and the possibility of a negative effect of muscle contractions on the graft, respectively (DeWard et al., 2014). In the case of intramuscular transplantation, it is also necessary to take into account the possibility of differentiation of the recipient's myosatellites in the osteogenic direction, which can distort the results of the experiment (Scott et al., 2012). A higher promise seems to be shown by experimental models of transplantation of MSCs into the subcutaneous connective tissue and under the kidney capsule, as well as the use of a closed system that involves the placement of transplanted cells in a diffusion chamber. ...
... However, due to the small number of blood vessels in the subcutaneous connective tissue and, as a result, insufficient blood supply to the graft, its cells may not fully show their ability to grow and differentiate. In particular, it is known that bone tissue is formed after implantation of osteoinductive materials under the skin in a smaller amount and later than in other experimental models of ectopic osteogenesis (Scott et al., 2012). ...
... The method of MSC transplantation under the kidney capsule is technically more complicated than subcutaneous transplantation, but, unlike the latter, it provides much better conditions for engraftment of donor cells due to abundant blood supply. An additional advantage is the ease of finding and identifying the graft, while it can be macroscopically difficult to distinguish from surrounding tissues under the skin and can migrate a considerable distance from the surgical site (Scott et al., 2012). As a rule, the recipient in this experimental model is a mouse, since the kidney capsule is quite strong and easily separated from the parenchyma in this species of animals. ...
The impact of endocrine-disrupting chemicals on the development and involution of the immune system is a possible reason for the increased incidence of disorders associated with inappropriate immune function. The thymus is a lymphoid and also an endocrine organ, and, accordingly, its development and functioning may be impaired by endocrine disruptors. The aim was to evaluate age-related thymus involution in mature rats exposed to the endocrine disruptor DDT during prenatal and postnatal ontogeny. Methodology included in vivo experiment on male Wistar rats exposed to low doses of DDT during prenatal and postnatal development and morphological as-sessment of thymic involution, including the immunohistochemical detection of proliferating thymocytes. The study was carried out at the early stage of involution. Results: DDT-exposed rats exhibited a normal anatomy, and the relative weight of the thymus was within the control ranges. Histological and immunohistochemical examinations revealed increased cellularity of the cortex and the medulla, higher content of lymphoblasts, and more intensive proliferation rate of thy-mocytes compared to the control. Evaluation of thymic epithelial cells revealed a higher rate of thymic corpuscles formation. Conclusion: The data obtained indicate that endocrine disrupter DDT disturbs postnatal development of the thymus. Low-dose exposure to DDT during ontogeny does not suppress growth rate but violates the developmental program of the thymus by slowing down the onset of age-related involution and maintaining high cell proliferation rate. It may re-sult in excessive formation of thymus-dependent areas in peripheral lymphoid organs and altered immune response.
... Ectopic bone formation refers to the ossification of tissues outside their usual origins (Scott et al., 2012). Ectopic bone formation has unique advantages over orthotopic environments, including a relative lack of bone cytokine stimulation and cell-to-cell interaction with host boneforming cells (Scott et al., 2012). ...
... Ectopic bone formation refers to the ossification of tissues outside their usual origins (Scott et al., 2012). Ectopic bone formation has unique advantages over orthotopic environments, including a relative lack of bone cytokine stimulation and cell-to-cell interaction with host boneforming cells (Scott et al., 2012). Subcutaneous implantation is the most commonly used ectopic bone formation method in bone tissue engineering (Scott et al., 2012). ...
... Ectopic bone formation has unique advantages over orthotopic environments, including a relative lack of bone cytokine stimulation and cell-to-cell interaction with host boneforming cells (Scott et al., 2012). Subcutaneous implantation is the most commonly used ectopic bone formation method in bone tissue engineering (Scott et al., 2012). Many types of scaffolds have been seeded with PDLSCs and transplanted subcutaneously, including polymers, calcium phosphate scaffolds and xenografts (He et al., 2011;Moshaverinia et al., 2013;Yu et al., 2014;Chen et al., 2016;E et al., 2016;Wang et al., 2016). ...
Objectives: Stem cell-based tissue engineering approaches are promising for bone repair and regeneration. Periodontal ligament stem cells (PDLSCs) are a promising cell source for tissue engineering, especially for maxillofacial bone and periodontal regeneration. Many studies have shown potent results via PDLSCs in bone regeneration. In this review, we describe recent cutting-edge researches on PDLSC-based bone regeneration and periodontal tissue regeneration.
Data and sources: An extensive search of the literature for papers related to PDLSCs-based bioactive constructs for bone tissue engineering was made on the databases of PubMed, Medline and Google Scholar. The papers were selected by three independent calibrated reviewers.
Results: Multiple types of materials and scaffolds have been combined with PDLSCs, involving xeno genic bone graft, calcium phosphate materials and polymers. These PDLSC-based constructs exhibit the potential for bone and periodontal tissue regeneration. In addition, various osteo inductive agents and strategies have been applied with PDLSCs, including drugs, biologics, gene therapy, physical stimulation, scaffold modification, cell sheets and co-culture.
Conclusoin: This review article demonstrates the great potential of PDLSCs-based bioactive constructs as a promising approach for bone and periodontal tissue regeneration.
