Abstract
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) bone-forming cells. This allows for relatively controlled in vivo experimental bone formation. A wide variety of ectopic locations have been used for experimentation, including subcutaneous, intramuscular, and kidney capsule transplantation. The method, benefits and detractions of each method are summarized in the following review. Briefly, subcutaneous implantation is the simplest method. However, the most pertinent concern is the relative paucity of bone formation in comparison to other models. Intramuscular implantation is also widely used and relatively simple, however intramuscular implants are exposed to skeletal muscle satellite progenitor cells. Thus, distinguishing host from donor osteogenesis becomes challenging without cell-tracking studies. The kidney capsule (perirenal or renal capsule) method is less widely used and more technically challenging. It allows for supraphysiologic blood and nutrient resource, promoting robust bone growth. In summary, ectopic bone models are extremely useful in the evaluation of bone-forming stem cells, new osteoinductive biomaterials, and growth factors; an appropriate choice of model, however, will greatly increase experimental success.
What Is Ectopic Bone Formation?
E
Ectopic Versus Orthotopic Bone Formation
The distinction between ectopic and orthotopic bone formation is an important one. Orthotopic bone formation is derived from the greek word orthos meaning “straight, right” and refers to studies in which bone is formed in its correct anatomical location. Such studies can either be nonsurgical (eg, the injection of materials into the long bone periosteum) or surgical (eg, a calvarial defect in which material is grafted in the defect site). In these instances, the distinct biochemical and mechanical environment of an orthotopic bone model should not be overlooked. Bone injury has long been understood to elicit a cascade of signaling pathway activation, including fibroblast growth factor [17], transforming growth factor-beta (TGF-β) [18], Hedgehog [19], and Wingless Protein (Wnt) signaling [20] among others. This upregulation of pro-osteogenic signaling cascades has been shown to be critical for successful MSC-mediated osseous repair of bone injury [21]. The bone morphogenetic protein (BMP) signaling pathway, for example, is acutely upregulated in a mouse calvarial defect model. This upregulation enables adipose-derived stem cells to successfully ossify a critical-sized defect [21].
The mechanical forces exerted on a graft site should be considered as well. MSC transplantation into a long bone defect is subject to significant stress/strain forces with weight-bearing and locomotion [22]. In contrast, ectopic models are largely void of mechanical force (perhaps slight compression of the implant depending on its size can be observed, especially in the kidney capsule model). Biomechanical forces are well-studied, involving a cascade of signaling events leading to bone formation, or “mechanotransduction” [22 –25]. It is important to realize that ectopic bone models allow for near-complete removal of this potential extraneous experimental variable. Thus, ectopic bone formation models reduce the number of variables involved in bone formation, eliminating (or reducing) the effects of bone stimulating cytokines, bone forming cells, endogenous stem cells, and potentially bone-stimulating mechanotransduction. Each of the commonly used models discussed below has significant advantages and drawbacks.
Models of Ectopic Bone Formation
Subcutaneous implantation
Subcutaneous implantation is the most simplistic of all experimental models of ectopic bone formation. Surgically, it is the easiest of all models and a novice can perform this procedure with success after learning basic suturing technique. Nearly any mammalian animal model can be chosen, ranging from mouse and rat to rabbit, dog, and pig among numerous others (see Table 1 for a review). Rodent models are preferable and most widely used due to their low cost, lax skin (which accommodates large-volume implants), and availability of immunodeficient rodents for xenograft-based experiments. Generally, incisions should be made on the dorsum of the rodent so as to prevent the animals from removing their own sutures. Alternatively, intradermal stitches can be placed which avoids the possible need to dress the wound. Classically, bone marrow derived mesenchymal stem cells (BMSCs) are the most commonly studied cell type (Table 1). The availability of immunodeficient rats and mice makes possible and practical the transplantation of human-derived cells for increased clinical relevance. Cells may be transplanted immediately after derivation, after culture expansion, or after predifferentiation [26]. In such cases, predifferentiation may ensure adequate in vivo bone formation. However, culture time and conditions may change the overall composition of a cell population [27,28]. Further, predifferentiation is less ideal for clinical translation as the barrier for regulatory approval is higher if cells are taken ex vivo before implantation. Mesenchymal cells have been shown to have the ability to form bone when placed in an osteogenic environment; however, subcutaneous implantation often requires cytokine supplementation or molecular modifications [29,30]. Cell delivery methods are widely variable, from stiff poly lactic glycolic acid, to composite gels and other matrices. As an alternative to material scaffolds, culture-expanded cell sheets can be rolled and implanted without a carrier, showing new bone formation [31]. In addition, various growth factors and other stimuli have been added such as BMP2 and vascular endothelial growth factor to name a few (see again Table 1).
bMBCP, macroporous biphasic calcium phosphate; BMP2, bone morphogenetic protein 2; hPDLSC, human periodontal ligament stem cell; BMSC, bone marrow derived mesenchymal stem cell; PLGA, poly-lactic-co-glycolic acid; TCP, tricalcium phosphate; PTH, parathyroid hormone; PLLA, poly-
One of the more important considerations for subcutaneous bone models is a technical one. The physical identification of the implant can be challenging, especially as newly-formed bone can be similar in color to the surrounding dermal tissues. Moreover, the lax skin of rodents allows for potentially significant migration of an implant, a difficulty that can be compounded by small implant sizes. The authors suggest the use of colored scaffolds or the labeling of cells/implants with a dye before implantation to facilitate identification upon removal. This becomes increasingly important with longer-term studies with months separating implantation and harvest.
Another consideration for subcutaneous bone formation is the theoretical lack of naturally bone-forming stem cells within the intradermal environment. This is in direct contrast to intramuscular bone formation (see below) in which striated muscle satellite progenitor cells are readily able to form bone, given an appropriate osteogenic stimulus. This lack of endogenous bone-forming cells may be a benefit or a disadvantage depending on the experimental design. For example, it may be a benefit if an exogenous stem cell is implanted, ensuring that in theory the predominant, newly-formed bone is from exogenous origin. On the other hand, it may be a detraction if the study is designed to expressly test a biomaterial scaffold, in which case an endogenous bone-forming stromal cell may be needed to ensure adequate bone formation. A similar caveat should also be considered in subcutaneous models: skin injury has been shown to result in the honing of circulating progenitor cells to the defect site [32], and one cannot definitively exclude these progenitor cell types from participating in bone formation.
Finally, subcutaneous models may show inferior bone-forming capacity in comparison to other experimental models of ectopic bone formation. In general, relatively greater ectopic bone formation is observed within the intramuscular compartment compared with intradermal compartment [33]. For example, in one study performed on dogs and pigs, bone formation could be histologically observed after 45 days after intramuscular transplantation in contrast to 60 days after subcutaneous implantation [34]. However, exceptions to this general observation do exist, where, for example, a particular nano-crystalline hydroxyapatite scaffold produced significantly more subcutaneous rather than intramuscular bone in minipigs [35]. In general, the reduced ectopic bone formation in subcutaneous models may be due to reduced vascularization and blood flow—which is especially striking in comparison to the infrarenal capsule model as discussed below. Therefore, a lack of robust subcutaneous bone formation and the need for extended in vivo “incubation” times may limit the utility of subcutaneous models.
Muscle pouch implantation
Muscle Pouch (or intramuscular) implantation has a rich history in bone formation. In fact, BMPs were first studied for their ability to induce bone formation in a muscle pouch model [36]. Like subcutaneous ectopic bone formation, intramuscular bone formation may also be used in nearly any animal model (see Table 2). Although the mainstay remains rodent models, intramuscular implantation is readily translatable to larger animals (dog, pig, goat, and sheep). Intramuscular implantation has also been used in the human patient to successfully generate bone. For example, in 2004 Warnke et al. reported the intramuscular growth of a replacement human mandible using autologous bone marrow and BMP7 [37]. As another example, Heliotis et al. determined that even without added stem cells, BMP7 with hydroxyapatite can lead to replacement mandible ossification [38].
ASC, adipose-derived stem cell; CA, carbonated apatite; FGF2, fibroblast growth factor 2.
In small animal models, the hind limb is preferentially used. Generally, the authors suggest implantation in the thigh complex that affords space for a large-volume implant. In the thigh muscles of a mouse, for example, a maximum volume of up to ∼150 μL can be implanted. By contrast, the lower leg can be used for smaller volume implants. Benefits of using the lower leg are a readily palpable implant that can be monitored for growth or imaged by surface ultrasound if desired. Intramuscular implantation in a large animal is most often in the dorsal musculature, including paraspinal muscles or trapezius.
In recent years, BMP2 and other BMPs have been widely studied in intramuscular ectopic bone models [39]. BMPs are known to stimulate the osteogenic differentiation of native skeletal muscle satellite cells. This reinforces an important distinction between the intramuscular implantation model and other ectopic bone models: the presence of a native skeletal progenitor cells. If another osteoprogenitor cell source is experimentally implanted in a muscle pouch environment, it becomes critical to identify the cells on later histological analysis. This can be achieved by transfection with a reporter system, by gender mismatch of host and donor, or by xenografting and detection of species-specific antigens among other techniques. These techniques will allow for definitive identification of hosts from donor-derived bone. This extra step in analysis is vital for valid interpretation of intramuscular bone formation and could be a consideration to opt for another ectopic bone formation assay.
Another critical distinction to make for intramuscular implantation experiments is that of the pro-osteogenic cytokine elaboration from sites of muscle injury. BMP signaling has been shown to be naturally upregulated postmuscle injury via Smad activation [40,41]. Normally this is important for the regulation of proliferation and myogenic differentiation of skeletal muscle satellite cells and their descendants. However, this heightened BMP signaling postmuscle injury theoretically represents a potentially confounding factor in intramuscular ectopic bone models. Like BMP signaling, other signaling pathways known to be important in osteogenesis are also upregulated by muscular injury. These include TGF-β1 and insulin-like growth factor 1 to name a few [42,43]. In light of these findings, it is important to utilize blunt dissection rather than traumatic sheering of muscle fibers when creating the potential space for implant insertion. A relatively atraumatic muscle pocket creation will theoretically minimize this natural upregulation of BMP and other pro-osteogenic cytokines. On that note, similar cytokines are upregulated after cutaneous injury (most prominently TGF-β1), and may also play a confounding role in subcutaneous ectopic bone models.
Kidney capsule implantation
The kidney capsule model is a less frequently used method of ectopic bone formation, primarily owing to its relative technical difficulty in comparison to subcutaneous or intramuscular transplantation. Material is placed between the thin, fibrous capsule of the kidney and the underlying renal parenchyma. This material can be inserted either directly by injection with a small gauge needle, or surgically by creating a small incision in the capsule and gently inserting the material underneath the capsule. For the insertion method, a space should be created using blunt dissection (eg, using a melted Pasteur pipette). Unlike in primates, the kidney of rodents is an intraperitoneal organ and so the peritoneum must be incised before visualization of the kidney. The surgical insertion method is more technically challenging than the injection method, so prior practice is advisable. Most importantly, the capsule should remain intact to ensure that the implant stays in place and that proper vascularization will ensue. Material should not be injected into the parenchyma of the organ. Both the peritoneum and skin should be sutured after implantation.
A list of recent citations using the kidney capsule model for bone formation can be found in Table 3. The majority of studies utilize mice (either wild-type or immunocompromised), while a few use rats. BMSCs are the most commonly studied cell type, while cells derived from the tooth are often studied as well. A wide array of materials can be used for implantation including gels, bone matrices, and biodegradable sponges. A cell suspension or cell pellet can be injected as well, without any scaffold or carrier. The majority of implants are thereafter analyzed from 1 to 4 weeks postimplantation, but studies even up to 10 months in length have been described [44].
EDTA, ethylenediaminetetraacetic acid.
Significant features of the renal capsule model include: (1) increased blood flow to the implant, (2) theoretical lack of endogenous bone-forming stem cells, (3) size limitations of the implant, and (4) compressive force on the implant. Each will be considered in turn below. First, implants placed in the subrenal capsular assay are exposed to significant blood flow and likewise blood-borne nutrients. This has led in reports to supraphysiologic bone growth in comparison to native bone samples [45]. However, this can be considered a benefit rather than a detraction as engrafted cells are likely to survive and proliferate once in place. Moreover, the highly vascular environment may allow for the development of complex tissue types such as tooth-like structures in the field of dental research [46 –48] and calvarial suture-like structures [45]. Secondly, the subrenal capsule microenvironment is theoretically free of bone-forming endogenous stem cells. While trafficking of endogenous stem cells is theoretical, it is almost certain that the engrafted cells are responsible for any observed bone formation. Third, limitations on the size of implant can be of concern, as larger volumes can inadvertently tear the capsule. While mice are most often used, rats can be substituted for larger implants (Table 3). Fourth and finally, the capsular tissue overlying the engrafted cells may be fairly taut, especially if large-volume implants are used. This can relay a compressive force onto the implant itself, which may predispose to cartilage over bone formation depending on the cell type [49 –51].
Summary
Experimentally induced ectopic bone formation is a well-studied, well-described entity with subcutaneous, intramuscular, and kidney capsule transplantation being the most common models. Briefly, the most pertinent concern for subcutaneous implantation is the potential lack of robust bone growth, potentially attributable to poor blood flow. Intramuscular implantation exposes the implant to satellite progenitor cells, which theoretically makes distinguishing host from donor osteogenesis difficult. The kidney capsule method allows for supraphysiologic blood and nutrient resource, allowing for robust bone growth despite being a technically challenging assay. All 3 models are valid experimental entities; however, their distinct differences should be taken into account when either constructing or analyzing an experiment. Analyses of such models are crucial to understand the osteogenic differentiation of cells independent from an osseous environment. Thus, as skeletal tissue engineering progresses and the use of osteogenic progenitor cells becomes more commonplace, such models will hopefully allow for optimization of bone formation. Conversely, understanding the biology of ectopic bone formation might also allow for improved treatments of debilitations such as HO.
Footnotes
Acknowledgment
A.W.J. was supported by 5T32DE007296-14.
Author Disclosure Statement
No competing financial interests exist.
