Drug-induced Physeal Abnormalities in Preclinical Toxicity Studies

Abstract

Most toxic physeal changes are characterized microscopically by altered chondrocyte development, proliferation, or maturation in the growth plate and eventually result in disordered appositional bone growth. Many therapeutic drugs directly or indirectly target proteins involved in chondrocytic differentiation and maturation pathways, so toxic physeal injury has become increasingly common in preclinical toxicologic pathology. While physeal dysplasia has been associated with several different drug classes including bisphosphonates, vascular endothelial growth factor receptor inhibitors, fibroblast growth factor receptor inhibitors, transforming growth factor beta receptor inhibitors, and vascular targeting agents, physeal changes often share similar morphologic features including thickening and disorganization of the hypertrophic layer, increased numbers of hypertrophic chondrocytes, altered mineralization of endochondral ossification, and/or increased thickness of subphyseal bone. Knowledge of genetic and nutritional diseases affecting bone growth has been important in helping to determine which specific target drugs may be affecting that could result in toxic physeal lesions. A pathophysiologic mechanism for most physeal toxicants has been determined in detail using a variety of investigative techniques. However, due to the signaling cross talk and the tight regulation required for chondrocyte maturation in the physis, several growth factor pathways are likely to be affected simultaneously with pharmacologic disruption of physeal homeostasis and inhibition of one factor necessary for chondrocyte function often affects others.

The long bone in rodents and juvenile dogs, pigs, monkeys, and humans consists of two epiphyses, two physes (or growth plates), two metaphyses, and a diaphysis. The physis is responsible for longitudinal bone growth and is therefore critical for normal pediatric development. In nonrodents, the physis disappears at the time of skeletal maturity, while in rodents, the physes may be open for the life of the animal. The physis itself is composed of parallel columns of chondrocytes embedded in a thick extracellular matrix made up of proteoglycans. Its blood supply is derived from the epiphysis, to which it is intimately connected, as well as a secondary supply through the metaphysis and a third vascular connection through the medullary canal of the diaphysis. The physis is divided into four zones: the resting or reserve zone, the proliferative zone, the hypertrophic zone, and the zone of endochondral ossification. In the last few cell layers of the hypertrophic zone, there is a thin area of provisional calcification of the extracellular matrix which transitions into the zone of ossification (Eastwood and de Gheldere 2011). In humans, the physis has been further defined by the zone of Ranvier and the perichondral ring of LaCroix, which connect to the epiphysis and metaphysis. The zone of Ranvier is a wedge-shaped zone at the periphery of the resting zone, containing germinal cells which resupply it, while the perichondral ring of LaCroix is continuous with the periosteum and encircles the zone of Ranvier. These layers are ill-defined in rodents.

Pathologic lesions in the physis can occur from genetic or nutritional diseases, infectious diseases (especially bacterial infections), trauma, and from a variety of toxic causes. Rickets, due to failure or delay in mineralization of the physis of long bones from calcipenic or phosphopenic dietary causes, results in widening and deformation of the physis and has been recognized in human medicine for thousands of years (Pettifor 2005). Altered physeal morphology is common to several distinct multicentric inherited human diseases including chondrodysplastic dwarfism, Jansen’s metaphyseal chondrodysplasia, mucopolysaccharidosis, osteogenesis imperfecta, Ellis–van Creveld Syndrome, and Meckel Syndrome (Frazier 2008; Lemyre et al. 1999). In achondroplasia, the zones of proliferation and hypertrophy are narrowed and may be disorganized with premature deposition of bone resulting in physeal closure, while in Ellis–van Creveld syndrome the physis is expanded with disorganization of cartilage columns and altered subphyseal mineralization (Superti-Furga et al. 1996). Osteogenesis imperfecta is due to collagen (Col) type I deficiency and also results in disordered maturation of the physis with thickened, irregular physeal plates (Cole 1994). In addition to human genetic diseases, physeal lesions have been recognized among a variety of inherited disorders in dogs, cats, horses, pigs, and in over 150 transgenic knockout mouse strains (Frazier 2008). Thus, while these genetic diseases have distinct histologic and morphologic features, they each share similar target tissue types in the bone and growth plate and also share some overlapping mechanistic processes with each other as well as with toxic or nutritional physeal disorders.

As a group, most toxic physeal changes are characterized microscopically by altered chondrocyte development, proliferation, and/or maturation within the growth plate and most eventually result in disordered appositional bone growth macroscopically. Many therapeutic drugs directly or indirectly target growth factors and proteins involved in these chondrocytic differentiation and maturation pathways (Frazier et al. 2007; Hall, Westwood, and Wadsworth 2006). As a result, physeal abnormalities have been encountered in toxicologic studies in animals with increasing frequency. In fact, there are a number of marketed drugs which can induce physeal changes in rodents that are utilized in adult human medicine with little or no consequences to their intended targeted patient population, including bisphosphonates, sunitinib, pazopanib, and many others (Ryan et al. 1999; Patyna et al. 2008).

Examples of Drug-induced Physeal Injury and Their Morphology

While the on- and off-target molecular and cellular targets of pharmaceutical intervention in the physis vary, the actual histologic presentation of physeal injury has been remarkably similar among a variety of agents, with either thickening or thinning of the physis as a primary feature. Physeal dysplasia is a catch-all morphologic term which has reached broad acceptance for many of the drug-induced lesions in the physis, although the recent “International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice” publication for bone uses the rather nondescript diagnosis “increased thickness, physis” (or “decreased”) as preferred terminology in cases where there are no obvious signs of alteration in chondrocyte organization (Fossey et al. 2016). However, while dysplasia and/or changes in thickness are highlighted phenotypes, specific morphologic features include a whole variety of microscopic changes. It should also be noted that dysplasia, in the context of bone malformations, denotes “an abnormal organization of cells” but not necessarily altered cellular phenotype and should not be considered a preneoplastic condition (Schofield and Cotran 1999).

Some of the first reports of physeal abnormalities noted from pharmaceutical intervention were described in rodents given bisphosphonates such as zoledronic acid. In addition to physeal expansion, studies on the effects of bisphosphonates on the physis of rodents noted the persistence of calcified cartilaginous columns after treatment (Camacho et al. 2001; Larsson and Larsson 1978; Schenk et al. 1973). Disruption in growth plate organization and cell morphology was also noted in animals receiving multiple weekly doses of another bisphosphonate, alendronate (Evans et al. 2003). Chondrocyte cell stacks within the physis appeared to lose their orientation parallel to the vertical axis of the bone. In some studies with bisphosphonates, particularly in rabbits, the thickness of the physis was decreased, rather than increased. This was due to markedly irregular collagenous columns, and these alterations were accompanied by reduced size of chondrocytes of the hypertrophic zone and up to a 60% reduction in the number of proliferating chondrocytes within the proliferative zone (Smith et al. 2005). Many animal studies with bisphosphonates have shown reductions in overall bone length. This may be because bisphosphonates cause retention of longitudinal cartilaginous septa at the chondro-osseous junction and increased local trabecular mass resulting in early physeal closure in rodents (Smith et al. 2005).

As specific pharmaceutical agents were developed to affect particular signaling pathways, physeal lesions were noted in preclinical species as a pharmacologically based effect of treatment. For instance, physeal changes were noted in cynomolgus monkeys administered a monoclonal antibody against vascular endothelial growth factor (VEGF; Ryan et al. 1999). Twice weekly injection of the anti-VEGF antibody (later marketed as bevacizumab) resulted in bilaterally symmetrical physeal dysplasia characterized by a dose-related increase in hypertrophied chondrocytes, subchondral bony plate formation, and inhibition of vascular invasion of the physis (Ryan et al. 1999). The specific physeal pathologic changes that were elegantly described in detail in that original report included “markedly thickened growth plate cartilage; degeneration of cartilage matrix; disorganization of chondrocyte columns which formed irregular packets or clusters; decreased numbers of primary bony trabeculae with inappropriate retention of hypertrophied chondrocytes; absence of metaphyseal capillary invasion into the zone of hypertrophied chondrocytes; and formation of a transverse subchondral bony plate” (Ryan et al. 1999, 80). Subsequently, a similar lesion focused on the hypertrophic layer of the physis was noted in rodents and other species from a variety of other small molecule VEGF pathway inhibitors, suggesting the lesion was a class effect related to pharmacologic activity of the drugs on angiogenesis (Hall, Westwood, and Wadsworth 2006; Patyna et al. 2008; Wedge et al. 2000). Physeal dysplasia occurred in both rats and monkeys given the VEGF receptor (VEGFr) inhibitor sunitinib and was also characterized by an increased number of hypertrophic chondrocytes that resulted in expansion of the zone of hypertrophy, increased calcification, reduction in bony trabeculae in the metaphysis, and the presence of focal areas of cartilage in the periosteum of the femur and tibia (Patyna et al. 2008). In monkeys given sunitinib, an osseous plate developed just subjacent to the physis and was associated with rare necrotic chondrocytes in the hypertrophic zone, all similar to lesions originally described with bevacizumab. A common morphologic feature of drug-induced physeal dysplasias in rodents with the VEGF or VEGFr inhibitors involves the frequent thickening of the subphyseal bone trabecula and often visualized microscopically as subcortical increased bone formation. Increased subchondral bone ossification at the transverse bony plate may help stabilize the underdeveloped and disorganized physis and may help prevent physeal fractures. In rodents and nonhuman primates, fractures are very uncommonly noted with toxic physeal injury, but rabbits appear to be the exception. Weakening and fracturing of the femoral head physis with dysplasia of the distal femoral articular cartilage and displacement of the femoral head has been described in rabbits as sequelae to physeal dysplasia associated with off-target inhibition of VEGFr and fibroblast growth factor receptor (FGFr) by fostamatinib (Hall et al. 2016).

Similar changes in physeal dysplasia were described about 10 years ago in rodents given activin-like kinase 5 inhibitors (ALK5—one of the transforming growth factor beta [TGFβ] receptors; Frazier et al. 2007). Physeal alterations with ALK5 inhibition were noted in rats as early as 4 days after administration and consisted of increased chondroid matrix, increased numbers of chondrocytes in the proliferative zone, increased thickness of the hypertrophic layer which progressed to disorganization of columns, and increased subphyseal bone formation over time (Frazier et al. 2007). Alterations were noted in proteoglycan composition in the hypertrophic zone, and reduced gelatinase proteolytic activity was noted across multiple zones. While there were slight increases in proliferative indexes via Ki67 and Topoisomerase II staining, apoptosis was decreased in the physis as gauged by reduced TUNEL and caspase-3 stains. The lesions were also considered pharmacologically based.

In addition to TGFβ signaling inhibition, disruption of a host of other growth factors may adversely affect physeal morphology. Administration of an FGFr kinase inhibitor resulted in cartilage and physeal dysplasia in rats with thickening of the growth plate and disorganization of the chondrocytes (Brown et al. 2005). Additional and somewhat unique features of this toxicity were thickening of the zone of mineralization and generalized soft tissue mineralization. Similar changes were noted with genetic disruption of FGFr-3 (Deng et al. 1996), but paradoxically FGF-2 administration, rather than inhibition, produced similar physeal expansile effects (Nagai and Aoki 2002). Increased physis thickness with FGF-2 treatment was accompanied by disruption and separation of the chondro-osseous junction between disordered chondrocytic columns and trabeculae in the metaphysis (Nagai and Aoki 2002).

Inhibition of matrix metalloproteinases (MMPs) has also resulted in physeal dysplasia with phenotypes that mimic those already described (Fossey et al. 2016; Lee et al. 1999; Renkiewicz et al. 2003; Stickens et al. 2004). Mice deficient in MMP9 or MMP13 have profound defects in physeal cartilage characterized by thickening and increased numbers of hypertrophic chondrocytes, and these effects have been replicated with MMP pharmaceutical inhibition using marimastat and other MMP inhibitors in rodents (Renkiewicz et al. 2003; Vu et al. 1998; Wu et al. 2002). Treatment of mice with galectin-3, which is an extracellular matrix protein and an MMP9 substrate found in the physis, produces a similar lesion (Ortega et al. 2005). It should be noted that more recent and selective MMP inhibitors have not shown the same physeal lesions as the earlier drug candidates (Baragi et al. 2009). Targeted inhibition of other proteases that affect cartilaginous matrix, such as cathepsin inhibitors, has also occasionally resulted in physeal changes in rodents during chronic toxicity studies, characterized by thickening and altered composition of the physeal extracellular matrix (unpublished data).

Physeal dysplasia has been noted with inhibition of parathyroid hormone -related peptide (PTHrp), in a phenotype mixed with a variety of other bone-related changes (Amizuka et al. 1994; Karaplis et al. 1994; Pateder et al. 2000). Finally, physeal dysplasia has been associated with other agents such as pp60 src kinase, chemokine receptor 4 antagonists, semicarbazide hydrochloride, and vascular disrupting agents such as ZD6126 (Fossey et al. 2016; Hall, Westwood, and Wadsworth 2006). The vascular disrupting compounds perturb the tubulin cytoskeleton of endothelium and have been reported to induce focal thickening of the physis with subphyseal osteocyte necrosis (Davis et al. 2002; Hall, Westwood, and Wadsworth 2006). Semicarbazide hydrochloride is a contaminant that can result from decomposition of nitrofuran drugs. Rats given dietary semicarbazide developed disarrangement and thickening of the physeal cartilage in addition to deformation and fissures of articular cartilage (Takahashi et al. 2010, 2014). In contrast, pp60 src kinase is an angiogenic signaling protein involved in the VEGF2 pathway, with a report of effects on chondrocytes of the hypertrophic layer of the physis similar to VEGF inhibitors (Coe et al. 1992). However, the proliferative effects on the physis were noted with pp60 src agonism rather than inhibition. The pp60 src protein forms part of a multimeric molecule involving gelsolin and phosphatidylinositide 3-kinase (Chellaiah et al. 2001). Inhibitors of the protein do not cause changes in the physis. In fact, transgenic null mice for both pp60 src and gelsolin instead are associated with an osteopetrosis phenotype limited to the metaphysis, probably as a result of effects on osteoclasts (Marzia et al. 2000; Miyazaki et al. 2000).

Decreased physeal thickness (as noted with some bisphosphonates) has been less commonly encountered in toxicologic studies than thickened or dysplastic physes but can occur with drug treatment, particularly in rodents. Like physeal dysplasia, it is noted most commonly in the femur and tibia and may be related to chronic inanition or from drugs that persistently affect chondrocyte differentiation such as doxorubicin or FGF21 inhibitors (Fossey et al. 2016).

Pathophysiologic Mechanisms of Toxic Physeal Injury

There are a variety of factors that may affect bone formation through a variety of mechanisms, including parathyroid hormone (PTH), 1,25-dihydroxy D3 (vitamin D), growth hormone, and a number of cytokines and prostaglandins. For instance, PTH stimulates a profound increase in osteoblasts and osteoclasts, while vitamin D affects bone mobilization, glucocorticoids decrease bone formation, and both insulin and prostaglandin regulate cartilage and bone growth. Growth hormone can increase longitudinal growth of bones via insulin-like growth factor-1 (IGF-1) or somatomedin C upregulation, and both estrogen and androgens can affect skeletal growth and maturation, albeit in different directions. However, while this wide variety of agents can affect bone formation indirectly through various pathways, these factors may not target or directly affect the physis at all. Instead, a common feature of drugs that does alter physeal morphology is that they have particular and selective effects on the growth plate chondrocytes themselves or on chondroblasts, rather than on osteoblasts or osteoclasts. Changes in woven bone are a secondary and downstream effect. In fact, most agents inducing physeal dysplasia (with the notable exception of semicarbazide hydrochloride) have little or no significant effects on the hyaline cartilage lining joints or the structural cartilage in other locations.

Study of human genetic diseases has been helpful in better defining the factors involved in the pathogenesis of physeal injury. For instance, mutations in Col 1 synthesis genes on chromosomes 7q or 17q are responsible for osteogenesis imperfecta, mutation of the diastrophic dysplasia sulfate transporter gene on chromosome 5 causes chondrodysplastic syndromes, and missense mutations in FGFr3 on chromosome 4p are associated with the inherited but lethal physeal disorder thanatophoric dysplasia (Frazier 2008). Unfortunately, the causal mechanisms in these syndromes rarely have involved only a single responsible protein and have generally involved a cascade of dysregulated proteins originating from the original gene mutation. There is currently a long list of factors that may affect physeal morphology and function including endochondral growth factors such as VEGF, TGFβ/ALK receptors and proteins, FGFrs and proteins, connective tissue growth factor and IGF; other regulatory factors such as PTHrp, hedgehog (HH), Wnt, and calreticulin-1; Col and proteases such as Col1, MMP9, and MMP13; and miscellaneous proteins such as matrilins, specific integrins, growth hormone, and histone deacetylase (Frazier 2008).

In-depth descriptions of specific molecular mechanisms of physeal alterations noted with previously described pharmaceutical agents or the list of proteins involved in each pathway is well beyond the scope of this review. There are several excellent comprehensive reviews of the pathophysiologic mechanism behind physeal changes (Frazier et al. 2007; Hall, Westwood, and Wadsworth 2006; Hosseinzadeh and Milbrandt 2011), and the reader is encouraged to seek the listed references for individual agents for more specific information around mechanism of physeal dysplasia with pharmaceutical intervention. However, there are several commonalities that can be expressed that provide general pathophysiologic information. Some mechanisms are straightforward. For instance, PTHrp effects are related to disruption of the PTH axis, while bisphosphonates target chondrocytes and also affect calcium–phosphorus metabolism. More often, multiple pathways are affected simultaneously.

A large number of growth factors and specific proteins are essentially involved in the sequential development and ossification of chondrocytes at the physis. Drugs such as the ALK5 inhibitors that directly or indirectly target chondrocyte development or maturation are going to have increased potential for physeal alterations. Lack of stimulatory factors at the proper time and context can result in lack of proliferation, lack of hypertrophy, and/or disorganization of cartilage layers. Apoptosis, vascularization, and mineralization are all precisely temporally and spatially regulated, and any modification to the ordered process can result in the histologic features pathologists note as physeal dysplasia. Perturbation or disruption of chondrocyte exposure to these factors at any point in their development can thus lead to uncoordinated interactions with each other or to altered effects on the extracellular matrix. It is important to realize that perturbation of one factor can result in dysregulation of several other essential factors. For instance, inhibition of ALK5 signaling in the physis resulted in marked expression patterns in a whole host of tightly regulated genes governing chondrocyte function such as basic fibroblast growth factor, bone morphogenic protein 7, Indian HH, IGF-1, PTHrp, and even VEGF (Frazier et al. 2007). Such widespread dysregulation can impact several aspects of chondrocyte differentiation and maturation (Alvarez et al. 2001).

Another example of these growth factor interrelationships is related to the physeal effects of FGF inhibition. Vascularization is critically important to endochondral bone growth, and FGFr inhibition is antiangiogenic. Thus, part of the physeal effects of FGF inhibition may mimic those related to VEGFr inhibition. FGFs are also directly involved in chondrogenesis and chondrocyte maturation and may share some secondary effects with ALK5 on chondrocytic growth factors (Bonaventure et al. 1996; Liu et al. 2002). However, dysregulation of FGF will also alter MMP expression, as they share common signaling pathways. Degradation of cartilage extracellular matrix by MMPs is necessary for vascular invasion at the zone of ossification, and these processes are under tight control by growth factors and cytokines (Baron et al. 1994; Borden et al. 1996; Stickens et al. 2004). Therefore, drugs affecting FGF receptors probably share some similar mechanistic pathways of physeal deformation with MMP inhibitors in addition to sharing pathophysiologic effects with antiangiogenic factors like VEGF inhibitors or factors that inhibit chondrogenic growth and maturation like TGFβ/ALK5 inhibitors. With such interconnected pathways, it may therefore be very difficult to delineate which pathophysiologic mechanism is most critically affected by a given compound.

VEGF couples chondrogenesis with osteogenesis, and the mechanistic effects of inhibition of VEGFr on the physis are largely due to retention of hypertrophic chondrocytes at the chondro-osseous border, as a result of reduced apoptosis and delayed angiogenesis/vascular invasion from the metaphyseal blood supply (Gerber and Ferrara 2000; Hall et al. 2016; Patyna et al. 2008; Wedge et al. 2005). This is the unifying mechanism of physeal injury by VEGF inhibitors such as bevacizumab, sunitinib, sorafenib, and pazopanib. Physeal thickening of the hypertrophic layer is accompanied by reduced primary trabeculae and increased thickness of secondary trabeculae in the ossification zone (Gerber et al. 1999). Lack of ossification also occurs (and hypertrophy of the other cartilaginous zones may ensue) if angiogenic factors at the subchondral bone surface are interrupted and necessary interactions with the vasculature at the zone of ossification are inhibited.

Many drugs that affect the physis appear to alter the nature of the cartilaginous matrix itself, through a variety of means. For example, hyaluronan synthesis by physeal chondrocytes is regulated by a number of factors, including growth hormone, IGF-1, PTH, and TGFβ1 (Pavasant, Shizari, and Underhill 1996). Interaction or inhibition of any of these pathways may therefore alter matrix synthesis and composition and further add to the physeal dysplastic phenotype (Frazier et al. 2007).

Most of the changes resulting in physeal abnormalities from growth factor inhibition or physis antiangiogenesis are reversible with time (>4 weeks). While complete recovery is possible with discontinuation of treatment, premature closure of the physis is a common and lasting side effect of prolonged physeal injury in juvenile rodents and can lead to shortened limbs. There may be other degenerative effects of physeal damage. Limited diffusion of oxygen to hypertrophied cartilage, insufficient exposure to growth factors regulating chondrocyte activity or division, or even structural side effects from increased physeal thickness can all result in secondary degeneration of cartilage and bone. It has been demonstrated that younger rodents are more susceptible than older rodents to many physeal changes (Frazier et al. 2007; Smith et al. 2005). The age sensitivity of rats to these effects and their prevalence in studies are directly related to bone proliferative activity in rodents at the age they are used in toxicologic studies. Rat femurs will grow approximately 5 mm or more in the 4 weeks after attaining 6 weeks of age, and the physis itself will increase in width by as much as 300 µm per day (Hansson et al. 1972). Rodent bones continuously grow, and most physes are still open at 2 years of age in rats or mice, but the rate of bone growth slows dramatically after about 36 weeks of age.

When physeal lesions develop in preclinical toxicologic studies, inevitably questions arise from project teams concerning translatability of risk from rodents or monkeys to humans, and the perpetual growth of rodent limbs and open physes is an important factor. Minimal changes to the thickness of growth plates, especially with chronic duration of treatment, probably have limited negative ramifications for the individual animal and its overall health, so these slight microscopic lesions to physeal chondrocyte organization may be considered potentially nonadverse. In contrast, physeal alterations that result in pronounced disorganization/deformation of the physis and/or result in premature physeal closure should obviously be considered adverse changes due to their effect on appendicular limb growth. Lesions in between these spectra fall into a gray area that must be up to the pathologists’ interpretation. However, adversity in a preclinical study does not necessarily imply clinical relevance. Physeal dysplasia induced by drug therapy is not expected to be a clinical problem in adult humans where most physes are completely closed after 21 years of age. While adult humans are therefore considered refractory to drug-related physeal perturbation in preclinical species, the theoretical risk remains for pediatric patients (Smith et al. 2005; Hall et al. 2016). However, in practice rodent or even monkey, cases of physeal dysplasia have not always resulted in clinical translation. Many agents that have caused lesions in rodents have not had similar bone liability in human pediatric patients due to species differences in sensitivity to the pharmacology. This includes most of the VEGFr inhibitors, and many of these agents remain in use in clinical trials for pediatric cancer without significant numbers of serious adverse events related to bone or physeal abnormalities. As with any other toxicity, a variety of factors may be involved in species sensitivity and these may be addressed case by case.

Methods of Investigation of Mechanism of Toxic Physeal Injury

While establishing a mechanism for preclinical physeal dysplasia may be important in determining human risk assessment for a drug in development, it may be difficult and is not necessarily a prerequisite for compound progression, given the fact that adult humans lack open physes. The first and most important step should be to carefully characterize the lesion morphologically so as to understand which layer of the physis is primarily being affected or if the lesion may instead be related to the periosteum or metaphysis. Clues to pathogenesis can be found in determining whether the lesions share features with known physeal toxicants. For instance, increased numbers of hypertrophic chondrocytes and increased subphyseal bone formation could indicate similarities in mechanism with the VEGF inhibitors. Review of receptor binding data and/or pathway analysis could hint at growth factor interactions and importantly provide circumstantial evidence of a pharmacologically based mechanism. Knowledge of similar lesions from genetic knockout or knockdown mice related to the same target is also extremely helpful in looking for a pharmacologic basis of mechanism and for pinpointing which pathways are crucial in developing the lesion.

There are a variety of special stains and procedures that may also aid in the mechanistic assessment, including TUNEL or caspase-3 immunostains for apoptosis, labeling indices such as Ki67 or topoisomerase II stains, and other stains that can provide assessment of bone calcification or proteoglycan composition such as Von Kossa or Movat’s staining, respectively (Frazier et al. 2007). Zymography is especially helpful in determining effects on MMPs or other matrix proteases. Proteomic or genomic analysis of the physis may reveal large changes in protein expression, but these may not point to a specific mechanism or original target of the drug. Even when there is a confirmed hypothesis regarding mechanism, the interconnectedness of signaling pathways and the tight regulation required for chondrocyte maturation in the physis suggest that several pathways are likely to be affected simultaneously. It may therefore be difficult to prove lack of human risk and/or rodent species selectivity even when a presumed pathophysiologic mechanism is demonstrated. Reasonable caution must therefore always be taken into consideration when a drug with physeal liability is given to juveniles or pediatric patients and guidelines related to clinical margin and risk–benefit certainly apply.