The proximal sesamoid bones (PSBs) are a pair of pyramidal-shaped bones on the palmar/plantar aspect of each metacarpo-/metatarso-phalangeal joint. Aside from their dorsal articular surfaces, the PSBs are embedded within extensive ligamentous attachments that collectively form the suspensory apparatus.1 The PSBs articulate with the palmar/plantar distal aspect of the third metacarpal (MC3)/metatarsal bones and serve as a fulcrum for the suspensory apparatus as well as a smooth gliding surface for the flexor tendons. Catastrophic fracture of the PSBs resulting in failure of the suspensory apparatus is the most common fatal musculoskeletal injury amongst Thoroughbred racehorses in the US and Hong Kong.2–6 Other pathological conditions of PSBs, including avulsion fractures, sesamoiditis, osseous cyst-like lesions, intersesamoidean ligament desmopathy, and suspensory ligament desmitis, remain common performance-limiting problems in many equine breeds and disciplines.7–9
Despite the clinical significance of PSB and suspensory ligament pathology in the horse, the development and maturation of the PSBs and associated soft tissue structures are still poorly understood. Sesamoid bones are considered a unique class of bones with limited formal investigation of their temporal development in or ex utero. A study investigating the appearance of ossification centers in the equine fetal appendicular skeleton reported radiographically detectable ossification centers of PSBs for both fore- and hindlimbs at 320 days of gestation.10 Sesamoid bones have a later ossification timeline as compared to primary ossification centers (POCs) and have therefore been historically categorized as secondary ossification centers (SOCs). However, unlike epiphyseal or apophyseal SOCs, which are attached to a parent bone,10–12 PSBs are separate entities.
Defining features of sesamoid bones include their location embedded within a tendon or ligament and their lack of a periosteum. Although sesamoid bones were previously thought to develop through mineralization of a tendon or a ligament in areas of mechanical stress, more recent studies13 suggest that sesamoid bones originate from a distinct pool of chondroprogenitor cells that detach from the cartilage template of a parent long bone and form a separate ossification center. Thus, although sesamoid bones are separate entities from their parent bones, the notion that they originate from a parent bone supports their designation as SOCs. However, their unique relationship to the tendons or ligaments in which they are embedded distinguish them from other SOCs. Characterization of the unique features of endochondral ossification in equine PSBs has not been described. Additionally, histologic studies that describe the development of the entheses enveloping sesamoid bones have not yet been performed, and it is uncertain whether enthesis tissue promotes or contributes to mineralization of these bones.
Herein, the development of equine PSBs is investigated from gestation to the postgestational period using radiography, microcomputed (micro)-CT, and histology. Our objective was to characterize the early development of PSBs, including their growth, ossification, and maturation. We hypothesized that the process of PSB ossification is similar to endochondral ossification of SOCs, including the presence of cartilage canals that arise within the cartilage template with the creation of a spherical growth plate. We also hypothesized that following ossification of the growth cartilage, maturation of articular cartilage and entheses, as well as modeling of their associated bone interfaces, would extend through postgestational stages of juvenile horses.
Methods
Specimen collection
Forelimb PSB specimens were obtained postmortem from horses that died or were euthanized at the Cornell University Equine Hospital for various reasons unrelated to this study (Supplementary Table S1).
Radiography
Preliminary assessment of PSB mineralization to determine whether the sample would undergo micro-CT analysis was performed by radiography (Faxitron X-Ray System [model 43855A]; Hewlett-Packard and Blue Basic Autorad film, 8 X 10"; GeneMate) using a 37-V exposure for 1 minute.
Microcomputed CT image acquisition and morphometric analysis
Images of the PSBs were obtained using high-resolution micro-CT Skyscan (model 1276; Bruker for IDs 5, 6, 11, and 12 and GE CT-120 for IDs 7 through 10) with an isotropic voxel of 50 μm (720 projection, 20-ms exposure time, 100 kV, 50 mA). Local adaptive thresholding techniques were used to segment bone from soft tissue and marrow space. A local window approximately 4 times the width of a trabecula was created, and voxels were identified as bone if the intensity was greater than the mean minus the SD times a custom multiplier in the local window in MATLAB (version R2016b; MathWorks).
Quantitative analysis was performed to assess the size, shape, and internal structure of the PSBs using the micro-CT images. Bone volume fraction (bone volume per tissue volume [BV/TV]) was determined in 2 regions and reported as a percentage of the tissue volume in each region. The 2 regions were defined as peripheral (outer shell) and center (the porous structure) bone. The outer 5% of the bone was defined as the peripheral bone and the remaining inner 95% as center bone, which allowed for comparisons between the different sizes of bones through development. The shape of the PSB was assessed by measuring the maximum height, width, and depth. Internal structure was assessed by measuring trabecular thickness and degree of anisotropy using BoneJ (ImageJ).14,15 Midplanar binarized images in the sagittal, dorsal, and transverse planes for each bone were used for descriptive analysis.
Histologic sectioning and staining
A midsagittal section of each PSB was prepared for histologic analysis. For the specimens where the entire forelimb was collected (IDs 1 through 4), the mid-diaphyseal region of MC3 and proximal phalanx were transected (Isomet Low-Speed Saw with diamond wafering blade; Buehler) to create a limb segment containing the metacarpophalangeal joint. The isolated limb segments were fixed in 10% formalin for 20 hours, rinsed in deionized water (4 baths of 15 minutes each), and transferred to a decalcification solution (1:1 solution of 20% sodium citrate and 49% formic acid). When possible, the limb segments (IDs 2 through 4) were cut into a medial and lateral sagittal section to improve penetration of the decalcification solution into the tissue. For isolated PSB specimens (IDs 5 through 12), a 2- to 3-mm-thick central sagittal section was cut (Isomet Low-Speed Saw with diamond wafering blade; Buehler) and fixed in formalin, rinsed, and decalcified as described. The duration of decalcification was determined by radiographic assessment. Following decalcification, specimens were rinsed and stored in 50% ethanol until processed. A tissue processor (Duplex processor; Shandon Elliott) was used to dehydrate, clear, and infuse the specimens with paraffin wax. Each specimen was embedded in a block of paraffin wax and sliced into 6- to 8-µm-thick sections with a microtome (Cut 4060 E microtome; Olympus) and affixed to frosted microscope slides (25 X 75 mm) with slide adhesive. Slides were stained with H&E and Safranin O/Fast Green stains. The slides were digitally archived using an Aperio CS2 scanner at 40X magnification (Leica Biosystems).
Descriptive and quantitative histologic analysis
Digitally archived slides of the PSBs for each animal (IDs 1 through 12) were viewed using Aperio ImageScope software (version 12.4.3.5008; Leica BioSystems) to describe the ossification process of PSBs. Quantitative histologic analysis was performed in samples where a distinctly mineralized ossification center was apparent and consisted of the ratio of cartilage template to the size of ossification center and cartilage thickness measurements of the articular surface of the PSB. Surface areas of the cartilage template and ossification center were obtained on midsagittal sections by tracing the perimeters of the PSB cartilage template and the ossification center using BoneJ (ImageJ).14,15 Cartilage thickness on the articular surface of the PSB was measured as the perpendicular distance from the ossification front to the dorsal extent of cartilage and measured at the apical, middle, and basilar regions along the articular surface. Three measurements were made at each site, and the average of the 3 measurements was reported as the cartilage thickness for each region. The average of all 9 measurements was used to report an average cartilage thickness of the articular surface for each PSB. Note that, depending on the stage of maturation, the cartilage that was measured included the growth cartilage, and therefore the measurement is denoted as cartilage thickness on the articular surface of the PSB.
Results
A total of 24 PSB specimens were harvested from the forelimbs of 12 equids (IDs 1 through 12). Seven were fetuses with the following gestational ages: 105-day fetus (ID 1), 150-day fetus (ID 2), 180-day fetus (ID 3), 210-day fetus (ID 4), 240-day fetus (ID 5), 290-day fetus (ID 6), and 340-day fetus (ID 7). Five were foals/yearlings with the following postgestational ages: 2-day foal (ID 8), 45-day foal (ID 9), 120-day foal (ID 10), 240-day foal (ID 11), and 540-day yearling (ID 12). A general schematic of the developmental process is illustrated in Figure 1.16
Equine proximal sesamoid bone (PSB) development schematic. Equine PSBs are illustrated in a midsagittal plane. Not drawn to scale. A—Equine PSBs develop from a hyaline cartilage template (pink). There is an articular surface (orange) and the cartilage-ligament interface (light pink). B—Cartilage canals invade the cartilage template from the cartilage-ligament interface. Chondrocyte morphology along the cartilage canals is organized in growth plate zones: reserve (R), proliferative (P), and hypertrophic (H). C—Cartilage canals undergo a quiescent angiogenesis (empty ovals) followed by a reactive angiogenesis with osteoid deposition (filled ovals), resulting in the formation of ossific nuclei. D–E—Multiple ossific nuclei coalesce to form a single ossification center eccentrically located at the base of the cartilage template. The margin of the ossification front is irregular as cartilage canals are successively incorporated into the ossification center. F—A spherical growth plate describes the growth of the ossification center due to the centrifugal arrangement of growth plate zones. Cessation of the growth plate activity (1) is followed by maturation of the articular cartilage and entheses (2). G—Coinciding with maturation of the articular cartilage and entheses, the subchondral plate develops on the articular surface and compaction of the underlying bone occurs along the bone-ligament interface, resulting in a rim of compact bone surrounding the trabecular bone. H—This rim of compact bone thickens. On the flexor surface, a population of fibrochondroblasts appears and forms a cartilaginous template for the boney ridge on the flexor cortex. I—As the PSB reaches maturation, further thickening of the regions of compact bone are seen along the ossification fronts and extending into the bone. The PSB becomes more angular in shape as it forms boney ridges at sites of entheses maturation.
Citation: American Journal of Veterinary Research 85, 10; 10.2460/ajvr.24.04.0101
Radiography
Preliminary radiographic assessment was performed for specimens collected from IDs 1 through 4. With the exception of the middle phalanx of the 105-day fetus, all long bones (POCs) were visible as mineralized structures corresponding to POCs. Aside from the tubercle of the scapula visible in IDs 2 through 4, SOCs were not radiographically visible. Proximal sesamoid bones were not radiographically distinct in the fetal samples of IDs 1 through 4 (Supplementary Figure S1).
Microcomputed CT quantitative analysis
Microcomputed CT images were obtained for all PSBs that could be individually isolated (IDs 5 through 12). Images obtained for PSBs from the 240-day and 290-day fetuses had insufficient mineralization for segmentation and postprocessing analysis, leaving PSBs from a total of 6 animals (IDs 7 through 12) that underwent the final micro-CT analysis. Overall, all measured parameters were greater with increasing age, indicative of an active maturation process during the postgestational period (Figure 2). Proximal sesamoid bones size, represented by tissue volume, was greater with age (ie, larger bones in older horses) as were the width, height, and depth dimensional measurements. Peripheral bone volume fraction ranged from 60.3% to 95.4% and was greater than center bone volume fraction, ranging from 53.0% to 79.3% in all ages. Although the fractional increases were nonlinear in earlier stages of development, peripheral BV/TV was greater than center BV/TV in every PSB. Trabecular thickness measurements were similar in earlier stages, ranging from 183.9 ± 32.8 μm to 276.6 ± 101.3 μm in the PSBs from the 340-day fetus to the 120-day foal but were substantially greater in the 540-day yearling PSBs, with thicknesses as large as 577.9 ± 269 μm. The measured values for degree of anisotropy ranged from 0.2 to 0.4 (Supplementary Table S2).
Quantitative analyses of proximal sesamoid bone (PSB) morphologic parameters. A—Cartilage template and ossification center surface area measurements from midsagittal histologic sections revealed an increase in cartilage template dimensions, which preceded and paralleled development of the ossification center development. B—Trabecular thickness (Tb.Th) measurements quantified using microcomputed CT remained similar from immediately pregestation PSBs to approximately 120 days postgestation and then increased from 120 to 540 days postgestation.
Citation: American Journal of Veterinary Research 85, 10; 10.2460/ajvr.24.04.0101
Microcomputed CT descriptive analysis
Directional organization of PSB trabecular bone was not identified in the 340-day fetus or 2-day foal but became apparent as early as in the 45-day foal. Consistent with mechanical loading by the metacarpal/metatarsal condyles, articular-flexor trabecular alignment was increasingly detected in the sagittal and transverse planes from the 45-day foal to the 540-day yearling. In addition, trabecular alignment in an apical-basilar orientation became apparent along the flexor one-third of the PSBs in the sagittal plane beginning in the 120-day foal, with progressive trabecular thickening from 240 days to 540 days postgestation. This apical-basilar trabecular alignment along the loading axis of the suspensory apparatus was also detected most prominently abaxially in the dorsal plane images of the 240-day foal and became so dense along the abaxial surface of the PSBs at the site of the suspensory branch insertion in the 540-day yearling that the alignment became difficult to discern (Figure 3). The development of a compact mineralized rim along the periphery of the PSBs in all 3 planes was detected starting with the 45-day foal and became more evident with age. In the 340-day fetus and 2-day foal, the margins of the rim were irregular with small circular depressions. In PSBs ≥ 45 days postgestation, these depressions were absent, and the peripheral margins of the mineralization front was smooth. In the 540-day yearling, the rim was thicker, extending deeper into the bone in all 3 planes but especially along the abaxial and flexor surfaces. Subjectively, with increasing age, the peripheral contour of the PSBs became more angular, with the 540-day yearling PSBs demonstrating the curvature and shape reminiscent of PSBs in adult horses.
Microcomputed CT images of the midplanar slice in the dorsal, sagittal, and transverse planes were used to analyze the developing proximal sesamoid bone (PSB) ossification center. Scale bar = 1 cm. The peripheral margin of the ossification center is irregular in the 340-day fetus and 2-day foal (A, G, M, B, H, and N), characterized by sites of cartilage canal invaginations and mineralized spicules. In the 45-day foal, the margin is smooth with a compact rim of bone that surrounds the ossification center (C), and directional organization of the trabeculae becomes apparent, particularly along the articular margin (I and O). Regionalized trabecular alignment in multiple directions within a single plane is demonstrated in (J), where an apical-basilar orientation of the trabeculae becomes apparent along the flexor one-third of the PSBs, whereas an articular-flexor orientation of the trabeculae is apparent at the articular margin. With increasing age (D, J, and P vs E, K, and Q vs F, L, and R), both the trabeculae and the peripheral compact bone thicken. The peripheral contour of the PSBs become more angular, with the 540-day yearling demonstrating the curvature and shape reminiscent of PSBs in adult horses (F, L, and R).
Citation: American Journal of Veterinary Research 85, 10; 10.2460/ajvr.24.04.0101
Histology
Equine PSBs develop from a hyaline cartilage template
Morphologically, PSB mineralization was initiated and progressed through the process of endochondral ossification (Figure 4). In the early-stage samples where PSBs were harvested in situ (IDs 1 through 4), the PSB cartilage template was visible as a distinct structure eccentrically embedded within the dorsal aspect of the primordial suspensory ligament, with the articular surface of the PSB forming the palmar aspect of the metacarpophalangeal joint cavity. The shape of the midsagittal section of the cartilage template in most samples was reniform with varying degrees of concavity on the articular surface, convexity on the flexor surface, and a tapered apex proximally. The cartilage template surface area was greater with increasing age, with a 10-fold difference between 210- and 240-day gestation (Figure 2). In the 105-day fetus, the PSB hyaline cartilage template comprised a well-demarcated homogenous cluster of chondroblasts embedded in a proteoglycan-rich extracellular matrix. The articular surface of this template directly abutted the cartilage template of the MC3 condyle without a clearly delineated joint space. In all subsequent specimens, the metacarpophalangeal joint cavity was present, distinctly separating the cartilaginous templates of the MC3 condyle and PSBs.
Equine proximal sesamoid bones (PSBs) develop by endochondral ossification characterized by cartilage canal formation and spherical growth plate activity in 105-day (A–C), 290-day (D and E), and 340-day (F–H) equine fetuses. All stains are Safranin O/Fast Green unless otherwise indicated. A—Metacarpophalangeal joint, including the cartilage templates of the third metacarpus (MC3), PSB, and proximal phalanx (P1). There is direct apposition with indistinct separation of the PSB and MC3 articular surfaces (arrowheads). The proximal, palmar, and distal surfaces of the PSB are embedded within the dense collagenous tissue of the suspensory apparatus (SA). Scale bar = 500 µm. B—The articular interface of the PSB and MC3 comprises a dense linear concentration of cells (arrowheads). H&E stain; scale bar = 100 µm. C—The interface of the PSB with the SA comprises a distinct band of incipient enthesis cells characterized by flattened morphology and collagenous extracellular matrix (arrowheads). H&E stain; scale bar = 100 µm. D—The PSB cartilage template has developed a flattened articular surface with cartilage canals extending hemicircumferentially from the enthesis interface into the template (frame). Scale bar = 3 mm. E—Higher magnification of framed region in (D) shows invagination of cartilage canals (cc) from the SA interface into the PSB cartilage template, morphologically organized into resting (r), proliferative (p), and hypertrophic (h) zones, compatible with endochondral ossification of a growth plate. Scale bar = 300 µm. F—The ossification center (OC) is eccentrically located at the base of the PSB, leaving a large apical region of unossified cartilage template (dashed frame) compared to the base and palmar regions (solid frame). Scale bar = 5 mm. G—The apical portion of the PSB cartilage template, corresponding to the dashed frame in (F), contains branches of cartilage canals (arrowheads) associated with zones of endochondral ossification, including r, p, h, and ossification (o). H&E stain; scale bar = 500 µm. H—Cartilage canals (arrowheads) extending from the SA persist in the basal and flexor portions of the template, corresponding to the solid frame in (F), through the different zones of endochondral ossification (ie, r, p, h, and o). Scale bar = 300 µm.
Citation: American Journal of Veterinary Research 85, 10; 10.2460/ajvr.24.04.0101
Cartilage canals invaginate from the tendon/ligament interface into the cartilage template
Interstitial growth of the cartilage template coincided with increasing numbers of cartilage canals identified within fetal samples from 150 days to 290 days in gestation (IDs 2 through 6). The distribution of the cartilage canals was preferentially along the flexor margin when compared to the articular margin and appeared to invaginate from the cartilage-ligament interface. In the 290-day fetus, mineralization of the cartilage canals was evident and characterized by the presence of hypertrophic chondrocytes with osteoid deposited along the canal margins.
Equine PSBs develop from a single, eccentrically located ossification center
Starting with the 340-day fetus and subsequent PSBs from postgestational horses (IDs 7 through 12), a single ossification center was identified within the cartilage template. The location of the ossification center within the cartilage anlagen was not central but eccentrically located toward the basilar and flexor margins, leaving a large cartilaginous apical region. Additional ossification centers were not identified within any of the specimens in this study.
Trabecular maturation and compaction occur in the postgestational period
In the 340-day fetus and 2-day foal, the ossification center was composed primarily of woven bone trabeculae with disorganized seams of osteocyte-rich osteoid surrounding spicules of mineralized growth cartilage. In the 45-day foal, the trabecular bone demonstrated progressive organization and compaction, including lamellar osteoid deposits, although inner cores of mineralized growth cartilage persisted. These mineralized cores of growth cartilage were minimally present in the 120-day foal and not identified in the 240-day or 540-day foal/yearling PSBs. Bone trabeculae were thicker in the 240-day foal, with progressive thickening of the subchondral and subligamentous osteonal plates in the 540-day yearling.
Activity of the spherical growth plate is not synchronous across ossification fronts
In the midsagittal plane, the apical, flexor, basilar, and articular ossification fronts were used to characterize the spherical growth plate activity. The zone of proliferation transitioned into the zone of hypertrophy via the formation of mineralized cartilage trabeculae that served as a template for ossification. The mineralized cartilage trabeculae were created from the mineralized cartilage matrix through chondroclastic resorption of interspersed chondrocyte arrays that were replaced by extensions of thin-walled vascular sinuses. The zone of hypertrophy transitioned into the zone of ossification via the deposition of woven eosinophilic osteoid seams by plump osteoblasts onto the lattice-like arrays of the mineralized cartilage template, thus forming primary trabeculae. Maturation progressed at each ossification front through the conversion of the distinct layer of mineralized growth cartilage through the zones of hypertrophy and ossification with progressive modeling and coalescence of deeper (ie, more mature) trabeculae into a compact plate of subchondral bone that subtended the articular cartilage along the dorsal aspect of the PSB and a hemicircumferential compact plate that subtended the ligamentous interface (ie, entheses).
Although with increasing age the ossification center encompassed a larger area of its corresponding cartilage template, incorporating cartilage canals into the ossification center, the activity across the ossification fronts was not identical, and growth in the size of the cartilage template and ossification center was not always synchronous.
Maturation of the articular cartilage occurs post cessation of growth plate activity
Maturation of the PSB articular cartilage occurred in the postgestational period between 240 days and 540 days postgestation (Figure 5). In the 340-day fetus, a tinctorial distinction seemingly separated incipient articular cartilage from the underlying growth cartilage. In the 240-day foal, the development of immature articular cartilage progressed through the expansion of the incipient articular cartilage layer and matrix with lacunar enlargement of isotropically distributed chondrocytes. A rudimentary tidemark was present in these PSBs, which corresponded to early, yet incomplete, closure of the growth cartilage that was incompletely capped by thin seams of osteoid. In the 540-day yearling, the tidemark was better defined and corresponded to complete capping of the growth cartilage by seams of lamellar and woven bone that converged to a compact subchondral bone plate. Maturation of the articular cartilage exhibiting distinct zones and columnar organization of chondrocytes was apparent in the 540-day yearling. The thickness of the articular cartilage across the sagittal plane varied depending on the location, with thinner articular cartilage at the midbody region compared to the apical and basilar regions (Supplementary Table S3).
Structural maturation of the osteochondral junction on the articular surface. A—Cartilage canals (dotted oval) are incorporated into the bone at the ossification front. The trabecular bone at the ossification front consists of immature mineralized spicules with remnants of mineralized cartilage matrix. A thin superficial layer of incipient articular cartilage cells (asterisk) is present. B–D—A mineralized cartilage layer (dotted line) becomes more evident at the ossification front, and the underlying trabecular bone begins to take on a more lamellar appearance. E—With cessation of the growth plate activity, a cortical subchondral plate forms, and the incipient articular cartilage layer has now expanded into a layer of scattered immature isotropic chondrocytes. A rudimentary tidemark (TM) is visible. F—Columnar organization of chondrocytes and a distinct TM characteristic of mature articular cartilage is seen. All stained with H&E; scale bars = 300 µm.
Citation: American Journal of Veterinary Research 85, 10; 10.2460/ajvr.24.04.0101
Maturation of the fibrocartilaginous entheses occurs post cessation of the growth plate activity
Fibrocartilaginous entheses were identified at the bone-ligament interface of the apical, flexor, and basilar ossification fronts of PSBs. Development of the entheses occurred in the postgestational period; however, the timeline of maturation was not synchronous across the 3 ossification fronts (Figure 6). In the 105-day fetus, a distinct rim of cells encasing the apical, flexor, and basal surfaces of the PSB growth cartilage was present. Isotropic fibrocartilaginous cells were present in the apical and basilar ossification fronts in the 2-day foal, and the 4 histologic zones of a fibrocartilaginous enthesis with a definitive tidemark was evident in the 240-day foal at the apical and basilar regions. Interestingly, on the flexor surface, fibrocartilaginous isotropic cells were not seen until the 240-day foal. In the 240-day and 540-day foal/yearling, a wide band of isotropic fibrocartilaginous cells were present, indicating an immature enthesis at these stages of PSB development.
Maturation across ossification fronts is not identical. The enthesis along the flexor surface is immature, characterized by the presence of proliferating fibrochondroblasts (asterisk) (A, D, and E). At the apical and basilar surfaces, the rudimentary tidemark (TM) distinguishes the calcified fibrocartilaginous and uncalcified fibrocartilaginous layers of mature fibrocartilaginous entheses (B, C, F, and G). H&E stain; scale bars = 300 mm (A) and 300 µm (B–G).
Citation: American Journal of Veterinary Research 85, 10; 10.2460/ajvr.24.04.0101
Discussion
This study describes the chronology and morphology of equine PSB development from early gestation to 18 months postgestation. Cartilage canal formation precedes formation of the ossification center at the base of the PSB hyaline cartilage template near the end of gestation. Subsequent eccentric radial growth plate activity leaves a large immature cartilaginous apical region in young foals. Our findings support the hypothesis that the processes of endochondral ossification in sesamoid bones and SOCs share similar features, including the formation of cartilage canals and radial progression of the mineralization front through a spherical-type growth plate. The maturation of these bones is primarily a postgestational process characterized by trabecular thickening and alignment, an increase in bone volume with regionalized peripheral compact bone formation, and concurrent maturation of the intimately associated surrounding tissues, including the articular cartilage and entheses. Interestingly, even at 540 days postgestation, the bone-ligament interface along the flexor cortex had not yet developed into the histological layers of a mature fibrocartilaginous enthesis.
Murine studies13,17 have demonstrated that sesamoid bones, including the patella and metapodophalangeal bones, mineralize through endochondral ossification of a distinct pool of chondroprogenitor cells that detach from the cartilage template of a parent bone. The spatial relationship of the PSB cartilage template to the cartilage template of the MC3 condyle in the 105-day fetus suggests a possible detachment model to explain PSB origin. In our equine samples, cartilage canals were first identified invading an uncalcified resting hyaline cartilage template in the 150-day fetus. From 150- to 290-day gestation, increasing numbers of cartilage canals populated the hyaline cartilage template, followed by chondrocyte hypertrophy and osteoid deposition.
The origin of cartilage canals in sesamoid bones is not well described. In epiphyses, cartilage canals originate as invaginations at the perichondrial surface, where mesenchymal cells and angiogenic tissues reside.18,19 It is speculated that the tendon or ligament that encases a sesamoid bone substitutes for the perichondrium and periosteum.19 In this study, we demonstrated that cartilage canals originate as invaginations from the cartilage-ligament interface, where a distinct rim of primordial enthesis cells reside. These findings suggest that the tendon or ligament, and more specifically the primordial enthesis tissue that exists at this interface, plays a substitutive perichondrial role by providing progenitor cells and angiogenic factors for cartilage canal invagination and endochondral ossification. Morphological development of these canals was similar with previously described stages of epiphyseal cartilage canal development.18,20,21 In epiphyses, the superficial cells of the incipient articular cartilage have been suggested to play a role in the spherical growth plate of epiphyseal articular cartilage.22,23 By demonstrating morphologic similarities between the osteochondral unit at the entheseal ossification fronts to that at the articular surface, we propose that the incipient enthesis may play a similar role as the incipient articular cartilage cells, providing a mechanism by which the ligament promotes sesamoid mineralization.
Our finding that PSB mineralization occurs toward the end of gestation (290-day to 340-day gestation) is consistent with prior radiographic studies.10,12 As with other SOCs, the initiation of sesamoid bone mineralization is a mechanically dependent process but differs from epiphyseal mineralization in that the osteogenic stimulus is thought to be more diffuse, resulting in multiple ossific nuclei that rapidly coalesce to form the ossification center.24 Failure of these ossific nuclei to coalesce may result in bipartition as has been reported in the apical region of foal PSBs.25,26 Discontinuous or ununited regions of mineralization are more likely to occur in the apical aspect of the PSB due to the eccentric position of the ossification center at the base of the cartilage template, leaving a proportionately large cartilaginous apical region that is presumably more susceptible to mechanical trauma and disruption of endochondral ossification. Our study is the first of its kind to provide reference on the size and location of the equine PSB ossification center in relation to its cartilage template. Establishing a reference for the PSB ossification center and cartilage template dimensions and localization of the ossification center within the cartilage template may have implications for understanding the pathologic processes of the PSB.27,28
Our study found that maturation of the PSB occurs predominantly in the postgestational period, which is a common feature of sesamoid bones. Maturation was characterized by internal and external osseous morphologic changes along with structural maturation of the articular cartilage and surrounding entheses. Alignment of trabecular bone along axes of mechanical loading was not obviously apparent until postgestation and became progressively more apparent after 45 days postgestation, consistent with load bearing from the condyle in the articular-flexor axis and the ligamentous structures in the apical-basilar axis. Although trabeculae tend to orient themselves in the principal direction of the applied load, sesamoid bone loading is complex as the bone is embedded within multiple entheses that generate variable forces in a single small bone, explaining why multiple alignments were present. This also explains why whole-bone measurements of anisotropy may be less useful for describing sesamoid bone maturation as compared to long bone maturation since trabecular orientation varies throughout the volume of bone, giving a false impression that the bone remains anisotropic or underdeveloped. Tissue volume, bone volume fraction, and trabecular thickness were all positively correlated with age. The regional differences between peripheral and central bone volume fractions are explained by the compaction of the trabeculae in the periphery, forming denser bone, with a cortical bone plate becoming visible by 45 days postgestation. Histologically, this coincided with the formation of the mineralized cartilage layer.
An interesting finding was that the maturation along the various ossification fronts of the PSB was not identical. We demonstrated the structural organization of the articular cartilage and fibrocartilaginous entheses at 240 days and 540 days postgestation. However, on the flexor surface, the enthesis was morphologically immature. Fibrocartilage is a transitional tissue that adapts to provide stress and strain reduction at the interface between 2 mechanically dissimilar tissues. The morphologic differences of the flexor enthesis compared to its apical and basilar counterparts suggest that regionalized differences in the mechanical environment play an intricate role in the developmental process.
The limitations of this study included a small sample size that limited developmental stages to 1 representative animal per stage. Some fetal samples were obtained from mares euthanized due to systemic illness, which could have affected fetal musculoskeletal development. Additionally, although decalcification is a well-accepted and, in most cases, necessary step to facilitate microtome sectioning, the authors acknowledge the that the decalcification process may alter histologic analyses of tissues that were mineralized prior to this processing method.
The descriptive detail and combined radiographic, micro-CT, and histological assessment provide valuable insight into the development and maturation of equine PSBs, including characterization of the developing articular cartilage and entheses. Our study determined that equine PSBs mineralize predominantly through an endochondral ossification process that radiates from a single ossification center, leaving a large immature cartilaginous apical region in young foals. Moreover, PSB ossification precedes maturation of the surrounding fibrocartilaginous entheses. Although further investigations of progressive PSB bone modeling and enthesis maturation are warranted, taken together, the regional variations in tissue development (ie, delayed mineralization at the apical region of the PSBs and delayed maturation of the flexor entheses) highlight developmental processes that may have clinical relevance in young foals and horses, including common pathologies, such as PSB avulsion fractures, suspensory ligament desmopathies, and other PSB-associated lesions.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors acknowledge Mary Lou Norman for proximal sesamoid bone sectioning and Ryan Peterson for scanning and imaging slides.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
Funding for this study was provided in part by the Harry M. Zweig Memorial Fund for Equine Research (HLR) and NIH S10OD025049 (Skyscan 1276 CT, Cornell Institute of Biotechnology Imaging Facility).
ORCID
H.L. Reesink https://orcid.org/0000-0001-8534-8839
J.B. Engiles https://orcid.org/0000-0001-9057-2093
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