An Atypical Degenerative Osteoarthropathy in Hyp Mice is Characterized by a Loss in the Mineralized...

ORIGINAL RESEARCH

An Atypical Degenerative Osteoarthropathy in Hyp Miceis Characterized by a Loss in the Mineralized Zoneof Articular Cartilage

Guoying Liang • Joshua VanHouten •

Carolyn M. Macica

Received: 18 January 2011 / Accepted: 6 May 2011 / Published online: 4 June 2011

� Springer Science+Business Media, LLC 2011

Abstract Patients with X-linked hypophosphatemia

(XLH) develop enthesophytes and osteophytes secondary

to articular cartilage degeneration and together are the

primary cause of morbidity in adult patients so afflicted.

We have previously characterized the enthesopathy in Hyp

mice, a murine model of XLH. We now extend these

studies to the synovial joint in order to characterize

potential cellular changes in articular cartilage that may

predispose patients to the osteoarthropathy of XLH. We

report that, despite highly elevated levels of alkaline

phosphatase activity throughout articular cartilage, there is

a complete loss in the mineralized zone of articular carti-

lage as assessed by von Kossa staining of mineral and as

quantified by EPIC-microCT analysis and evidence of

vascular invasion. We also identify the downregulation of

extracellular matrix (ECM) factors identified as regulators

of terminally differentiated mineralizing articular chon-

drocytes. There is also a striking increase in the histo-

chemical staining of sulfated proteoglycans, a change that

may reflect the loss of a transitional tissue that reduces

mechanical stress at the interface between cartilage and

subchondral bone. The failure of mineralizing articular

chondrocytes to develop in the hypophosphatemic state

suggests that phosphate may be a key regulator of

chondrocyte mineralization. Accordingly, we find that the

appropriate zonal arrangement and phenotypic markers

of articular cartilage are significantly reestablished by

phosphate-replacement therapy. Given the turnover and

maintenance of articular cartilage ECM, the identification

of early and abnormal cellular changes unique to XLH

will undoubtedly aid in a more effective management of

this disease to minimize the onset of degenerative

osteoarthropathy.

Keywords X-linked hypophosphatemia � Articular

cartilage � Hypophosphatemia � Degenerative

osteoarthropathy � Mineralization

X-linked hypophosphatemia (XLH), an X-linked dominant

disorder, is the most common form of familial hypophos-

phatemic rickets, affecting an estimated 1 in 20,000 [1].

The hypophosphatemia of XLH occurs in response to

elevated levels of the phosphatonin, fibroblast growth

factor-23 (FGF23). FGF23 is inappropriately high in

patients with XLH and significantly elevated in Hyp mice

as a consequence of inactivating mutations of the PHEX

(phosphate-regulating endopeptidase homolog, X-linked)

gene product. FGF23 contributes to diminished bone

mineralization by increasing urinary phosphate excretion

and suppressing 1,25(OH)2D3 production, acting via the

renal FGFR1/klotho receptor [2–5].

Generalized and severe mineralizing enthesopathy and

osteoarthropathy characterized by osteophytes are hall-

marks of XLH [2, 6–12]. Indeed, both of these complica-

tions come to dominate the clinical picture of XLH,

accounting for a great deal of the disease’s morbidity in

adulthood [6, 12]. We have previously reported that the

paradoxical mineralizing enthesopathy of XLH involves

The authors have stated that they have no conflict of interest.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00223-011-9502-4) contains supplementarymaterial, which is available to authorized users.

G. Liang � J. VanHouten � C. M. Macica (&)

Section of Endocrinology and Metabolism,

Department of Internal Medicine, School of Medicine,

Yale University, New Haven, CT 06520, USA

e-mail: [email protected]

123

Calcif Tissue Int (2011) 89:151–162

DOI 10.1007/s00223-011-9502-4

http://dx.doi.org/10.1007/s00223-011-9502-4

multiple tendon/ligament insertion sites and occurs early in

adulthood [2]. Correcting the mineral ion product can

effectively treat the rickets and osteomalacia of XLH;

however, such therapy appears to have little effect on the

development of the enthesopathy and may or may not

influence the progression of osteoarthropathy and osteo-

phyte formation [2, 9, 13]. In addition, most clinical

intervention has focused on childhood management of the

disease, with very little emphasis on management of the

contributing factors of morbidity during adulthood [1].

Degenerative osteoarthropathy has been reported to be

prevalent in young patients with XLH and common to all

older patients, characterized by thinning of the articular

surface of ankle and knee joints and subchondral sclero-

sis, both characteristic of degenerative joint disease

[6, 12]. We have reported a 41% incidence of spinal

enthesophytes, and vertebral spondylosis, a degenerative

osteoarthritis of the spine, has been reported to affect

approximately 40% of XLH subjects studied [2, 6, 14].

There is an especially strong relationship between the

formation of osteophytes, lateral outgrowths of bone at

the margins of the synovial joint, and articular cartilage

degeneration. Osteophytes are thought to arise as an

adaptive response to injury to remodel the articular sur-

face [15, 16]. While the degenerative osteoarthropathy of

XLH may be a consequence of the abnormal mechani-

cal forces of rickets and osteomalacia, it is currently

unknown if the articular cartilage of patients with XLH is

inherently abnormal. With little understanding of the

factors that contribute to the osteoarthropathy of XLH,

there is currently no clinical strategy to intervene with the

premature and pervasive pathological skeletal changes

that ultimately dominate this disease.

To gain insight into understanding the cellular changes

that may occur in articular cartilage, we conducted exper-

iments in Hyp mice, a murine model that genocopies and

phenocopies human XLH and one that we have previously

used to explore and characterize the enthesopathy of XLH

[17–19]. Specifically, we performed equilibrium partition-

ing of an ionic contrast (EPIC)-microCT analysis to obtain

quantitative, high-resolution, three-dimensional images of

articular cartilage of 7-month-old control and Hyp mice to

determine if articular cartilage thinning is characteristic of

Hyp mice. We also examined articular cartilage at skeletal

maturity to determine if Hyp mice display intrinsic carti-

lage abnormalities that might predispose them to degen-

erative osteoarthropathy and compared these findings to

Hyp mice undergoing conventional treatment for XLH

during long bone growth. Our findings provide a rationale

for both early and long-term treatment in order to preserve

both the anatomical and biochemical characteristics that

define the unique articular cartilage architecture that has

evolved to accommodate mechanical loads.

Materials and Methods

Chemicals

All chemical reagents were obtained from Sigma-Aldrich

(St. Louis, MO) unless otherwise indicated. Osteopontin

antibody was obtained from American Research Products

(Belmont, MA; 18621), matrix metalloproteinase-13 anti-

body was from Thermo Scientific (Fremont, CA; MA1-

38191), and von Willebrand factor antibody was from

Dako (Carpinteria, CA; A0082).

Animals and Tissue Processing

Hyp mice of the C57BL/6 strain (and aged-matched litter-

mate C57BL/6 controls) were obtained in-house in the Yale

University School of Medicine Animal Care Facility using

animals obtained from Jackson Laboratories (Bar Harbor,

ME). All animals were maintained on normal rat chow and in

accordance with the NIH Guide for the Care and Use of

Laboratory Animals. Serum calcium and phosphate levels

were confirmed in control and Hyp mice and at death by

orbital bleed and analyzed at the Yale Mouse Metabolic

Phenotyping Center. Serum was collected in treated Hyp

mice before treatment and at death. Treated Hyp mice were

maintained on high-phosphate drinking water (1.93 g ele-

mental phosphate/L starting at weaning, ad libitum) and

injected subcutaneously with calcitriol (1,25[OH]2D3;

Cayman Chemical, Ann Arbor, MI) from weeks 3 to 12

(0.175 lg/kg daily) every other day. At death, the legs were

rapidly dissected and fixed in 4% buffered paraformaldehyde

for 1–2 h on ice for immunohistochemistry and alkaline

phosphatase activity or for 48 hours for safranin O staining.

Selected bones were decalcified in daily changes of 7%

EDTA/PBS solution at pH 7.1 for 14 days at 4�C and washed

with PBS/50 mM MgCl2 overnight. Tissues were paraffin-

embedded and sectioned to a thickness of 8 lm, with the

section thickness used as an indicator of relative tissue depth

for comparison between Hyp and control. For von Kossa

staining, the leg was fixed in ethanol, embedded in plastic,

and sectioned at 4 lm. Each condition was repeated in at least

10 age-matched mice and eight aged-matched mice for

phosphate/vitamin D studies. All analysis was conducted on

the tibiofemoral articulation at the most heavily loaded

region of the mouse knee (the anterior one-half of the medial

tibial plateau with the central contact region defined by the

anterior and medial meniscus).

Equilibrium Partitioning of an Ionic Contrast Agent

via EPIC-microCT of Articular Cartilage

EPIC-microCT was performed at the Yale Core Center for

Musculoskeletal Disorders (YCCMD) microCT facility using

152 G. Liang et al.: Atypical Degenerative Osteoarthropathy in Hyp Mice

123

a MicroCT 35 (Scanco Medical, Bruttisellen, Switzerland).

Analysis was performed on tibial articular cartilage of control,

Hyp, and a subset of Hyp-treated mice. Remaining treated

mice (n = 5) were used for safranin O staining, von Kossa

staining, alkaline phosphatase staining, and immunohisto-

chemistry. Tibias were labeled with 50% Hexabrix, a charged

X-ray-absorbing contrast agent for 30 min, and imaged in air

at 6 l isometric voxel size with the X-ray tube set at a peak

electric potential of 45 kVp. Regions of interest were drawn to

accurately segment the articular cartilage from bone, essen-

tially as in Xie et al. [20], around the outside of the unmin-

eralized and mineralized cartilage, and passed through the

underlying subchondral bone to exclude inner bone cavities

(see supplementary data). Within the region of interest, the

lower and upper thresholds were adjusted to allow segmen-

tation of the unmineralized and mineralized cartilage from

each other and from subchondral bone; a gaussian filter with a

sigma of 2 and support of 4 was applied in order to enhance

image structures at different threshold values by smoothing

out the difference between gray levels of neighboring voxels.

Using this smoothing algorithm, three separate peaks were

resolved to reflect unmineralized cartilage, mineralized car-

tilage, and subchondral bone. Each corresponding peak was

assigned a range of threshold values to segmented cartilage

and bone. On a 1/1,000 scale, unmineralized cartilage was

considered to lie in the range of thresholds values of 91 and

313 for 12-week-old mice and 91 and 455 for 7-month-old

mice, while mineralized cartilage was between 313 and 545

for 12-week-old mice and between 455 and 606 for 7-month-

old mice. The attenuation of subchondral bone was above the

thresholds of 545 and 606, respectively, thus allowing segre-

gation of the subchondral bone from the articular cartilage in

both 12-week- and 7-month-old mice. Three-dimensional

data also allowed a thickness map of cartilage tissue volume

and is presented as a pseudocolor image of the gray-scale

value (see Fig. 3).

Immunohistochemistry

For immunohistochemistry, deparaffinized tissue sections

were processed as previously described with immuno-

staining visualized using the ABC staining system (Vector

Labs, Burlingame, CA) followed by incubation with a

peroxidase substrate (diaminobenzidine) for 3–5 min [2].

Epitope retrieval of paraffin sections was performed using

Decal� retrieval solution (Biogenex, San Ramon, CA)

according to the manufacturer’s instruction.

Alkaline Phosphatase Activity and Analysis: von Kossa

and Safranin O Staining

Alkaline phosphatase activity was performed on deparaff-

inized sections as previously described [2]. Briefly,

alkaline phosphatase-stained cells of tibial articular carti-

lage were quantified by analysis of three identical rectan-

gular areas per section from three stained sections per

animal. Images of articular cartilage were converted to

gray scale in Adobe Photoshop (Adobe, Mountain View,

CA) and analyzed using ImageJ software (NIH, Bethesda,

MD), as previously described [21]. From histograms of

eight-bit gray-scale images, a luminance value of 40 was

defined as the upper threshold that defined positive staining

of cells based on the separation of peaks between stained

and unstained cells. The total sum of data points within this

range of the histogram was calculated, and data are

reported as the density of alkaline phosphatase-stained

articular chondrocytes and expressed as a percentage of

alkaline phosphatase-positive articular chondrocytes to

total articular chondrocytes.

Mineral staining was conducted by the YCCMD,

employing a von Kossa staining procedure with toluidine

blue counterstaining. Histochemical staining of sulfated

proteoglycans was assessed using safranin O/fast green

staining. For safranin O, deparaffinized sections were

stained with Weigert’s iron hematoxylin working solution

(1% hematoxylin/7.25% acidified ferric chloride; Electron

Microscopy Sciences, Hatfield, PA) followed by 0.001%

fast green solution (Acros Organics, Morris Plains, NJ),

rinsed with 1% acetic acid solution, and then stained

in 0.1% safranin O solution for 15 min (Polysciences,

Warrington, PA).

Statistical Analysis

Data are expressed as mean ± standard error. Statistical

significance (P \ 0.01) was determined using one-way

ANOVA (GraphPad� software; GraphPad, San Diego,

CA).

Results

Evidence of Degenerative Osteoarthropathy

in Hyp Mice

Degenerative osteoarthropathy is characterized by the

thinning of articular cartilage; we therefore examined the

articular cartilage of 7-month-old Hyp mice, the age at

which we found extensive evidence of enthesopathy in Hyp

mice [2]. Quantitative EPIC-microCT analysis was per-

formed to quantify and compare high-resolution three-

dimensional images of articular cartilage of control and

Hyp mice [20]. The lower and upper thresholds of Hexa-

brix-labeled articular cartilage of tibias were adjusted to

segment the unmineralized and mineralized cartilage from

each other and from subchondral bone. EPIC-microCT

G. Liang et al.: Atypical Degenerative Osteoarthropathy in Hyp Mice 153

123

analysis revealed a 50% decrease in the total articular

cartilage thickness compared to controls, as well as a

striking absence of mineralization of articular chondrocytes

in Hyp mice (Table 1). Total data are shown in Fig. 1.

Together, these data define the osteoarthropathy of Hyp

mice to be one that is characterized by a thinning of

articular cartilage as well as an apparent loss of mineral-

ized cartilage.

Elevated Chondrocyte Alkaline Phosphatase Activity

We next examined the articular cartilage of Hyp mice at

skeletal maturity (12 weeks) to identify cellular changes

that might ultimately predispose articular cartilage to

thinning. Degenerative osteoarthropathy, while character-

ized by degeneration of articular cartilage, may be

accompanied by reactivation and duplication of the tide-

mark, with the advancing zone of calcified cartilage

expressing typical markers of hypertrophic chondrocytes

including alkaline phosphatase [22–24]. To determine if

the articular cartilage of Hyp mice is accompanied by

advancement of the mineralized zone of articular cartilage,

we first examined tissue alkaline phosphatase activity. We

have found that chondrocyte alkaline phosphatase activity,

including articular cartilage and growth plate chondro-

cytes, is considerably higher in 12-week-old Hyp mice

(Fig. 2a, b vs. c, d). Notably, alkaline phosphatase activity

of Hyp mice encroaches upon the entire articular surface,

just short of the superficial zone, suggestive of inappro-

priate mineralization (Fig. 2c). The density of alkaline

phosphatase-stained cells of tibial articular cartilage,

expressed as a percentage of alkaline phosphatase-positive

articular chondrocytes to total articular chondrocytes, was

analyzed and found to be 35 ± 4% in control mice vs.

80 ± 6% in Hyp mice (n = 10), confirming a significant

induction of tissue alkaline phosphatase activity in Hyp

mice.

Defective Cartilage Mineralization

Consequently, we measured mineral deposition in articular

cartilage by von Kossa staining. Mineral staining of control

mice corresponded to the alkaline phosphatase-positive

zone of articular cartilage typically seen in the tibial pla-

teau (Fig. 2e). However, mineral deposition was clearly

absent in much of the articular cartilage of Hyp mice,

despite chondrocyte alkaline phosphatase activity (Fig. 2f).

To better characterize the disparity between alkaline

phosphatase activity and mineral deposition, EPIC-

microCT analysis was performed. The thickness of total

unmineralized articular cartilage in Hyp mice was found to

be twice that of control mice (Table 2). In addition, like

that of 7-month-old Hyp mice, EPIC-microCT revealed an

absence of mineralized cartilage in Hyp mice (Fig. 3a vs.

b), consistent with von Kossa staining shown in Fig. 1f and

as shown in the three-dimensional rendering of mineralized

and unmineralized cartilage layers of control and Hyp mice

(Fig. 3d vs. e).

Table 1 EPIC-microCT of articular cartilage (AC) in 7-month-old

mice

Mineralized AC Unmineralized AC Total AC n

Control 0.046 ± 0.004 0.050 ± 0.011 0.096 ± 0.015 4

Hyp 0* 0.051 ± 0.006 0.051 ± 0.006* 3

* P \ 0.01 control vs. Hyp

Fig. 1 Articular cartilage thickness of 7-month-old control vs. Hyp

mice, as measured by EPIC-microCT. Scatter graph comparing the

thickness of unmineralized and mineralized articular cartilage of

control (a) and Hyp (b) mice. c Scatter graph comparing the total

articular cartilage thickness of 7-month-old control vs. Hyp mice. See

also Table 1


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Defective Expression of Noncollagenous Matrix

Proteins

Several noncollagenous matrix proteins are involved in the

remodeling and mineralization of articular cartilage, the

expression of which coincides with the onset of chondro-

cyte mineralization [25–27]. Osteopontin (OPN), a min-

eral-binding secreted extracellular matrix glycoprotein, is a

marker of hypertrophic chondrocytes, while collagen

matrix degradation by matrix metalloproteinase-13

(MMP13) is required for the terminal differentiation of

hypertrophic chondrocytes [28–31]. To determine if pro-

teins specific to mineralized articular cartilage are dis-

rupted, immunoreactive OPN and MMP13 of 12-week Hyp

mice were compared to those of control mice. Low-power

images comparing a representative control vs. Hyp mouse

are shown in Fig. 4a, f. We report that OPN precisely

colocalizes with the alkaline phosphatase-positive miner-

alized zone of articular cartilage in control mice (Fig. 4b,

c). In contrast, OPN is significantly downregulated in

articular cartilage of Hyp mice and does not colocalize

with alkaline phosphatase-positive cells (Fig. 4g, h).

MMP13 immunoreactivity is likewise confined to the

mineralized zone of articular cartilage in control mice

(Fig. 4b, d). However, we find that MMP13 is notably

absent in cartilage of Hyp mice (Fig. 4g, i). Of significance

is that the expression of OPN and MMP13, which are also

secreted matrix proteins of osteoblasts, is unaffected in

osteoblasts of Hyp mice, despite an equivalent metabolic

backdrop (Fig. 4h, i) [27, 32]. These findings were highly

Fig. 2 Comparison of alkaline

phosphatase activity and von

Kossa staining of 12-week-old

control and Hyp mouse.

Alkaline phosphatase activity of

articular cartilage (a) and

growth plate chondrocytes

(b) of control mouse; alkaline

phosphatase activity of articular

cartilage (c) and growth plate

chondrocytes (d) of Hyp mouse.

The density of alkaline

phosphatase-stained cells of

tibial articular cartilage,

expressed as a percentage of

alkaline phosphatase-positive

articular chondrocytes to total

articular chondrocytes, was

35 ± 4% in control mouse vs.

80 ± 6% in Hyp mouse

(n = 10). e, f Representative

sections showing von Kossa

staining revealing a decrease in

mineral deposition in cartilages

of a Hyp mouse (f) relative to a

control mouse (e)


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reproducible and performed in 10 littermate control and

Hyp animals.

These data are further corroborated by histochemical

staining of sulfated proteoglycans by safranin O, which

revealed a characteristic pattern in control mice of staining

most heavily above the tidemark boundary between the

unmineralized and mineralized cartilage. In contrast, car-

tilage of Hyp mice was heavily stained by safranin O, with

staining diffusely distributed throughout the articular sur-

face, intercalating with the subchondral bone and with no

evident tidemark. Representative sections of safranin O

staining are shown in Fig. 4e, j (n = 10) and are consistent

with increased cartilage proteoglycan content. Taken

together, these data demonstrate that the significant loss of

the mineralized zone of articular cartilage is intrinsic to the

disease and typifies the murine model of XLH.

Evidence of Articular Cartilage Vascular Invasion

in Hyp Mice

With the loss of a mineralized zone of articular cartilage,

the cement line (the interface between calcified articular

Table 2 EPIC-microCT of articular cartilage (AC) in 12-week-old mice

Mineralized AC Unmineralized AC Total AC n Serum P (mg/dL) Serum Ca (mg/dL) n

Control 0.051 ± 0.004 0.083 ± 0.011 0.134 ± 0.015 6 9.0 ± 0.3 9.3 ± 0.5 20

Hyp 0* 0.158 ± 0.004* 0.158 ± 0.004 3 4.9 ± 0.2* 9.0 ± 0.1 20

Hyp txpre ND ND ND 3 4.8 ± 0.1* 9.3 ± 0.6 8

Hyp txpost 0.048 ± 0.008 0.109 ± 0.035 0.157 ± 0.027 3 7.8 ± 0.4 9.4 ± 0.7 8

ND not determined

* P \ 0.01 control vs. Hyp

Fig. 3 Thickness of

unmineralized and mineralized

articular cartilage in 12-week-

old control vs. Hyp mice as

measured by EPIC-microCT.

Scatter graph comparing the

articular cartilage thickness of

control (a) and Hyp (b) mice.

(c) Scatter graph showing the

articular cartilage thickness of a

Hyp mouse treated with oral

phosphate and calcitriol. (d–

f) Three-dimensional renderings

were generated using Scanco

microCT software of

mineralized (orange) and

unmineralized (transparent)cartilage layers in control (e),

untreated Hyp (f), and treated

Hyp (g) mice, revealing

recovery of a mineralized zone

of cartilage. See also Table 2


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cartilage and subchondral bone) is inherently absent. To

determine if the resulting compromise in the cement line

influences the avascularity of articular cartilage, we looked

for evidence of vascular invasion in 7-month-old Hyp mice

using an antibody directed against von Willebrand factor,

an endothelial cell glycoprotein. Indeed, compared to the

avascular articular cartilage of control mice, evidence of

vascular invasion was observed in representative sections

from Hyp mice (Fig. 5a vs. b, c).

Restoration of Zonal Arrangement of Articular

Cartilage by Phosphate

We next conducted experiments designed to mimic the

clinical management of the disease of combined phosphate

and 1,25(OH)2D3 treatment. To determine if normalization

of serum phosphate restores the normal architecture

of articular chondrocytes, Hyp mice were treated with

high-phosphate drinking water (1.93 g elemental phos-

phate/L starting at weaning, ad libitum) and calcitriol

(1,25[OH]2D3) from weeks 3 to 12 (0.175 lg/kg daily) to

facilitate intestinal reabsorption of phosphate and to min-

imize the risk of secondary hyperparathyroidism of sole

phosphate supplement [13, 33]. Treatment of Hyp mice

with phosphate and calcitriol resulted in normalization of

serum phosphate, with no evidence of hypercalcemia

(Table 2). In addition, compared to untreated Hyp mice,

Hyp mice showed significant improvement of the tibial

growth plate and of epiphyseal and metaphyseal hyperos-

teoidosis (Fig. 4k vs. f). In support of data showing an

improvement of articular cartilage architecture, we also

find that while alkaline phosphatase activity of articular

cartilage remains slightly elevated, there is a significant

normalization of expression and localization of OPN and

MMP13 immunoreactivity (Fig. 4l–n). In addition, the

intensity of proteoglycan staining by safranin O diminishes

to within the normal range typically seen in control mice

(Fig. 4o). This, along with restoration of the tidemark,

confirms the critical role of phosphate in the regulation of

mineralization [34]. Finally, the defects of articular carti-

lage including alkaline phosphatase, noncollagenous pro-

teins, and proteoglycan staining are also duplicated in

growth plate chondrocytes, as shown in images of the tibial

epiphysis (supplementary data).

EPIC-microCT was performed to quantify potential

changes in articular cartilage of Hyp mice treated with oral

phosphate in conjunction with calcitriol. We found that

treatment resulted in a significant recovery in the zone of

mineralized articular cartilage, similar to that of control

levels, shown in Table 2 and Fig. 3c. Recovery of miner-

alization is illustrated in Fig. 3f as a three-dimensional

rendering of mineralized and unmineralized cartilage lay-

ers of control and Hyp-treated mice. Nonetheless, the total

thickness of articular cartilage was not normalized, owing

to persistence in expanded unmineralized cartilage.

Discussion

XLH Gives Rise to an Atypical Form of Degenerative

Osteoarthropathy

Patients with XLH have radiological evidence of articular

cartilage degeneration and subchondral sclerosis, as well as

pervasive osteophyte formation [6, 12]. Using a murine

model of XLH, we demonstrate the development of the two

prototypical features of degenerative osteoarthropathy that

are precursors to osteophyte formation: thinning of artic-

ular cartilage and vascular invasion of a tissue that is

normally avascular by virtue of a rigid interfacial structure

[15, 35, 36]. However, unlike the cellular mechanisms

thought to underlie these features in prototypical osteoar-

thropathy, we present evidence that they arise by com-

pletely different mechanisms in Hyp mice. Degenerative

osteoarthropathy typically evolves with a disrupted balance

of matrix turnover, with breakdown exceeding synthesis.

This is evidenced by observations of proteolytic cleavage

of matrix molecules mediated by upregulation of MMP13

expression with degradative loss of proteoglycans from the

matrix in osteoarthritis [36–40]. Tearing, fibrillation, and

thinning of the unmineralized zone of cartilage occur and

may be accompanied by vascular invasion and duplication

of the tidemark [22–24].

In contrast, we describe a thinning of articular cartilage

and see evidence of vascular invasion that is unlike the

prototypical form in that it instead involves a complete loss

of the mineralized zone of articular cartilage and is not

associated with upregulation of MMP13. Our finding that

MMP13 is significantly reduced makes it more likely that

cartilage vascular invasion is the result of other factors

involved in maintaining a tissue resistant to angiogenesis.

While these factors may include changes in the local pro-

duction of angiogenic and antiangiogenic factors, the

structural components of articular cartilage including col-

lagen fibrils, mineral, and the nonfibrillar matrix proteins

such as OPN may also play a role in compromising the

cement line. In addition to acting as a transitional tissue,

the mineralized zone of articular cartilage is thought to

minimize diffusion from subchondral bone. For example,

OPN plays a role in adhesion between opposing substrates

such as cartilage and subchondral bone by virtue of its

matrix-binding properties and ability to form a noncollag-

enous protein network in complex with calcium [41, 42].

Thus, matrix loss of mineral and OPN likely affect the

structural integrity of articular cartilage against vascular


123

invasion and may initiate the endochondral osteophyte

formation that typifies XLH [2, 6, 10–12].

Disruption in the Mineralized Zone of Cartilage May

Stimulate Proteoglycan Biosynthesis

We speculate that the observed increase in proteoglycan

biosynthesis is a compensatory response to an increase in

stress transduced to subchondral bone that would be pre-

dicted to occur with loss of the mineralized zone. Miner-

alized articular cartilage effectively functions as a

transitional tissue to reduce the stress at the boundary

between unmineralized cartilage and subchondral bone

because it has an elastic modulus that is an order of mag-

nitude less than that of subchondral bone [43]. This is

because the extracellular matrix defines the biomechanical


123

properties of articular cartilage. It is both negatively

charged and hydrated, and the mutual repulsive forces of

polyanionic proteoglycans embedded within a collagen

matrix determine its compressive stiffness. In addition, the

solid matrix of proteoglycans and collagen provides a

resistance to the flow of interstitial water upon loading

[44–46]. Together, these biophysical properties define the

effective compressive stiffness of the tissue and determine

deformation in response to load. Thus, a compensatory

increase in proteoglycan synthesis would offset the

increase in stress to the underlying subchondral bone by

both increasing deformation-dependent stiffness and

decreasing permeability. Indeed, we have shown that pro-

teoglycan synthesis, which is responsive to biomechanical

stimuli, is normalized in response to treatment (Fig. 3)

with recovery of the normal architecture of articular car-

tilage [47].

What Underlies the Decrease in Articular Cartilage

Thickness in Hyp Mice?

The absence of mineralized cartilage is a persistent feature

of Hyp mice and largely contributes to the net loss in the

Fig. 5 Evidence of vascular invasion of articular cartilage in

7-month-old Hyp mice. a Articular cartilage of a control mouse,

which is normally resistant to vascular invasion. b, c Representative

images from different Hyp mice showing vascular invasion (arrows)

of the articular surface as detected by immunohistochemical staining

with von Willebrand factor antibody

Fig. 4 Localization of alkaline phosphatase and markers of miner-

alizing articular chondrocytes of representative 12-week-old control,

Hyp, and Hyp mice treated with oral phosphate and calcitriol. Verticalbar shows the area between the edge of the articular cartilage (AC)

surface and its juxtaposition to subchondral bone (SCB). a Tibia of

12-week-old control mouse, shown at low power. b–e Control mouse,

localization of alkaline phosphatase (ALP) activity in articular

cartilage of femoral and tibial plateau below the tidemark (TM), the

interface between unmineralized and mineralized zones of cartilage,

and in subchondral bone (b). Immunoreactive osteopontin (OPN) and

matrix metalloproteinase 13 (MMP13) secreted into the chondrocyte

matrix of a control mouse (c, d). Note also immunostaining of

osteoblasts in subchondral bone (arrows). Histochemical staining of

sulfated articular cartilage proteoglycans by safranin O/fast green

staining, showing characteristic staining intensity that is heavier

above the tidemark, marked by arrowheads (e). f Tibia of 12-week-

old Hyp mouse, shown at low power; note widened growth plate

(rickets), metaphyseal splaying, and high levels of tissue alkaline

phosphatase. g–j Hyp mouse, alkaline phosphatase activity in

articular cartilage of femoral and tibial plateau and in subchondral

bone (g); absence of immunoreactive OPN and MMP13 secreted into

the chondrocyte matrix of a Hyp mouse (h, i). Images are positioned

to show immunoreactive OPN and MMP13 in subchondral bone

(arrows). j Safranin O staining of articular cartilage proteoglycans of

a Hyp mouse, heavily stained throughout the articular surface, with no

evidence of a tidemark. k Tibia of a 12-week-old Hyp mouse treated

with oral phosphate and calcitriol, shown at low power; note loss of

rachitic lesions. l–o Treated Hyp mouse, alkaline phosphatase activity

in articular cartilage of femoral and tibial plateau and in subchondral

bone (l); immunoreactive OPN and MMP13 secreted into the

chondrocyte matrix showing significant recovery of OPN and

MMP13 immunoreactivity (m, n), redefining the tidemark (arrow-heads). o Safranin O staining of articular cartilage proteoglycans of a

treated Hyp mouse, showing recovery in the intensity of staining of

sulfated proteoglycans (arrowheads)

b


123

total thickness of articular cartilage. In addition to the loss

of a mineralized zone of cartilage, the relative thickness of

cartilage in Hyp mice is significantly higher at 12 weeks

than at 7 months, reflecting a net loss of 70% of articular

cartilage thickness. It is unlikely to be mediated by phos-

phate-dependent apoptosis of chondrocytes since phos-

phate-replacement therapy corrects rachitic growth plate

structure by restoring (increasing) the terminal apoptosis of

hypertrophic chondrocytes by phosphate-dependent mod-

ulation of caspase-9 activity [48, 49]. In addition, unlike

growth plate chondrocytes, mineralizing chondrocytes of

articular cartilage do not undergo apoptosis and occur only

in a cell-nonspecific manner in osteoarthritis [49–53]. The

large increase in loss of articular cartilage is also reflective

of a thicker zone of cartilage in Hyp mice at 12 weeks. It

may be that the increase in total thickness at 12 weeks is

largely reflective of an increase in unmineralized matrix,

much like the accumulation of unmineralized osteoid in

bone seen in XLH. However, the ultimate loss of articular

cartilage thickness may be a reflection of the failure of

chondrocytes to differentiate into hypertrophic mineraliz-

ing articular chondrocytes, which have a larger cell volume

than do the chondrocytes of the upper zones of cartilage

[54].

Treatment of XLH Restores the Mineralization

of Articular Chondrocytes: Implications for the Short-

and Long-Term Management of XLH

The restoration of a mineralizing zone of articular chon-

drocytes (and markers of mineralized chondrocytes) with

phosphate replacement is likely akin to the restoration of

bone mineralization with normalization of hydroxyapatite

formation since extracellular phosphate is a key regulator

of mineralization [55–57]. 1,25(OH)2D3 may also play a

direct role in the regulation of mineralizing chondrocytes,

which is added by necessity to the phosphate-replacement

therapy of XLH to prevent hypocalcemia and secondary

hyperparathyroidism [33, 48]. It would be difficult to

ascertain the chronic effects of either of these agents alone

as contributing to restoring a zone of mineralized cartilage

in a clinical trial. However, unlike osteoblasts and growth

plate chondrocytes, the vitamin D receptor is not expressed

in normal articular chondrocytes but has been identified in

cartilage of ‘‘prototypical’’ osteoarthritis [58, 59].

Whether our findings in the articular cartilage of Hyp

mice are reproduced in patients with XLH and whether

they translate into destabilization of the joint are currently

under investigation. However, data obtained from Hyp

mice provide a novel rationale for early and long-term

management of the disease. Our cumulative data suggest

that early treatment may be critical to the development

of the appropriate zones of articular cartilage and to

minimizing the cellular changes that predispose patients to

degenerative osteoarthropathy.

While treatment during long bone growth in children

with XLH is the standard of care, long-term treatment

following closure of the growth plate is variably recom-

mended [60]. However, the unique zones of cartilage are

likely dependent upon the maintenance of a normal bio-

chemical milieu throughout adulthood since they are met-

abolically active cells, synthesizing an extracellular matrix

that is continually being turned over in response to load,

growth factors, and hormones [37, 61, 62]. If reversion to

the untreated articular cartilage phenotype occurs in the

absence of adequate phosphate supplementation, long-term

treatment during adulthood may be an important aspect of

therapy. Notwithstanding, the optimal treatment regimen of

calcitriol and phosphate is difficult to titrate [60]. In

addition, while we show a clear trend toward recovery of

the articular cartilage in response to treatment, the recovery

does not appear to be absolute. This may be due to the

direct potentiation of FGF23 production by both phosphate

and 1,25(OH)2D3 and the inability to completely correct

the hypophosphatemia [63, 64]. Newer interventions tar-

geted at limiting the phosphaturic actions of FGF23 may be

required to effectively maintain long-term serum phosphate

concentrations while minimizing the toxicities associated

with standard therapies [65].

Conclusions

The Hyp mouse has proved invaluable in elucidating cel-

lular changes within the context of XLH and provides an

excellent model for future studies aimed at better under-

standing the differentiation of articular chondrocytes into

two unique zones of cartilage. In addition, our data suggest

that disruption of extracellular factors in the hypophos-

phatemic environment severely impacts the distinct archi-

tecture of articular cartilage. We provide evidence that

management of serum phosphate may also be important in

both the early intervention and the long-term management

of the osteoarthritis-related complications of the disease.

Acknowledgments The authors thank Drs. Thomas Carpenter,

Arthur Broadus, and Pete Amos for helpful discussion. We also thank

Ali Nasiri, Nancy Troiano, and Christiane Coady for excellent tech-

nical assistance.

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