Connective Tissue Growth Factor (CTGF/CCN2) is Required for Normal Intramembranous and Endochondral
Ossification
1
1
Lambi, A G; 2Pankratz, T L; 1Hendesi, H; 1Pixley R A; 1Barbe, M F; 2Richtsmeier, J T; 1Popoff, S N.
Temple University School of Medicine, Philadelphia, PA, 2Pennsylvania State University, University Park, PA
Senior author: [email protected]
Introduction:
Connective tissue growth factor (CTGF, CCN2) has emerged as an
important growth factor for skeletogenesis. Its importance in
skeletogenesis is seen in global CTGF knockout (KO) mice, which
demonstrate defects in growth plate chondrogenesis and skeletal defects
including kinked ribs, tibiae, radii and ulnae, and craniofacial
abnormalities. Ultimately, global ablation of CTGF results in neonatal
lethality from respiratory failure. This study addressed the effects of
global CTGF ablation on skeletogenesis using quantitative techniques,
such as micro-CT. Furthermore, to discern any differences in the
separate processes of ossification affected by CTGF ablation, specific
bone sites and cells were studied.
Methods:
To address our goals, we used: (a) micro-CT analysis of long bones
and skulls to assess phenotypic differences, (b) histological analyses to
assess parameters of endochondral bone formation, and (c) ex vivo
studies to assess bone cell function.
CTGF heterozyogous mice (CTGF+/LacZ) were used as breeders to
obtain the mice for this study. Animal care and use was monitored by
the University Animal Care and Use Committee to assure compliance
with Federal and NIH regulations. Newborn animals used for this study
were sacrificed at birth (P0).
Micro-CT scanning was accomplished using a Skyscan 1172, 11
MPix camera model, high-resolution cone-beam micro-CT scanner. For
trabecular analysis, distal femora and proximal tibiae were scanned at a
pixel size of 5.2µm and a distance of 250µm from the growth plate was
taken. For skull analyses, entire skulls were scanned at a pixel size of
9.4µm. For landmark analysis, 3D coordinate locations of cranial
landmarks was recorded using eTDIPS software
(www.getahead.psu.edu).
For histologic analysis, plastic-embedded sections of tibiae and
femora were stained with von Kossa, Safranin-O, and Masson’s
trichrome for mineralization, cartilage, and a general bone stain,
respectively. Immunostaining of paraffin sections was also conducted
for osteoblast (Runx2, Osterix, Osteocalcin) and osteoclast (Cathepsin
K, actin, TRAP) markers. Histologic quantification was accomplished
with BioquantOsteo program.
Ex vivo studies of bone cell shape and function was carried out with
proliferation, differentiation, and adhesion studies. Primary osteoblasts
were isolated from newborn mouse calvariae and used for either cell
proliferation assay, differentiation under osteogenic culture conditions,
or to assess the ability of cells to adhere to various matricellular
components (fibronectin, vitronectin).
Results:
To quantify differences in bone resulting from the process of
endochondral ossification, we first performed analysis on distal femoral
and proximal tibial metaphyses. Our results demonstrated a decrease in
trabecular bone volume in CTGF KO mice compared to WT littermates.
In CTGF-null tibial diaphyses (the region of the structural kink), total
TV, BV and bone perimeter (B.Pm.) were markedly increased,
compared to WT tibiae.
CTGF-null skulls demonstrated decreases in cranial bone mineralization
(being absent in some sites), increases in suture and fontanel size, and a clear
failure of midline convergence of the maxillary and palatine processes to form
the palate. This was also accompanied by asymmetry of the vomer, a consistent
feature found in CTGF KO skulls.
Histologic analyses of WT and
CTGF-null tibiae showed differences
in various markers of endochondral
bone formation, including decreased
osteoblast cell numbers immediately
below the growth plate, decreased
bone matrix formation, and increased
hypertrophic zone thickness. We also
demonstrated a unique dichotomy of
bone formation in the kinked tibiae,
where ossification at the kink
appears to form by a mechanism
distinct from the region under the
growth plate.
Ex vivo studies from isolated
primary osteoblasts revealed
differences in cell function
comparing CTGF-null cells to WT.
We demonstrated that CTGF-null
osteoblasts have decreased ability to
adhere to bone matricellular proteins,
such as fibronectin. Furthermore,
these cells also show decreased
ability to proliferate, a feature
consistent over various time periods
and cell concentrations.
Figure Legend. Boxes 1,2, and 3 in histologic sections correspond to the
graphically depicted regions ‘Proximal Metaphysis’, ‘Proximal Diaphysis’, and
‘Mid Diaphysis’, respectively. Scale bars are equal to 500µm.
Discussion:
CTGF KO mice demonstrated quantitative defects in skeletogenesis, through
both endochondral and intramembranous ossification processes, as well as at the
cellular level. Micro-CT and histologic assays revealed abnormal endochondral
ossification of long bones, such as decreased trabecular bone formation and
osteoblast cell number in proximal tibial metaphyses, as well as increases in
hypertrophic zone length in proximal tibial growth plates. In addition, a unique
dichotomy of bone formation within the kinked tibiae alone was seen.
Three dimensional landmark analysis of CTGF KO skulls revealed decreased
ossification of skull flat bones, indicative of disruptions in intramembranous
ossification. Furthermore, craniofacial aberrations in CTGF KO mice included a
failure of palatine bones to converge toward the midline, and asymmetry of the
vomer. Lastly, CTGF-null osteoblasts demonstrated decreased ability to adhere
and proliferate in ex vivo culture. While the defects in osteoblast function could
explain, in part, the gross decreases in bone formation in CTGF KO mice,
findings such as the dichotomy of bone formation within the tibiae suggest a
more complex disruption of normal skeletogenesis is occurring in these mice.
Significance:
This study is crucial in elucidating the role of CTGF in bone development,
because for the first time it addressed the effects of CTGF ablation on bone
development using quantitative techniques, such as micro-CT analyses. These
analyses have generated novel information regarding the global skeletal effects
of CTGF ablation (i.e. kinked long bones), information necessary to
comprehensively define the role of CTGF in bone formation and provide
physicians with better tools for preventing and treating patients with conditions
of bone loss and/or gain.
Figure Legend. *p<0.05 and **p<0.01, compared to WT.
Acknowledgement: This work was supported by NIAH-NIAMS grant to SNP
(AR047432) and PA Department of Health grant to AGL.
Poster No. 0535 • ORS 2012 Annual Meeting