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Bone Biology Group, Division of Integrative Biology, Roslin Institute, Roslin, Scotland, United Kingdom EH25 9PS
Address all correspondence and requests for reprints to: Dr. Colin Farquharson, Bone Biology Group, Division of Integrative Biology, Roslin Institute, Roslin, Scotland, United Kingdom EH25 9PS. E-mail: colin.farquharson{at}bbsrc.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A series of elegant gene knockout and overexpression experiments have clearly shown that PTHrP together with the morphogen Indian hedgehog (Ihh) and their respective receptors regulate chondrocyte terminal differentiation in the early embryonic cartilage anlage in a highly coordinated manner (6, 7). These researchers have proposed a model for the molecular regulation of chondrocyte terminal differentiation in embryonic tissue. In this model, Ihh is produced by prehypertrophic chondrocytes committed to hypertrophy and acting through its receptor patched (Ptc), which is expressed by cells in the adjacent perichondrium, increases the expression of PTHrP in the periarticular region. PTHrP then diffuses beyond the proliferating cells, binds to the PTHR expressed on prehypertrophic chondrocytes, i.e. before their conversion to Ihh-expressing cells, and blocks their further differentiation. As the population of committed cells progresses to the hypertrophic phenotype, they stop expressing Ihh, thereby attenuating the negative feedback loop and allowing the further differentiation of uncommitted prehypertrophic cells. Further refinement of this model has indicated that bone morphogenetic protein-6 (BMP-6) and Sox-4 are likely intermediates in the PTHrP-Ihh regulatory feedback loop (8, 9).
The existence of the PTHrP-Ihh feedback loop regulating chondrocyte hypertrophy is attractive in early embryonic tissue, where distances over which PTHrP and Ihh must diffuse are relatively small. However, given the much larger distances involved, the existence of such a mechanism in the postembryonic growth plate has been questioned (10). Immunocytochemical and in situ hybridization studies indicate that PTHrP, Ihh, and their receptors are expressed by chondrocytes of the chick, rat, and mouse postembryonic growth plates (11, 12, 13, 14), suggesting that the components involved in the PTHrP regulation of terminal differentiation are intrinsic to the growth plate itself. In contrast, however, recent data from in vitro studies using cocultures of chick epiphyseal and growth plate chondrocytes support the concept that PTHrP is produced by the epiphyseal chondrocytes, thereby limiting the rate of growth plate chondrocyte terminal differentiation (15).
The aims of this current study were, therefore, to clarify and provide further information on the existence of the PTHrP-Ihh feedback loop in regulating chondrocyte terminal differentiation in the postembryonic chick growth plate. To this end we examined the spatial and temporal expression patterns of PTHrP, PTHR, Ihh, Ptc, and BMPs in growth plate sections and maturationally distinct chondrocyte fractions. In addition, a series of in vitro chondrocyte and growth plate explant studies provided functional data on the autocrine/paracrine actions of PTHrP signaling. This multidisciplinary approach provides new insights into the autocrine/paracrine control of chondrocyte differentiation and endochondral ossification.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chondrocyte isolation and Percoll fractionation
Growth plates from 3-wk-old male chicks were dissected free from
the proximal tibiotarsus and diced into 1-mm cubes. Chondrocytes were
isolated by incubating the tissue for 4 h at 37 C in 0.4%
collagenase, 0.2% hyaluronidase, and 0.01% deoxyribonuclease as
previously described (16, 17). Cell number was determined
using a hemocytometer, and 50–60 x 106
cells were resuspended in 1 ml Mg2+- and
Ca2+-free PBS. Discontinuous Percoll gradients
were prepared, which consisted of six density gradient steps
(1.04–1.09 g/ml). The isolated cells were carefully layered onto each
gradient, centrifuged at 400 x g for 30 min, and the
cells in the resultant five fractions were harvested by aspiration
(16, 17). After washing, cell viability was assessed by
trypan blue staining and was greater than 95% in all fractions. The
cells were either used in culture experiments (see below) or pelleted
by centrifugation and stored at -70 C until required.
Functional analysis of PTHR in Percoll- fractionated
chondrocytes
Chondrocytes from each Percoll fraction were resuspended in DMEM
containing 10% FBS, plated in 24-well plates at a density of 200,000
cells/well, and maintained at 37 C under an atmosphere of 95% air/5%
CO2. After an overnight incubation to allow cell
attachment, PTHrP (10-8 M;
Bachem, Essex, UK) was added to the cells of each
fraction (in quadruplicate for each assay and treatment group), whereas
others received vehicle only. As ascorbic acid is required for
chondrocyte differentiation, but severely inhibits avian chondrocyte
proliferation, this vitamin (ascorbic acid-2-phosphate) was added (100
µmol/liter) to the medium of the differentiation experiments only.
After 24 h the medium was removed and stored at -70 C until
assayed for cAMP using RIA (IDS, Boldon, UK). The cells were assessed
for alkaline phosphatase (ALP) activity, a recognized marker of
terminal differentiation, by measuring the cleavage of
p-nitrophenol phosphate at 410 nm. Total ALP activity was
expressed as nanomoles of p-nitrophenol phosphate hydrolyzed
per min/mg protein (16). For the determination of
chondrocyte proliferation, 0.2 µCi/ml
[3H]thymidine (37 MBq/ml; Amersham Pharmacia Biotech, Little Chalfont, UK) was added for the last
18 h of culture, and the amount of radioactivity incorporated into
trichloroacetic acid-insoluble precipitates was measured
(16).
Responses of epiphyseal and growth plate chondrocytes to
PTHrP
Chondrocytes from both epiphyseal and growth plate cartilages
were isolated and plated in 24-well plates as described above. After an
overnight incubation to allow cell attachment, PTHrP
(10-6–10-10
M) or vehicle was added, in quadruplicate, to each cell
type for 24 h, and [3H]thymidine uptake
and cAMP concentration in conditioned medium were measured as
previously described. In separate experiments growth plate chondrocytes
were cultured at a density of 7.5 x 106/T75
culture flasks as previously described. After an overnight incubation,
PTHrP (10-8 M) was added and after
1, 6, and 24 h the medium was removed, and the flasks were stored
at -70 C for RNA extraction and RT-PCR analysis of BMP expression. In
some cases freshly isolated epiphyseal and growth plate chondrocytes
were frozen directly at -70 C for RNA extraction and RT-PCR analysis
of PTHrP and PTHR expression.
Cloning of chick PTHR
To obtain information about the sequence of chick PTHR, we used
RT-PCR to amplify a 360-bp cDNA from chick chondrocyte RNA using the
degenerate primers TTYGGNTGGGGNYCCNGC and
GGAGGATCCRAANARCATYTCRTARTGGAT. These correspond to the amino acid
motifs FGWGLPA and MHYEMLFN, which are conserved within the human, rat,
and opossum PTHR sequences. The cDNA fragment was cloned into the
T-vector pCRII to generate the clone pAH1. The predicted amino acid
sequence of the pAH1 insert had 85% identity to the human PTHR
sequence and 59% identity to the human PTHR2 sequence, confirming that
a fragment of the gene for the type 1 receptor had been cloned.
RNA extraction
Total RNA was extracted from chondrocytes by repeated aspiration
through a 25-gauge syringe needle in 1.5 ml Ultraspec (Biotecx,
Houston, TX). After extraction with chloroform, RNA in the aqueous
phase was precipitated with isopropanol and bound to RNA Tack resin
(Biotecx) following the manufacturer’s protocol. After washing with
75% ethanol, the RNA was eluted in 100 µl ribonuclease-free water
(17). In each case the 260/280 ratio was 1.9–2.0,
confirming the purity of the RNA. All preparations were diluted to a
concentration of 50 ng/µl and stored at -70 C.
Semiquantitative RT-PCR
Gene expression in the five Percoll fractions and PTHrP-treated
chondrocytes was analyzed by semiquantitative RT-PCR
(16, 17, 18). Aliquots of 500 ng RNA (or an equivalent volume
of water as a control) were reverse transcribed in 20-µl reactions
with 200 ng random hexamers and 200 U Superscript II reverse
transcriptase using the Superscript preamplification protocol
(Life Technologies, Inc., Paisley, UK). PCR was performed
in 20-µl reactions containing cDNA equivalent to 10 ng RNA and 200
nM gene-specific primers (Table 1
) in 11.1 x PCR buffer
(19). The cycling profile was 1 min at 92 C (first cycle,
2 min), 1 min at 55 C, and 1 min at 70 C. The number of cycles
performed was carefully titrated to ensure that the reactions were in
the exponential phase (Table 1). Reaction products were analyzed on
1.5% agarose gels in the presence of ethidium bromide (250
µg/liter), and a digital image of each gel was captured using a gel
documentation system (Bio-Rad Laboratories, Inc., Hemel
Hempstead, UK).
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Statistical analysis
All in vitro cell culture experiments were performed
a minimum of three times, and although slight variations in absolute
values between individual experiments existed, the trends shown in the
representative figures were noted in all experimental repeats. All data
are expressed as the mean ± SEM (of four
observations within each experiment), and statistical analysis was
performed using t test. In the gene expression studies the
results shown are representative of the expression levels in two
separate PTHrP treatment experiments and four independent Percoll
fractionations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The
chondrocytes within the proliferating and hypertrophic zones of the
postembryonic growth plate all reacted positively (Figs. 1, B and C),
with strongest staining observed in the terminally differentiated,
hypertrophic chondrocytes. The chondrocytes within the epiphyseal
cartilage juxtaposed to the underlying growth plate cartilage were also
strongly stained with the PTHrP antiserum (Fig. 1D). No staining was
evident in control sections reacted in the absence of the primary
antibody (Fig. 1E).
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).
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). As assessed by semiquantitative analysis the levels of
expression of PTHrP, Ihh, and PTHR were similar in all Percoll
fractions, and no obvious expression trends with respect to the
chondrocyte maturational stage were noted in four independent Percoll
fractionations. However, the expression of Ptc was consistently lower
in hypertrophic chondrocytes (fractions 1 and 2) compared with
fractions 3–5. The levels of expression of BMP-2 and -7 were similar
in all chondrocyte fractions, whereas the expression of BMP-4, -5, and
-6 varied with the degree of chondrocyte differentiation (Fig. 2C).
BMP-4 showed a biphasic response and was most abundantly expressed in
fractions 2 and 3. BMP-5 expression was highest in the proliferating
cells and decreased with chondrocyte differentiation, whereas BMP-6
expression was highest in the hypertrophic chondrocytes and lower in
the proliferating cells.
Treatment of cultured chondrocytes with PTHrP markedly down-regulated
the expression of BMP-4 and BMP-6. After 6 h, expression was lower
and was undetectable by 24 h posttreatment (Fig. 3A). The levels of expression of BMP-2,
-5, and -7 were very low in cultured growth plate chondrocytes and were
not affected by PTHrP at the time points studied (data not shown).
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). In contrast,
PTHR expression was only detectable in chondrocytes of the whole growth
plate (Fig. 3B).
Response of fractionated chondrocytes to PTHrP
The RT-PCR analysis indicated that the PTHR was expressed by all
growth plate chondrocytes (Fig. 2B). However, although receptor mRNA
was observed, this was not sufficient to determine whether functional
receptors were present. Therefore, the response of each cell fraction
to exogenous PTHrP was assayed. PTHrP increased thymidine uptake and
cAMP production and decreased the ALP activity of all fractionated
chondrocytes, indicating the presence of functional cell surface
receptors for PTHrP on all growth plate chondrocytes (Fig. 4). However, the magnitude of the effect
of PTHrP on proliferation, differentiation and cAMP production varied
with maturational phenotype. In general, the fold difference (Table 2) between treated and control cultures
was less for the hypertrophic chondrocytes (fractions 1 and 2) than
those of a more proliferative phenotype (fractions 3–5). It is
possible that a proportion of the response within each fraction may be
due to a small number of contaminating cells in each fraction. This
would, however, not alter our conclusion that proliferative cells are
more responsive (with respect to thymidine uptake, ALP activity, and
cAMP production) than hypertrophic cells. For example, if the small
PTHrP response noted in fractions 1 and 2 was partly due to
contamination by proliferating chondrocytes, this would imply that the
hypertrophic cells, which are the principle cell in these fractions,
are even less responsive to PTHrP. The same argument holds for the gene
expression data shown in Fig. 2.
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and 3B), it was of interest to determine whether the response
of epiphyseal chondrocytes to PTHrP was similar to that of growth plate
chondrocytes. Experiments with monolayer cultures showed that PTHrP
significantly stimulated the thymidine uptake of growth plate
chondrocytes (P < 0.001) and their production of cAMP
(P < 0.001), whereas no significant effect of PTHrP
was observed on either parameter in epiphyseal chondrocytes (Fig. 5). The effect of PTHrP on growth plate
chondrocytes was concentration dependent; thymidine uptake and cAMP
production were stimulated to similar levels by
10-6 and 10-8
M PTHrP, whereas the response to
10-10 M PTHrP was less for
both parameters (Fig. 5).
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and 2D) but did not respond to exogenous PTHrP in
culture (Fig. 5) suggested that the function of epiphyseal-derived
PTHrP may be to control the rate of growth plate chondrocyte
differentiation in a way similar to that proposed for embryonic
skeletal growth (7). This hypothesis was tested using a
growth plate explant system previously described by us
(16). In these explant studies the distinction between
proliferative and hypertrophic chondrocytes was based entirely on
morphology, as our previous explant studies have clearly indicated that
chondrocytes with a hypertrophic, but not a proliferative, morphology
synthesized a cartilaginous matrix rich in a collagen type X
(16). The area of growth plate taken for explant culture
contained the entire proliferating zone and the upper part of the
hypertrophic zone (d 0; Fig. 6A). After
4 d in culture, the chondrocytes situated within the original
proliferating zone of the control explants were clearly hypertrophic
(Fig. 6B), which is in accord with our previous observations
(16). However, chondrocytes from PTHrP-treated explants
maintained the flattened ellipsoid shape characteristic of
proliferating chondrocytes (Fig. 6C). This strongly suggests that PTHrP
inhibits the terminal differentiation of chondrocytes. Explants that
had been cultured in medium containing PTHrP for 4 d and then
maintained for an additional 4 d in medium free of exogenous PTHrP
developed cells that had a hypertrophic phenotype (Fig. 6D) and were
indistinguishable from those in Fig. 6B. This indicates that the cells
within the explants were still viable after 4 d of PTHrP
treatment. In explants comprising both epiphyseal and growth plate
cartilage, a band of chondrocytes within the proximal region of the
growth plate and close to the junction with the epiphyseal cartilage
maintained their proliferative phenotype with no evidence of
chondrocyte hypertrophy (Fig. 6, E and F). Cellular morphology was
remarkably similar to that of the PTHrP-treated explants (Fig. 6C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This observation is in contrast to the PTHrP distribution noted during early embryonic endochondral bone formation in the mouse, rat, and chick, where in situ hybridization studies have indicated that PTHrP is principally located to the perichondrial cells closest to the articular surfaces (periarticular region) and, with the exception of faint staining in the hypertrophic cells of one study (20), is essentially absent from the growth plate (7, 20, 21, 22). These data suggest age-dependent differences in PTHrP localization, although the apparent differences in the exact cells that express PTHrP may result from studying various species of different ages by a number of disparate techniques. PTHrP shares many of the features common to members of the early response gene family that are characterized by unstable and rapidly turned over mRNA (23). This highlights the importance of the sensitivity of the detection technique employed, and this is exemplified by the results of the study by Medill et al. (13), who found PTHrP to be expressed by proliferating chondrocytes using RT-PCR (as in this present study), but not using immunocytochemistry. An alternative explanation for the widespread distribution of PTHrP protein within the postembryonic growth plate is that it localizes to chondrocytes that express the PTHR rather than at sites where it is synthesized. This argument has previously been used to explain the discordant distribution of PTHrP (22) and also IGF (24), nerve growth factor (25), and TGFß1 (26). However, we detected the expression of PTHrP mRNA throughout the growth plate, and this provides direct evidence that PTHrP is indeed produced by chondrocytes of all maturational stages.
Regardless of the source of PTHrP in the growth plate, the specific target cells for this ligand have not yet been unequivocally identified. As PTHR expression is highest in the early differentiating chondrocytes (6, 20), these cells have been regarded as the target for PTHrP signaling (7). However, recent data obtained from chimeric mice containing both wild-type and PTHR-negative (-/-) cells, indicate that PTHrP acts directly on the low level of PTHR present on proliferating cells (27) to delay and synchronize their differentiation (28). This observation would explain why proliferating chondrocytes are most noticeably affected in PTHrP knockout (7) and PTHrP-overexpressing mice (29). Our present data show that cells of all growth plate fractions express PTHR and respond in culture to exogenous PTHrP. This indicates that the PTHR is functional in all growth plate maturational zones. Significantly, we found that PTHrP elicited the greatest response in chondrocytes of a more proliferative phenotype (fractions 3–5). This observation is consistent with the view that it is these cells that are both the target of and principal responders to PTHrP (28). It cannot be ruled out, however, that the hypertrophic cells may elicit a greater response to PTHrP than the proliferating chondrocytes in the synthesis and secretion of collagen, proteoglycans, and other matrix molecules. This aspect of the response to PTHrP requires further study.
The original in situ hybridization studies by Vortkamp and colleagues (7) indicated that Ihh was expressed by prehypertrophic chondrocytes in both embryonic and postembryonic chick growth plates and that the perichondrium flanking the Ihh expression domain was the location of the Ptc receptor (7). Recent studies, however, failed to localize Ptc protein to the perichondrium of postnatal rat bone (14), and other reports have now shown Ptc expression by proliferating cells (27, 30). Our observations that Ptc mRNA expression was higher in the proliferating chondrocytes is in agreement with these data, and together they suggest that Ihh acts directly on chondrocytes, and not via the perichondrium, to regulate the pace of chondrocyte differentiation. A dual action of Ihh on the chondrocyte differentiation process has been proposed (31). The attachment of cholesterol moieties to the C-terminus of the cleaved N-terminal fragment of Ihh (32) results in both its tethering to the plasma membrane and sequestration at the site of the Ptc receptor (33). The resultant accumulation of hedgehog protein may lead, through short range and PTHrP-independent effects, to promotion of the terminally differentiated phenotype (31, 34, 35, 36). These prodifferentiating actions of Ihh are in contrast to those elicited by the long-range diffusion of Ihh and activation of the PTHrP signaling pathway (31). Recent data indicated that Ihh also drives chondrocyte proliferation in a pathway that is essentially independent of PTHrP signaling (36), and therefore, it is likely that not all actions of Ihh are mediated by PTHrP and result in delayed chondrocyte differentiation. The above evidence suggests a more complex control of chondrocyte differentiation by Ihh and PTHrP than previously recognized. The precise contribution of each pathway to the differential fate of growth plate chondrocytes remains to be determined.
The mechanisms for relaying Ihh over many cell diameters to its target cells are unclear (37). The lipophilic character of Ihh presents special problems for long-range signaling, but clues to the mechanisms involved have come from analysis of the Drosophila gene, dispatched (disp) (38). The disp protein is phylogenetically related to a family of proteins containing a sterol-sensing domain and in disp mutant cells, cholesterol-modified hedgehog protein is retained within the cell, indicating that disp functions to release cholesterol-tethered hh protein (38). As entries with homology to disp exist in vertebrate EST databases (37), similar mechanisms for Ihh mobilization are likely to function in vertebrates. It is, however, unknown whether disp and other proposed hedgehog- signaling pathways (39) can propel Ihh over sufficient distances in the embryonic cartilage anlage. As a consequence of the large increases in size during vertebrate development, such problems will be exacerbated in the postembryonic growth plate.
The importance of BMPs in endochondral ossification is unequivocal, and various studies have localized BMP-1 to -7 to all maturational zones of the growth plate (40, 41, 42). BMPs have been shown to promote both chondrogenesis (43) and chondrocyte terminal differentiation (44), and a blockade of BMP receptor signaling results in the delayed differentiation of chick limb bud cells (45). We observed distinct expression patterns of the genes encoding BMPs. BMP-2 and -7 were expressed uniformly throughout the growth plate, whereas BMP-5 was expressed most abundantly in the proliferating chondrocytes. In agreement with earlier workers we noted that BMP-6 expression is progressively up-regulated during chondrocyte terminal differentiation (7). Interestingly, we found that the expression of both BMP-4 and BMP-6 by cultured growth plate chondrocytes was dramatically inhibited by PTHrP. The rapidity of the response observed in the present study (within 6 h) and the absence of any increase in the expression of these BMPs in control cultures argue that this is a direct effect and is not secondary to the inhibition of chondrocyte differentiation by PTHrP. As BMP-6 stimulates chondrocyte maturation and up-regulates the expression of Ihh, Grimsrud and colleagues (8) proposed that the inhibition of chondrocyte maturation by PTHrP is indirect and is mediated by the suppression of BMP-6 expression.
As an alternative fate to terminal differentiation, postmitotic chondrocytes can rapidly undergo apoptosis (46). BMP-4 has been shown to be expressed in populations of apoptotic cells as well as directly promoting apoptosis during embryonic pattern formation (47). Our finding that BMP-4 expression was highest in the prehypertrophic chondrocytes indicates that BMP-4 expression also coincides with the onset of apoptosis within the growth plate (46). PTHrP inhibits chondrocyte apoptosis as well as terminal differentiation (48) and therefore the inhibition of BMP-4 by PTHrP suggests a mechanism by which PTHrP could prevent chondrocyte apoptosis. Thus, by inhibiting the expression of both BMP-4 and BMP-6, PTHrP may control the two developmental fates available to postmitotic chondrocytes.
The infection of chick limb buds with a constitutively active form of the BMP receptor 1A results in the stimulation of PTHrP expression and the inhibition of chondrocyte differentiation (45). Although this suggests that some BMPs may mediate the effects of Ihh in regulating PTHrP expression, it is unlikely to be BMP-7, whose inhibitory actions on terminal differentiation have been shown to be independent of PTHrP expression (49). The results of our study are in agreement with this conclusion, as PTHrP had no effect of on BMP-7 expression in cultured chondrocytes.
In addition to positive PTHrP immunolocalization to growth plate chondrocytes, we noted the presence of PTHrP in the epiphyseal chondrocytes situated proximal to the growth plate. Analysis by RT-PCR confirmed this expression pattern and further indicated that PTHrP mRNA expression by epiphyseal chondrocytes was higher than that by growth plate chondrocytes. Although this substantiates earlier data (15), no evidence exists for an autocrine role for this epiphyseal chondrocyte-derived PTHrP. Unlike their growth plate counterparts, the epiphyseal chondrocytes showed no PTHR mRNA expression and neither a cAMP nor a proliferative response to exogenous PTHrP in culture. Alternatively, coculture experiments with both chondrocyte types have supported the suggestion that PTHrP diffuses from the epiphysis into the growth plate and acts in a paracrine manner to regulate the rate of chondrocyte maturation (15). We have confirmed and extended these results using an organ culture system that more closely parallels the complex tissue interactions that occur in the growing bone. These results are analogous to developmental models in which PTHrP is expressed by chondrocytes in the periarticular region of embryonic long bones and regulates the pace of growth plate chondrocyte maturation (7). However to rule out the role of other potential mitogens, further experimentation, possibly involving PTHrP-neutralizing antibodies, will be needed to confirm that PTHrP is indeed the paracrine factor central to this regulation.
The results of our study show that the known components of the PTHrP-Ihh autoregulatory loop are all present within the postembryonic chick growth plate and suggest that the long diffusion distances required for the interaction of PTHrP and Ihh with their receptors (7) may not be required for the control of chondrocyte terminal differentiation. This situation, however, may not be so far removed from the events that occur in the developing cartilage anlage. It has recently been implied that both Ihh and PTHrP interact directly with their receptors located on proliferating chondrocytes to control the rate of chondrocyte differentiation (28, 30). It is therefore likely that local mechanisms intrinsic to the growth plate exist to control the rate of longitudinal bone growth, although a role for epiphyseal chondrocyte-derived PTHrP in regulating proliferating chondrocyte differentiation cannot be ruled out.
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Acknowledgments |
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Footnotes |
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Abbreviations: ALP, Alkaline phosphatase; BMP, bone morphogenetic protein; disp, dispatched; Ex-FABP, extracellular fatty acid binding protein; Ihh, Indian hedgehog; Ptc, patched; PTHR, PTH receptor.
Received February 15, 2001.
Accepted for publication May 24, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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