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Published in final edited form as: Matrix Biol. 2009 Sep 17;29(1):3–8. doi: 10.1016/j.matbio.2009.09.003

ADULT HUMAN BONE MARROW STROMAL CELLS REGULATE EXPRESSION OF THEIR MMPs AND TIMPs IN DIFFERENTIATION TYPE-SPECIFIC MANNER.

Joshua Mauney 1, Vladimir Volloch 2,*
PMCID: PMC6817335  NIHMSID: NIHMS146449  PMID: 19765656

Abstract

Previously, we described a profound impact of structural conformation of collagen matrix on osteogenic and adipogenic differentiation of bone marrow stromal cells. Thus, a marginal p38-independent adipogenesis on native collagen I matrix contrasts with an efficient p38-dependent differentiation on denatured collagen I. An efficient Hsp90-dependent osteogenesis occurs on native collagen I matrix but not on its denatured counterpart where it is insignificant and proceeds in an Hsp90-independent manner. Whereas only marginal osteogenesis and no detectable adipogenesis of bone marrow stromal cells occur on native collagen IV, the same matrix supports a highly efficient adipogenesis in denatured structural state. The present study addresses the opposite direction in the flow of cell-matrix interaction, namely the cells’ influence on structural state of collagen matrix, and tests the possibility that differentiating bone marrow stromal cells may adjust the expression phenotype of MMP and TIMP in such a way that, if translated into matrix modification, would facilitate the maintenance of collagen matrix in or its modification into structural state optimal for the ongoing differentiation process. The results obtained indicate that this is indeed the case. In bone marrow stromal cells stimulated to undergo adipogenesis the expression of MMP increases and that of TIMP decreases. In cells induced to undergo osteogenesis the opposite is true: MMP/TIMP expression is adjusted in a manner that, if translated into matrix modification, could promote the native structural conformation optimal for this type of differentiation. The results obtained also indicate that the observed adjustment in MMP/TIMP expression phenotype might be an early differentiation event and that differentiation stimulation alone might be sufficient to trigger it even on matrices not favorable to a given type of differentiation. The findings of the present study raise significant questions and indicate directions for further experimentation.

INTRODUCTION.

Earlier, we demonstrated that structural conformation of extracellular collagen I, the major organic matrix component in musculoskeletal tissues, plays, along with differentiation stimuli, a crucial role in differentiation of human bone marrow stromal cells into osteogenic and adipogenic lineages (Mauney and Volloch, 2009a). Thus, on native collagen I matrix a marginal inefficient adipogenesis of bone marrow stromal cells is p38-independent, whereas on collagen I matrix in denatured structural conformation an efficient adipogenesis is primarily regulated by p38 kinase. Inversely, osteogenic differentiation occurs efficiently on native, but not on denatured collagen I matrix, with a low commencement threshold on the former and a substantially higher one on the latter (Mauney and Volloch, 2009a). As with adipogenesis, the profound difference in the efficiency of osteogenic differentiation on matrices in different structural states reflects distinctly different regulatory pathways. Osteogenesis on collagen I matrices in both structural conformations is fully dependent on ERK. However, whereas on native collagen I matrix osteogenic differentiation of is Hsp90-dependent, on denatured collagen I matrix it is Hsp90-independent (Mauney and Volloch, 2009a).

It appears, therefore that it is advantageous for cells to undergo adipogenesis on collagen I matrix in denatured structural conformation, and osteogenesis on its native counterpart. Our previous study implicated a mechanism capable of assuring such an advantage for osteogenesis and possibly operating during bone remodeling (Mauney and Volloch, 2009a). This study indicated that bone marrow stromal cells require significantly higher osteogenic inducer concentration (termed osteogenic commencement threshold) on denatured collagen I matrix than on its native counterpart due to distinctly different regulatory mechanisms operating on these two types of matrices (Mauney and Volloch, 2009a). Therefore, it appears that when mesenchymal stem cells recruited from the bone marrow cavity arrive to a bone remodeling site and encounter an extracellular matrix consisting, in its organic component, primarily of denatured collagen I transitioned into denatured conformation during the resorption stage of remodeling cycle, they do not, despite constitutive presence of anabolic stimuli, commence osteogenic differentiation because of the high osteogenic commencement threshold. Instead, interaction with denatured collagen I matrix promotes the expansion of the mesenchymal stem cell population and facilitates the retention of their differentiation potential (Mauney et al., 2004). Proliferating mesenchymal stem cells deposit native collagen I matrix. Consequently, through the enabling of the Hsp90-dependent mechanism, osteogenic commencement threshold is lowered, and osteogenic differentiation is initiated in its efficient mode (Mauney and Volloch, 2009b).

Thus, on one hand, cells undergo an efficient differentiation into adipogenic and osteogenic lineages in a collagen I matrix’s structural conformation-dependant manner, with the former being promoted by denatured matrix and the latter being facilitated by the native one. Moreover, it appears that cellular mechanisms exist that are designed to ascertain that cells undergo differentiation only when collagen I matrix is in structural state which is optimal for a given type of differentiation. Furthermore, similar dependency on matrix’s structural conformation is observed in adipogenic differentiation on collagen IV, the major matrix component associated with differentiating adipocytes in adipose tissue (Mauney and Volloch, 2009c). Whereas only marginal, if any, adipogenesis is seen on native collagen IV matrix, an extremely efficient adipogenic differentiation occurs on collagen IV in denatured structural conformation; this is due to differential structure-specific presentation of integrin binding sites. On the other hand, in addition to responding to the matrix’s structure, cells are capable of influencing it, in terms of maintenance or modification, through adjustments in the expression phenotype of MMPs and their inhibitors, TIMPs (Birkedal-Hansen et al., 1993; Borden and Heller, 1997; Fini et al., 1998; Nagase and Woessner, 1999). This opens up a potential possibility that cells undergoing adipogenic or osteogenic differentiation might be capable of adjusting the expression of MMP and TIMP in such a way that, if translated into matrix modification, would facilitate the maintenance of collagen matrix in or its modification, in terms of alteration of either preexisting or newly deposited ECM, into structural state optimal for a given type of differentiation. The MMPs most likely to be involved in such a process are mammalian collagenases, a class of MMPs comprised of MMP-1, MMP-8, and MMP-13 (also known as collagenases 1, 2, and 3) capable of cleaving native triple-helical collagens into defined fragments that denature at physiological temperature (Birkedal-Hansen et al., 1993; Veis and George, 1994; Kähäri and Saarialho-Kere, 1997; Knäuper et al., 1997; Messent et al, 1998). Indeed, an increase in the activity of secreted MMP collagenases, which can occur either through elevation of levels of MMPs or reduction of levels of TIMPs or both, would, if translated into matrix modification, promote the transition of native collagen matrix into denatured state optimal for adipogenesis (Birkedal-Hansen et al., 1993; Veis and George, 1994; Kähäri and Saarialho-Kere, 1997; Messent et al, 1998). Inversely, a reduction in the activity of secreted MMP collagenases would, if translated into matrix modification, facilitate the maintenance of newly deposited collagen I in native conformation optimal for osteogenesis. The expression of many MMPs, including all three mammalian collagenases, and of their inhibitors, TIMPs, was shown to be inducible, i.e. regulated in large degree on transcriptional level (Woessner, 1991; Birkedal-Hansen et al., 1993; Borden and Heller, 1997; Fini et al., 1998; Nagase and Woessner, 1999; Vincenti, 2001; Vincenti and Brinckerhoff, 2002; Vikman et al., 2007). Therefore, to assess the possibility of differentiation-specific adjustment of MMP/TIMP balance, the present study investigated the expression phenotype of MMP collagenases and of TIMPs in bone marrow stromal cells induced to undergo adipogenesis or osteogenesis on collagen matrices in native or denatured structural conformations and compared it with that seen in undifferentiated controls.

RESULTS.

During adipogenic stimulation of bone marrow stromal cells on collagen I matrices in both native and denatured structural states the expression of MMP increases and that of TIMP decreases.

The experiments described below were designed to test the notion that bone marrow stromal cells stimulated to undergo adipogenesis on either native or denatured collagen I matrices might adjust the expression of their MMP and TIMP genes in such a manner that, if translated into matrix modification, would promote the maintenance of matrix in or its modification into denatured structural conformation shown to be optimal for adipogenic differentiation. Bone marrow stromal cells were maintained as untreated controls or induced to undergo adipogenesis on native collagen I matrix by the addition of adipogenic stimulants as described in Experimental Procedures section. After fourteen days, MMP and TIMP expression levels were measured by real time PCR. As shown in Figure 1A, MMP/TIMP expression balance indeed changes during adipogenic stimulation of bone marrow stromal cells on native collagen I matrix, with expression levels of MMP significantly increasing and those of TIMP strongly decreasing. In a parallel experiment shown in Figure 1B, the same pattern was observed in bone marrow stromal cells undergoing adipogenic differentiation on denatured collagen I matrix. It should be mentioned that in the experiment depicted in Figure 1 and in all experiments described in the present study transcript levels of all three MMP collagenases (MMP-1, MMP-8, and MMP-13) and of all four TIMPs were assessed but only those that underwent statistically significant changes were presented.

Figure 1. During adipogenic stimulation of bone marrow stromal cells on both native and denatured collagen I matrices the expression of MMP increases and that of TIMP decreases.

Figure 1.

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C: untreated controls; AD: adipogenic stimuli; NC: native collagen I matrix; DC: denatured collagen I matrix; MMP: matrix metalloproteinase; TIMP: tissue inhibitor of metalloproteinase. [*= p<0.05]: significantly different in comparison to the same values in untreated controls.

During adipogenic stimulation of bone marrow stromal cells on collagen IV matrix the expression of MMP increases and that of TIMP decreases.

Bone marrow stromal cells undergo adipogenic differentiation in a manner dependent on matrix’s structural conformation not only on collagen I but also on collagen IV. Whereas very little, if any, adipogenesis is seen on native collagen IV matrix, adipogenic differentiation is highly efficient on collagen IV matrix in denatured structural conformation which presents 15 αVβ3 binding sites that are instrumental, when engaged, in activation of p38 needed for efficient adipogenesis (Engelman et al., 1998; Jaiswal et al., 2000; Salasznyk et al., 2004), per molecule versus only one in native conformation (Mauney and Volloch, 2009c). Therefore, adipogenesis of bone marrow stromal cells on denatured collagen IV matrix can be used as a model system to test whether the adjustment of MMP/TIMP expression described above is limited to collagen I or is utilized on another type of collagen matrix. For this purpose, cells were either maintained as untreated controls or induced to undergo adipogenesis on denatured collagen IV matrix by the addition of adipogenic stimulants. After nine days, MMP and TIMP expression levels were measured by real time PCR. As shown in Figure 2, in cells undergoing adipogenic differentiation on denatured collagen IV matrix, expression levels of MMP significantly increase and those of TIMP strongly decrease in the same manner as seen in cells undergoing adipogenesis on collagen I matrices. Similar trend (not shown) was seen with bone marrow stromal cells stimulated to undergo adipogenesis on native collagen IV matrix where no detectable adipogenesis was observed (Mauney and Volloch, 2009c).

Figure 2. Levels of MMP increase and of TIMP decrease during adipogenic differentiation of bone marrow stromal cells on denatured collagen IV matrix.

Figure 2.

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DCIV: denatured collagen IV matrix; AD: adipogenic stimuli; MMP: matrix metalloproteinase; TIMP: tissue inhibitor of metalloproteinase. [*= p<0.05]: significantly different in comparison to the same values in untreated controls.

During osteogenic stimulation of bone marrow stromal cells on both native and denatured collagen I matrices the expression of MMPs decreases and that of TIMPs increases.

To test a possibility that bone marrow stromal cells stimulated to undergo osteogenesis might adjust the expression of their MMP and TIMP genes in such a manner that, if translated into matrix modification, would promote the maintenance of matrix in native structural conformation shown to be optimal for osteogenic differentiation, cells were either maintained as untreated controls or induced to undergo osteogenesis on native collagen I matrix by the addition of osteogenic stimulants as described in Experimental Procedures section. After fourteen days, MMP and TIMP levels were measured by real time PCR. As shown in Figure 3A, MMP/TIMP expression phenotype does change, with expression levels of MMPs receding and those of TIMPs advancing during osteogenesis of bone marrow stromal cells on native collagen I matrix. During osteogenic stimulation of bone marrow stromal cells on denatured collagen I matrix, similar adjustment in MMP/TIMP expression levels is activated (Figure 3B).

Figure 3. Levels of MMP decrease and of TIMP increase during osteogenic differentiation of bone marrow stromal cells on both native and denatured collagen I matrices.

Figure 3.

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C: untreated controls; OS: osteogenic stimuli; NC: native collagen I matrix; DC: denatured collagen I matrix; MMP: matrix metalloproteinase; TIMP: tissue inhibitor of metalloproteinase. [*= p<0.05]: significantly different in comparison to the same values in untreated controls.

DISCUSSION.

Previously, we described a profound impact of structural conformation of collagen matrix on osteogenic and adipogenic differentiation of bone marrow stromal cells (Mauney and Volloch, 2009a; 2009b; 2009c). The effect of structural state of collagen matrix is unequivocal. Thus, a marginal p38-independent adipogenesis on native collagen I matrix contrasts with an efficient p-38-dependent differentiation on denatured collagen I whereas an efficient Hsp90-dependent osteogenesis occurs on native collagen I matrix but not on its denatured counterpart where it proceeds in an Hsp90-independent manner (Mauney and Volloch, 2009a; 2009b). The drastic impact of matrix’s structural state on bone marrow stromal cells differentiation is seen also on collagen IV. Whereas both osteogenesis and adipogenesis of bone marrow stromal cells occur only marginally on native collagen IV matrix, in its denatured structural conformation collagen IV supports a highly efficient adipogenesis (Mauney and Volloch, 2009b; 2009c). It is apparent, therefore, that one structural state of collagen matrix is clearly advantageous, or “optimal”, over the other for a given type of bone marrow stromal cells’ differentiation. Thus, native collagen I matrix is the optimal one for osteogenic differentiation whereas collagen I and collagen IV in denatured structural conformation are optimal matrices for adipogenic differentiation of bone marrow stromal cells.

The present study addresses the opposite direction in the flow of cell-matrix interaction, namely the possibility of bone marrow stromal cells undergoing differentiation influencing structural state of collagen matrix. It was initiated in order to test whether bone marrow stromal cells may adjust the expression phenotype of MMP collagenases and TIMPs in such a way that, if translated into matrix modification, would facilitate the maintenance of collagen matrix in or its modification into structural state optimal for the ongoing differentiation process. The results obtained indicate that this is indeed the case. In bone marrow stromal cells stimulated to undergo adipogenesis the expression of MMP collagenases increases and that of TIMP decreases thus facilitating, if translated into matrix modification, the denatured structural state of collagen matrix optimal for this type of differentiation. In bone marrow stromal cells stimulated to undergo osteogenesis the opposite is true: MMP/TIMP expression phenotype is adjusted so as to facilitate, if translated into matrix modification, the native structural conformation optimal for this type of differentiation. The observation that the adjustment in MMP/TIMP expression phenotype occurs in cells on both collagen I and collagen IV matrices indicates that the observed phenomenon is not limited to a specific type of collagen matrix but might be of a more general nature. The observation that such an adjustment is seen also on matrices where only marginal differentiation takes place suggests that it might be an early differentiation event and that differentiation stimulation alone is sufficient to trigger it even on matrices not favorable to a given type of differentiation. Finally, the results obtained in the present study indicate that both MMP collagenases and TIMPs appear to be differentially up- or down regulated in matrix’s structure-specific as well as in differentiation type-specific manners. Indeed, in cells undergoing both adipogenic and osteogenic differentiation, different MMP/TIMP subsets exhibit substantial transcript level changes on native versus denatured matrices. Likewise, the expression levels of distinct MMP collagenases/TIMP subsets are changed in adipogenic versus osteogenic types of differentiation.

The crucial question is, of course, whether the observed adjustment in the MMP/TIMP expression phenotype translates into matrix modification. The direct approach to answering this question would be to analyze structural state of collagen matrix. This, however, is a challenging proposition. If the translation takes place, the changes would occur only in the upper layer, i.e. a very small fraction, of a rather thick collagen film, making the determination trough direct analysis of total collagen not feasible. Therefore, the analysis has to be performed in situ. The best way to do it would be to employ atomic force microscopy, which would not require the removal of cells. This technique, however, is still in developing stages for this type of application. Another in situ approach is to use collagen structure-specific antibodies. This, however, would require the removal of cells coupled with precise mapping of their position, which would be difficult considering movements of film in relation to plate and the elasticity of film itself.

In light of the above, the best practical approach might be a functional assay. If changes in MMP/TIMP expression do translate into matrix modification, it is a relatively slow process (otherwise there would be, for example, no differences in adipogenesis of bone marrow stromal cells on native and denatured collagen matrices). Therefore, the following experimental strategy can be suggested. Cells should be seeded on native collagen I matrix and induced to undergo adipogenic differentiation. After two or three weeks cells should be removed and the extent of adipogenesis determined. The same collagen film should be then seeded with fresh cells and experiment repeated. If a proportion of collagen matrix was modified toward denatured state, the extent of adipogenesis would increase. The same cycle might be reenacted more than once with an expected gradual increase (or plateau) of the extent of adipogenesis and its shift from a p38-independent to p38-dependent process (Mauney and Volloch, 2009a). In a parallel experimental set, following one or more cycles of adipogenesis, fresh cells should be seeded on the same film and induced to undergo osteogenesis. In this setting, if the changes in MMP/TIMP expression do translate into matrix modification, the extent of osteogenesis should decline in comparison to that seen in cells on native collagen I matrix, eventually reaching levels characteristic for cells on denatured collagen I matrix; the osteogenic process itself should shift from Hsp90-dependent to Hsp90-independent (Mauney and Volloch, 2009a; 2009b). This approach has its own challenges, for example collagen film, especially in native state, is highly sensitive to manipulations when in liquid and can easily detach; it is, however, more feasible and potentially more informative than an in situ analysis. In conclusion, the findings of the present study raise significant questions and indicate directions for further experimentation.

EXPERIMENTAL PROCEDURES.

Bone marrow stromal cells preparation.

Human bone marrow stromal cells were obtained from commercially available bone marrow aspirates (Cambrex, Walkersville, MD) from five male donors; similar trends in cell responses were observed with all donors. Cells were expanded on tissue culture plastic to passage 1 (P1) utilizing previously reported methods (Altman et al., 2002; Mauney et al., 2004; 2005). Briefly, whole bone marrow aspirates were plated at 8–10 μl aspirate/cm2 on 185 cm2 tissue culture plates and cultivated until confluency (~12–14 days) in 40 ml of expansion medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100U/ml penicillin, 100 μg/ml streptomycin, 0.1 mM nonessential amino acids, and 5 ng/ml of basic fibroblast growth factor (bFGF) (Life Technologies, Rockville, MD). Cultures were maintained in a humidified tissue culture incubator at 37°C with 5% CO2 and 95% air. Bone marrow stromal cells were selected on basis of their ability to adhere to tissue culture plastic (TCP); non-adherent hematopoietic cells were removed during medium replacement after approximately 7 days in culture. Medium was changed twice per week thereafter. P1 cells were frozen in liquid nitrogen at 5 mln/ml in 90% fetal calf serum and 10% DMSO and utilized in subsequent experiments. Viable cell recovery after thawing was over 80%. Differentiation potentials of so obtained cell populations for both osteogenic and adipogenic lineages have been verified both in vitro and in vivo (Mauney et al., 2006; 2007). These differentiation-competent cells are commonly referred to either as adult mesenmchymal stem cells, defined as adult bone marrow-derived nonhemopoietic stromal cells capable of differentiation into various lineages, or as bone marrow stromal cells, a designation adopted in the present manuscript.

Preparation of collagen matrices.

Collagen I solutions were prepared and utilized to cast collagen films using methods previously described (Volloch and Kaplan, 2002). Briefly, sterile rat tail-derived collagen type I (Roche, Indianapolis, IN; cat. #1179179) was dissolved at 0.5 mg/ml in 0.1% acetic acid and either maintained in its native conformation (NC) or denatured (DC) by incubation at 50°C for 8 hrs as previously reported (Volloch and Kaplan, 2002); at these conditions collagen was shown to be denatured and have only a small degree of fragmentation (Volloch and Kaplan, 2002).. To prepare films, native or denatured collagen solutions were added to 96 well tissue culture plates at 250 µg/cm2, dried under a laminar flow hood for 4–5 days and lyophilized for 1–2 days to enhance film stability during cell cultivation. Similar trends in cell responses were observed when rat tail collagen was compared with human placenta-derived collagen type I (Sigma-Aldrich, cat. #C7774) or when films were cast in 6 or 24 well tissue culture plates. Reported results were obtained with rat tail-derived collagen, except when human placenta-derived collagen type IV (Sigma-Aldrich, cat. #C7521) was used and compared with human collagen I mentioned above.

Osteogenic and adipogenic differentiation of bone marrow stromal cells.

Cells on native or denatured collagen I matrices were treated with either OS or AD or maintained as untreated controls as previously described (Mauney et al., 2004; 2005), and analyzed for osteogenic or adipogenic markers. Briefly, cells were plated at 5×103 cells/cm2 on native or denatured collagen I matrices in medium consisting of DMEM supplemented with 10% FCS, 100U/ml penicillin, 100 μg/ml streptomycin and 0.1 mM nonessential amino acids. 12 hours later either osteogenic (OS) or adipogenic stimulants (AD) were added and subsequently replaced with each medium change every 3–4 days. Osteogenic stimulants included 100 nM dexamethasone, 10 mM β-glycerophosphate, and 0.05 mM L-ascorbic acid-2-phosphate as previously reported (Mauney et al., 2004). Adipogenic stimulants consisted of 0.5 mM 3-isobutyl-1-methyl-xanthine, 1 μM dexamethasone, 5 μg/ml insulin, and 50 μM indomethacin as previously reported (Mauney et al., 2005). Unless otherwise stated, experiments continued for 14 days. Cultures were maintained as described above and medium exchange was carried out twice per week. Parallel control cultures were maintained similarly in the absence of stimulants.

Real time RT-PCR Analysis.

Markers of interest were analyzed by real time RT-PCR as previously described (Mauney etal., 2004; 2005). Briefly, RNA was extracted using Trizol reagent (Life Technologies), and cDNA were synthesized using High-Capacity cDNA Archive kit (ABI Biosystems) following the manufacturers instructions. Reactions were performed and monitored using an ABI Prism 7000 Sequence Detection System (Applied Biosystems). The PCR master mix was based on AmpliTaq Gold DNA polymerase (Applied Biosystems) and matrix metalloproteinase 1 (MMP1), MMP8, MMP13, tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2, TIMP3, and TIMP4 were measured. Analysis was performed using commercially available primers and probes from ABI Biosystems Assays-on-Demand™ Gene Expression kits (MMP1, cat.# Hs00233958_m1; MMP8, cat.# Hs01029055_g1; MMP13, cat.# Hs00233992_m1; TIMP1, cat.# Hs00171558_m1; TIMP2, cat.# Hs00234278_m1; TIMP3, cat.# Hs00165949_m1; TIMP4, cat.# Hs00162784_m1) following the manufacturer’s instructions; the housekeeping gene GAPDH was analyzed using primers and probes as previously described (Frank et al., 2002). Data analysis was performed using the ABI Prism 7000 Sequence Detection Systems version 1.0 software (Applied Biosystems, Foster City, CA). For each cDNA sample, the Ct value was defined as the cycle number at which the fluorescence intensity of each target gene was amplified within the linear range of the reaction. Relative expression levels for each gene of interest were calculated by normalizing the quantified gene of interest transcript level (Ct) to the GAPDH transcript level (Ct) as described previously (2∆Ct formula, Perkin Elmer User Bulletin #2).

Statistical Analysis.

All measurements were collected with N=3–5 independent samples per data point and expressed as means ± standard deviations. Data were analyzed with Microsoft Excel software utilizing a Student’s two tailed t-test assuming equal levels of variance. Statistically significant values were defined as p<0.05.

AKNOWLEDGEMENTS.

This research was supported by NIH, NIBIB through P41-EB002520 grant.

Abbreviations.

TCP

tissue culture plastic

NC I

native collagen I matrix

DC I

denatured collagen I matrix

MMP

matrix metalloproteinase

TIMP

tissue inhibitor of metalloproteinase

OS

osteogenic stimuli

AD

adipogenic stimuli

Footnotes

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