Patent Application: US-45433099-A

Abstract:
an isolated and essentially purified cell matrix plaque of initial bone formation comprising of α v β 3 integrin and rapid assays using such cell matrix plaques to measure potentials of factors , regimens or tissues for stimulation and / or inhibition of bone formation .

Description:
the contents of each of the references cited herein are incorporated by reference . in accordance with the present invention it has been discovered that primary bone cells adhering to vitronectin and type i collagen , demonstrate a unique form of cell - matrix interaction and a major upregulation of the α v β 3 integrin following mechanical strain . the α v β 3 integrin localized to plaque - like sites of cell / substratum interaction along with osteopontin which was expressed as a macromoieculc , possibly cross - linked by tissue transglutaminase which was also localized to the plaques . such bone plaque sites , not previously isolated or essentially purified , are herein shown to be key indicators of bone formation and mineralization at the sub - cellular level . such an indicator can be used , as described in detail below , to evaluate a candidate medium , or regimen ( for example , employing an environmental stimulus such as mechanical strain alone or in combination with other factors ) for its physiological or therapeutic potential as a stimulator or inhibitor of osteogenesis . in general , bone cells transduce responses through the process of mechanotransduction . however , the mechanisms of mechanotransduction have been essentially unknown . to address this issue , applicants perfoined studies in primary human bone cells at three defined stages of differentiation : the osteoblasts , preosteoblasts and osteoprogenitors grown on collagen / vitronectin coated supports . applicants discovered that a mechanical strain regimen of 70 , 000 μe at 0 . 05 hz stimulated the expression of the α v β 3 integrin in irregular plaque - like areas of integrin - extracellular matrix protein interaction . the plaque - like areas were distinct from another type of cell matrix interaction expressed in ostcoblastic cells represented by fine and regularly spaced focal adhesions ( fa ) that expressed α v and β 1 integrins and colocalized with vinculin and other fa markers . the unique sites of cell - matrix interaction containing α v β 3 colocalized with focal adhesion kinase but not with other fa markers . mechanical strain increased the number and size of the plaques defined by surface expression of α v β 3 strain also increased secretion of matrix proteins into the plaque - like areas colocalizing with the α v β 3 integrin there . osteopontin was secreted as a cross - linked macromolecular complex , likely through the action of tissue transglutaminase which was also found in the plaques of the α v β 3 integrin - cell matrix interaction . in addition , mechanical strain increased mineralization of the extracellular matrix by osteoblasts . since the plaque - like areas of cell - matrix organization exhibit macromolecular assembly and mineralization , they represent subcellular indicators of bone formation , and α v β 3 integrin activation represents one mechanism by which mechanical strain stimulates bone formation . utilizing this discovery , a method of measuring new bone formation using a rapid in vitro assay has been developed . the method involves extracting bone marrow stromal cells from bone marrow cavities and growing them to semi - confluence ; exposing the cells to a candidate medium or regimen for osteogenesis or inhibition of osteogenesis ; determining the mineralization / development of bone plaque ; and , optionally , comparing the mineralization / development of bone plaque generated using the candidate medium or regimen with the mineralization / development of bone plaque using a preselected control such as bone morphogenetic factor - 7 ( bmp - 7 ), also known as osteogenic factor - 1 ( op - 1 ). alternatively , the assay can be used as a diagnostic screen for healthy and / or deficient bone formation , making the variable the cells being observed for production of mineralization / bone plaque formation . using the techniques detailed below , a rapid , relatively high throughput screen for large - scale evaluation of bone formation has been devised . for example , using techniques known in the art , immunocytochemical / fluorescent - activated cell sorting can be employed , in which the area of plaque growth is examined under the microscope , photographed and digitized for precise measurements . examples of such techniques for assaying for the presence of the bone plaques isolated by applicants are detailed below . the following examples illustrate the invention , but are not to be taken as limiting the various aspects of the invention as illustrated . the anti - α v β 3 integrin and anti - osteopontin antibodies were kindly provided by dr . david griggs ( monsanto ). the anti - α v β 3 integrin complex monoclonal antibody ( p112 ) behaves almost identically to the lm609 antibody ( 31 ), it appears to bind the same epitope and operate through the same mechanism of action ( personal communication ) 1 . the anti - α v integrin subunit ( l230 ) and anti - β 6 integrin subunit ( a10 ) antibodies were generous gifts from drs . scott blystone and eric brown ( washington university ) and drs . dean sheppard and john chen ( ucsf ), respectively . the anti - tissue transglutaminase antibody was a kind gift from dr . daniel aeschlimann ( university of wisconsin ). the anti - β 1 , - β 5 and - α 2 integrin antibodies were obtained from chemicon international inc . the anti - fak ( pp125 fak ) antibody , anti - goat cy3 - conjugated igg and fetal calf serum were purchased from sigma . the cy3 - and cy2 - conjugated affinipure goat anti - mouse and anti - rabbit igg were from jackson immunochemicals . the protein a - horscradish peroxidase conjugate was obtained from transduction research . complete ™ protease inhibitor cocktail was purchased from boehringer mannheim . flex i and flex ii cell culture dishes were obtained from flexcell corporation . the omeacell ™ 10 ml disposable membrane cells were from pall filtron . immobilon transfer membrane for western blotting applications was purchased from millipore . ponceau s solution was obtained from sigma . all other chemicals utilized were reagent grade . the primary human bone marrow stromal cell cultures were prepared as described previously . briefly , bone marrow stromal cells were collected from bone marrow cavities , treated with 1 μg / ml dnase and 10 units / ml heparin in dme / f - 12 medium , then pclleted , resuspended in α - mem supplemented with 10 % heat - inactivated fcs and separated by ficoll / hypaque density gradient . the interphase obtained by centrifugation was collected and resuspended in α - mem supplemented with 10 % heat - inactivated fcs , then grown to confluency . at that time , bone marrow stromal cells were plated on collagen type i - coated flex i and flex ii cell culture dishes at a density of 40 , 000 cells / well and maintained in culture for 7 - 14 days , changing medium every 3 - 4 days . these cell cultures are 1 personal communication with dr . jeffrey w . smith at program on cell adhesion and extracellular matrix . the burnham institute 10901 north toltey pines road , la jolla , calif . 92037 . referred to as osteoprogenitors ( 3 ). these osteoprogenitor cell cultures are characterized by low , but detectable , alkaline phosphatase activity of 400 nmole pnpp / min ./ μg protein ; osteopontin expression ; absence of osteocalcin expression ; and mineralization of the extracellular matrix after 20 days of culture in osteogenic media . bone marrow stromal cells grown to semi - confluence and then exposed to osteogenic medium containing 10 mm of β - glycerophosphate , 50 μg / ml ascorbic acid and 40 ng / ml of osteogenic protein op - 1 ( 32 ) also referred to as bone morphogenetic protein - 7 ( bmp - 7 ), for 48 hours were defined as preosteoblasts . these prcosteoblast cultures are characterized by alkaline phosphatase activity of 80 , 000 nmole pnpp / min ./ μg protein , osteopontin expression , absence of osteocalcin expression , and mineralization after 14 days in osteogenic media . for the purposes of this paper , bone marrow stromal cells exposed to op - 1 - containing osteogenic medium for 14 days were defined as “ mineralizing preosteoblasts ” or osteoblasts . the osteoblast cultures are characterized by alkaline phosphatase activity of 60 , 000 nmole pnpp / min ./ μg protein , osteopontin expression , osteocalcin expression and mineralization of the extracellular matrix . near - confluent human preosteoblasts , osteoprogenitors , and osteoblasts were placed on flexcell strain apparatus that vacuum - stretches the flexible silicone bottoms of flex culture dishes . applicants applied cyclic strain at three cycles / minute ( 10 seconds on / 10 seconds off ; frequency of 0 . 05 hz ) for 48 hours . one characteristic of the flexcell strain apparatus is the nonuniformity of the strain applied across the surface of the cell culture well . the magnitude of strain delivered in these experiments ranged from approximately 120 , 000 microstrain ( μe ) at the edge of the plate ( 12 % maximal displacement ) to 0 μe at the center of each cell culture well . strain applied to cells in analyzed areas ( approximately 2 - 5 mm from the edge of the well ) was estimated at 70 , 000 μe using the strain curve described by gilbert and colleagues ( 35 ). the strain parameters were as follows : strain was applied in the form of a square wave with an amplitude of 70 , 000 μe with approximately 0 . 5 second rise time associated with each cycle . since the observations regarding strained cells were made approximately 2 - 5 mm from the edge of each well , applicants presume that the cells portrayed as “ strained ” in the data contained herein received about 7 % stretch . each strained 6 - well flex i plate was accompanied by a non - strained 6 - well flex ii control . immunocytochemical analysis essentially was performed as previously described ( 34 ). briefly , primary human progenitors and preosteoblasts were rinsed with phosphate - buffered saline ( pbs ), and fixed for 30 minutes at room temperature ( rt ) with 4 % ( v / v ) paraformaldehyde in pbs . cells were rinsed twice with pbs , incubated in 50 mm nh 4 cl solution in pbs for 15 minutes to quench the fixative , and then permeabilized with 0 . 2 % triton x - 100 in pbs for 15 minutes at rt . potential sites for nonspecific antibody binding were blocked by a 30 minute incubation with 2 % bovine serum albumin ( bsa ), 0 . 2 % triton x - 100 and 0 . 04 % sodium azide in pbs at rt . each immunological co - localization study involved two primary antibodies raised in different species against two distinct antigens . primary antibodies were typically diluted 1 : 100 ( or used at 1 - 10 μg / ml ) in 2 % bsa , 0 . 2 % triton x - 100 ,), 04 % sodium azide in pbs and incubated with the sample for 1 hour at rt . this was followed by four 15 minute washes with 0 . 02 % triton x - 100 in pbs and a 1 hour incubation with an appropriate combination of a secondary indocarbocyanine ( cy3 )- and cy2 cyanine ( cy2 )- conjugated goat anti - mouse , goat anti - rabbit or rabbit anti - goat igg ( 1 : 100 dilution in 2 % bsa , 0 . 2 % triton x - 100 and 0 . 04 % sodium azide in pbs ). the cells were then washed as before with pbs containing 0 . 2 % triton x - 100 . the silicone cell culture supports were then excised and mounted on glass microscope slides with aqua polymount . the results were analyzed either on zeiss fluorescent microscope ( axioscope ) using a 63 × oil immersion lens or on a zeiss laser confocal microscope using a 63 × lens . the magnification of the optical system was 630 . following the enzymatic dissociation , cells were resuspended in serum - free medium and pelleted at 4 ° c . the pellet was then resuspended in serum - free medium and kept on ice . equal numbers of strained and of non - strained control cells , ranging from 1 - 2 × 10 6 cells and resuspended in 1 ml of serum - free medium were pipetted into polypropylene test tubes . cells tested for the cell surface integrin expression were incubated with either 10 μg / ml ( anti - α v β 3 antibody ) or with 1 : 100 antibody dilution ( anti - β 1 and anti - β 5 integrin subunit antibodies ) or with 1 : 10 dilution of anti - β 6 integrin subunit antibody for 1 hour on ice . the parallel negative control tubes were incubated with an irrelevant mouse antibody under identical conditions . following the incubation with a primary antibody , cells were washed extensively in serum - free medium and incubated with the appropriate fluorescein isothiocyanate ( fitc )- conjugated secondary goat anti - mouse or goat anti - rabbit igg at 20 μg / ml for 1 hour on ice . cells were then washed twice in serum - free medium and finally resuspended in 1 ml of serum - free medium . the fluorescence intensity was analyzed using a becton dickinson facs scan flow cytometer . primary human cell cultures were mechanically strained on the flexcell strain apparatus for 48 hours immediately following a replacement of cell culture medium . after the strain , medium was collected and pooled separately from the strained and non - strained wells , and concentrated approximately 6 fold using omegacell ™ 10 ml disposable membrane cells . the protein content in the concentrated medium was determined using a standard bio - red method . medium samples from strained and control cells with equal total protein content were loaded onto a 6 % sds - page gel . immediately following the electrophoresis , proteins were transferred onto an immobilon transfer membrane . the efficiency of protein transfer was judged after a 5 minute membrane incubation in ponceau s solution . nonspecific antibody binding sites on immobilon membranes were then blocked by immersing the membrane in a 5 % milk and 0 . 1 % tween 20 in pbs solution overnight at 4 ° c . osteopontin was detected by incubating the membranes for 2 hours with anti - osteopontin rabbit polyclonal antibody diluted 1 : 1000 in 0 . 1 % tween 20 in pbs at rt , followed by washing the mcmbrane once for 15 minutes and three times for 5 minutes with 0 . 1 % tween 20 in pbs . applicants then incubated the membrane with protein a - horseradish peroxidase conjugate diluted 1 : 2000 in 0 . 1 % tween 20 in pbs for 1 hour at rt . the final washes included one 15 minute wash and four 5 minute washes in 0 . 1 % tween 20 in pbs . the antibody binding was detected by exposure to enhanced chemiluminescence using 10 mls of the ecl reagents mix for 60 seconds and then visualized on autoradiography film ( hyperfilm - ecl ). human bone marrow stromal cells were plated in flex i and flex ii type i collagen - coated cell culture dishes at 40 , 000 cells / well and maintained in α - mem supplemented with 10 % fcs in culture until confluent . cells were then exposed to osteogenic medium containing α - mem supplemented with 10 % fcs , 10 mm of β - glycerophosphate , 50 mg / ml of ascorbic acid and 40 ng / ml of osteogenic protein op - 1 for 20 days , replacing medium every 3 - 4 days . for the purposes of this paper , bone marrow stromal cells exposed to these conditions were defined as osteoblasts . following the incubation in osteogenic medium , cells plated in flex i dishes were mechanically strained on the flexcell strain apparatus for 48 hours , and then both strained cells and their non - strained controls were examined for the formation of mineralized nodules . applicants applied a slight modification of the alizarin red - s histochemical staining described previously by stanford and colleagues ( 35 ). briefly , cells were washed 3 times with pbs and then fixed in 70 % ice - cold ethanol for i hour at 4 ° c . following 3 water rinses , cells were stained with 0 . 5 % ( w / v ) alizarin red - s for 10 minutes at room temperature , and then extensively washed with water and pbs . alternatively , matrix mineralization was examined via calcein staining . osteoblasts differentiated in osteogenic medium were washed 3 times with pbs and then fixed in 4 % paraformaldehyde for 30 minutes at room temperature . following 3 pbs rinses and a 30 minute incubation with 2 % bsa / pbs , cells were stained with 1 mm calcein in 2 % bsa / pbs for 1 hour , and then extensively washed with pbs . the present studies were carried out using primary human bone marrow stromal cell cultures at distinct functional stages of osteoblast differentiation : progenitors , preosteoblasts and osteoblasts ( mineralizing preosteoblasts ). the human bone marrow stromal cells cultured under conditions described in methods , were defined as the osteoprogenitors . in these heterogenous cell cultures , the cells expressing osteopontin , low alkaline phosphatase levels and responding to mechanical strain with an increase in the α v β 3 integrin level were considered to be the osteoprogenitors . preosteoblasts were defined as cells expressing high alkaline phosphatase levels , low or absent osteocalcin and absence of matrix mineralization . osteoblasts were defined as cells expressing high alkaline phosphatase and osteocalcin levels that were mineralizing their extracellular matrix . these cells were produced by extending the culture of the preosteoblasts . mechanical strain enhances the expression of the α v β 3 integrin on the cell surface of osteoprogenitors and preosteoblasts in order to examine whether mechanical strain has a qualitative and / or quantitative effect on the localization and expression level of integrins , near - confluent human preosteoblasts and osteoprogenitors were strained on flexcell strain apparatus , as described in methods . the applied mechanical strain was the nonuniform strain applicants have previously described ( 15 ) using this apparatus , using the immunocytochemical approach to immunostain bone cells with an anti - human α v β 3 monoclonal antibody , applicants determined that the cells in human osteoblast lineage utilize α v β 3 as a cell surface integrin in our experimental conditions , and that osteoprogenitors ( fig1 a and 1 b ), preosteoblasts ( fig2 a and 2b ) and osteoblasts ( not shown ) expross the α v β 3 integrin . in the presence of triton x - 100 , which leads to cell permeabilization and thus , allows examination of the distribution of intracellular as well as cell surface molecules , the uvp , integrin , as detected by an anti - human α v β 3 antibody , was localized along actin fibers and in small , granular structures adjacent to actin fibers ( fig1 a , ib , 2 a and 2 b ). mechanical strain increased the expression of the α v β 3 integrin in progenitor cells ( fig1 b ), as compared to non - strained controls ( fig1 a ). specifically , the number of the α v β 3 - containing granular structures and the intensity of fluorescence were noticeably increased after 48 hours of cyclic strain . the effect of mechanical strain on the α v β 3 integrin expression was particularly well appreciated when cells were stained with tihe anti - α v β 3 , integrin antibody in the absence of triton x - 100 ( fig1 c and 1 d ). such a protocol dramatically diminished cell pertneabilization defined by fluorescent 70 , 000 mw dextran staining ( data not shown ), and it produced improved focus for the examination of the integrin localization concentrated on the plasma membrane at sites of matrix interaction . applicants demonstrated that the α v β 3 , integrin on the cell surface was expressed in large , plaque - like sites of cell - matrix interaction , and it was not expressed in focal adhesions as discussed below . upon exposure of progenitor and preosteoblast cells to 48 hours of mechanical strain , the number and size of the plaque - like sites increased significantly , and these complexes assumed an organized , directional pattern ( fig1 d and fig2 d ). in preosteoblasts , the effect of mechanical strain was in addition to a stimulation by op - 1 of the number of the plaque - like cell / matrix areas of the α v β 3 expression ( compare fig1 c with fig2 c ). to better characterize the large , plaque - like sites of matrix organization , applicants examined colocalization of an anti - α v β 3 integrin antibody with antibodies directed against focal adhesion kinase ( fak ), talin and vinculin in non - perneabilized cells . as shown in fig3 fak ( fig3 a ) and α v β 3 integrin ( fig3 b ) localized to the blotchy plaques on the cell surface of human bonc cells . each plaque containing the α v β 3 integrin also contained fak ( fig3 c ), but the precise pattern of expression of each protein in a single plaque was different . microscopic analysis of the cell surface plaques revealed abundant α v β 3 integrin present throughout the surface of the plaques ( fig3 b ), whereas the expression of fak was confined to the boundaries of each blotchy plaque ( see arrows in fig3 c ). vinculin and talin were not localized to the plaque - like areas of cell surface α v β 3 integrin expression ( data not shown ), but they were found in sites of cell - matrix interaction which were defined as osteoblastic focal adhesion complexes ( see fig9 a ). flow cytometry confirmned thiat mechanical strain increased the cell surface expression of the α v β 3 integrin in progenitors , preosteoblasts , and osteohiasts the enhancement of the α v β 3 integrin expression on the cell surface of ostcoprogenitors , preosteoblasts and osteoblasts , as detected by immunocytochemical techniques , was confirmed by flow cytometric analysis . fig4 shows two representative plots of flow cytometric analysis of the α v β 3 integrin expression . in the right panel , applicants demonstrated a shift in the fluorescence from non - strained ( b ′) to mechanically strained preosteob lasts ( d ′) shown in the overlay panel , which indicates an increase in the preosteoblast cell surface expression of the α v β 3 with strain . as shown in the left panel of fig4 a similar shift in fluorescence was detected after osteoprogenitors were mechanically strained , hence showing that strain also increased the cell surface expression of the α v β 3 in these cells and thus corroborating our immunocytochemical results . as demonstrated by flow cytometry in three to five experiments / cell type using , an anti - human α v β 3 integrin antibody to react with single cell suspensions of osteob last lineage cells , the mean fluorescence of the integrin on the cell surface of osteoprogenitors was increased significantly from 49 . 3 ± 2 . 7 in non - strained cells to 78 . 3 ± 6 . 7 in mechanically strained cells ( table 1 ). similarly , in preosteoblasts , applicants detected a remarkable mean fluorescence chance from 45 . 3 ± 8 . 5 in control cells to 90 . 0 ± 6 . 6 in strained cells . further differentiation of preosteoblasts in op - 1 - containing osteogenic medium to cells expressing osteocalcin and mineralizing the extracellular matrix ( osteoblasts ) lead to an increased fluorescence intensity due to increased α v β 3 integrin expression in strained cells ( 59 . 8 ± 10 . 6 ) as compared to non - strained cells ( 43 . 2 ± 7 . 0 ). table 1 . changes in mean fluorescence as a measure of the α v β 3 integrin stimulation by mechanical strain . the flow cytometric analysis was performed in single cell suspensions of cells mechanically strained for 48 hours as well as in their non - strained controls . osteoprogenitors , preosteoblasts and mineralizing preosteoblasts were incubated with a mouse monoclonal antibody directed against anti - human α v β 3 integrin , followed by an incubation with a secondary fitc - conjugated anti - mouse antibody , as described in methods . each data point is a mean of at least 3 experiments . *, p ≦ 0 . 01 compared to cells not strained . table 2 . summary of specific integrin localization sites in bone cells . in addition to their expression in the cytosol , the α v β 1 integrin is apparently present in focal adhesions , the α v β 3 integrin is evident in blotchy cell - matrix interactions , and the α v β 5 integrin is found in cellular projections . using immunocytochemical techniques , applicants demonstrated that human bone cells express ostcopontin , a ligand for the α v β 3 integrin ( fig5 a ). in addition , applicants observed a high degree of colocalization ( fig5 c ) between osteopontin ( fig5 a ) and the α v β 3 integrin ( fig5 b ) in non - permeabilized bone cells . osteopontin co - localized with the α v β 3 integrin in the large plaque - like areas of cell matrix interaction . this finding coupled with the elevated level of α v β 3 integrin found in mechanically loaded cells suggests that in response to mechanical strain , osteopontin may serve as an integrin ligand . applicants also addressed the issue whether osteopontin levels , both intracellular and secreted into cell culture media , were affected by mechanical strain . by performing western blot analyses using anti - human osteopontin antibodies , applicants detected several forms of osteopontin that were secreted into bone cell culture medium ( fig6 a ). the predominant osteopontin band had a molecular size of approximately 168 kda and may represent either a cross - linked osteopontin - matrix protein macro - molecule or an osteopontin homomultimer ( fig6 a ). in cell lysates , the osteopontin bands included predominant 75 kda and a 78 kda bands ( fig6 b ). the 75 kda band has been frequently seen in several cell types as a mature opn ( 36 , 37 ). fig6 a is representative of five experiments demonstrating stimulation of osteopontin secretion into the medium of mechanically strained cells in the osteoblast differentiation program . the strain over control stimulation index determined by scanning densitometry of the 168 kda bands was more than 2 . 0 in osteoprogenitors and approximately 1 . 0 for preosteoblasts . the level of osteopontin secretion in non - strained osteoprogenitors in the presented blots seems lower than that in non - strained preosteoblasts exclusively due to technical reasons ( as not to overexpose the band representing strained progenitors ). thus , the effect of strain was apparent only in the osteoprogenitors . in contrast to the findings in cell culture mediums , western blotting of cell lysates revealed predominant osteopontin bands of 75 and 78 kda and no differences in osteopontin levels between strained and non - strained bone cells , regardless of the cell differentiation stage ( fig6 b ). these data suggest that the effect of strain includes stimulation of secretion of the 168 kda molecule such that it does not accumulate in the cell . to further analyze the basis of the large osteopontin molecular sizes , applicants analyzed whether mechanical strain affected osteoblastic expression of tissue transglutaminase , which cross - links proteins into homo and heteromultimers through the formation of γ - glutamyl - σ - lysine cross - links between substrate proteins . bone osteopontin and osteonectin are tissue transglutaminase substrates ( 38 - 40 ). based on the immunocytochemical analysis , tissue transglutaminase in osteoprogenitors is expressed throughout the cytoplasm in punctate subcellular structures , as shown in detergent - permeabilized cells ( fig7 a ). interestingly , in cells fixed in the absence of triton x - 100 , tissue transglutaminase was also found in large , plaque - like sites ( fig7 b and fig7 c ) that closely resemble the cell surface α v β 3 integrin plaques ( fig7 d ). moreover , the immunocytochemical approach demonstrated a colocalization of tissue transglutaminase and α v β 3 integrin in well defined plaques in osteoprogenitors ( fig7 e ). since osteoblasts are known to produce , deposit and mineralize the bone matrix , applicants examined the influence of mechanical strain on the fonnation of mineralized nodules . alizarin red s histochemical staining of osteoblasts derived from bone marrow stromal cells demonstrated that although mineralization was detected in both strained ( fig8 b , 8 d , 8 f ) and non - strained cultures ( fig8 a , 8 c , 8 e ), the intensity of nodules produced by mechanically strained cells was dramatically enhanced at least twofold , as compared with controls . these osteoblastic cells were maintained in osteogenic medium containing ascorbate , β - glycerophosphate and op - 1 for 18 days prior to 48 hours of mechanical strain ( days 18 - 20 ). furthermore , on the basis of colocalization of another mineralization marker , calcein with the anti - α v β 3 integrin antibody ( fig8 g ) in osteoblasts derived from bone marrow stromal cells , applicants observed that the α v β 3 integrin plaques colocalized with calcein - labeled mineralization nodules . moreover , in mineralizing cultures the α v β 3 plaques assumed a nodular shape ( fig8 g ) indicated by their expansion in the z axis ( vertically ), unlike their level , flatter shape observed in non - mineralizing cells ( compare with fig1 d and fig2 d ). application of a monoclonal anti - human α v antibody in our immunocolocalization protocol followed by confocal analysis demonstrated that the α v subunit epitopc detected by monoclonal antibody l230 was present in the plaque - like areas of cell - matrix interaction discussed above , but it was predominantly found in fine , regularly and densely spaced structures morphologically similar to focal adhesions described in other cell types ( 41 - 43 ). these focal adhesions were observed in each of the osteoblast differentiation stages examined ( fig9 a ). besides their characteristic shape , the structures containing the α v subunit were defined as focal adhesions on the basis of their co - localization with known focal adhesion markers vinculin and talin . interestingly , an identical pattern of α v localization was obtained in both permeabilized and non - permeabilized cells , thus suggesting that this α v subunit epitope is primarily exposed during cell surface / substratum interaction . moreover , our results indicate a high level of focal adhesion expression in resting osteoblastic cell cultures obscuring the effects of mechanical strain on the level of α v integrin expression in the focal adhesion of human bone cells . studies showing a cell surface colocalization of the α v subunit ( l230 antibody ) with osteopontin in plaque - like structures provided proof that this α v subunit was also found in the α v β 3 integrin plaques , since osteopontin colocalized closely with the α v β 3 in those plaques ( fig5 c ). to identity the β subunit of the α v β heterodimer detected in the focal adhesions of our bone cell cultures ( fig9 a ), applicants immunologically colocalized the anti - α v antibody with each of the potential α v β candidate heterodimers of the osteoblast . the anti - β 5 and the anti - β 6 integrin subunit antibodies revealed a cytoplasmic distribution of these integrins distinct from the α v subunit — containing fine focal adhesions , as shown in fig9 b and 9d , respectively . interestingly , the colocalization of the β 5 integrin subunit and the α v β 3 integrin revealed that our human bone cell cultures were characterized by long cellular projections containing both the α 5 subunit and the α v β 3 integrin ( fig9 b and fig9 c ). these cellular projections abut other cell bodies at their end , thus , suggesting the involvement of the α v β 5 integrin in cell - cell communication ( see arrows in fig9 c ). as demonstrated by flow cytometry , mechanical strain increases the expression of the α 5 integrin subunit by approximately 25 % on the surface of preosteoblasts , but not on the osteoprogenitor plasma membrane . in contrast to the β 5 subunit , the β 6 integrin subunit expression level , as measured by flow cytometry , was not affected at all by mechanical strain . the immunolocalization studies demonstrated that the primarily cytosolic staining pattern of the β 6 integrin subunit was different from that of fine focal adhesions containing the α v subunit ( fig9 d ). this is the first demonstration of the α v β 6 integrin in osteoblasts . finally , applicants compared the distribution of the β 1 integrin subunit with the staining pattern of fine focal adhesions ( fig1 ). as shown in fig1 b , the β 1 integrin subunit was expressed in a rather diffuse staining pattern , as expected from the large number of osteoblastic b 1 integrins . however , the immunochemical analysis indicated that a significant portion of the β 1 integrin subunit colocalized closely with the α v integrin subunit , as shown in fig1 c . careful microscopic analysis of a large number of samples demonstrated that the α v and β 1 integrin subunits are present in the focal adhesions of osteoprogenitors , preosteoblasts and osteoblasts . applicants have demonstrated strain - induced osteoblastic mineralization in vitro . it provides several important new clues as to the molecular basis of the cellular events that lead to mechanical strain induced bone formation . the general conclusion from this investigation is that in response to mechanical strain , the α v β 3 integrin of the osteoblast lineage is upregulated and that it may participate in the organization of the osteopontin - containing extracellular matrix in unique plaque - like areas of cell - matrix interaction . applicants have presented evidence that primary human cells of the osteoblast lineage express at least two types of cell - matrix interactions . one type represents fine and densely spaced cell surface structures that colocalize with vinculin and represent osteoblastic focal adhesions . a second type of relatively large , plaque - like areas of integrin / extracellular matrix protein interaction colocalizes with focal adhesion kinase ( fak ) but not with vinculin . applicants have demonstrated that the α v β 3 integrin specifically colocalizes to the second type of cell - matrix interaction sites and that its expression was further stimulated by mechanical strain . upon mechanical strain , both types of cell - matrix interactions assumed an organized , directional pattern ( fig1 d and fig9 a ). in agreement with earlier reports by buckley and colleagues ( 49 ), this new orientation was perpendicular to the vectors of strain . fak has been previously shown to colocalize with several components of cellular focal adhesions , such as tensin , vinculin and talin ( 45 ). furthermore , these plaque - like areas are specific to the osteoblast lineage in that applicants have failed to demonstrate them in other cell types ( i . e . human melanoma cells ), and cells differentiating from hematopoetic progenitors ( i . e . macrophages and osteoclasts ). applicants also demonstrated that the α v β 5 integrin once considered a dominant osteoblast adhesion molecule is largely cytoplasmic in primary human bone cells , and that it is also expressed along extended cellular projections that contact distant osteoblast cell bodies . applicants believe that these cellular projections participate in cell — cell communication . applicants have also identified at least one matrix protein , osteopontin as a ligand for the α v β 3 integrin contained in the plaque - like cell - matrix organization sites . osteopontin in our studies in the osteoblast may act as an autocrine matrix protein , secreted in the focal adhesion in response to regulatory stimuli and important in the proliferation and differentiation of cells during the osteoblast program . based on previous reports , osteopontin is present in human milk and melanoma cclls , as well as in rat osteosarcoma cells as an approximately 75 kda protein , and in osteoclasts as a protein of 55 - 65 kda ( 36 , 37 , 47 - 48 ). applicants have demonstrated the presence of several osteopontin bands of different molecular sizes in the cell culture medium of osteoprogenitors and osteoblasts . the osteopontin band of highest intensity was detected at approximately 168 kda , thus representing a cross - linked macromolecular complex of either osteopontin and other matrix proteins or of osteopontin oligomers . applicants have discovered that in human cells of the osteoblast lineage , osteopontin colocalizes with the α v β 3 integrin in plaque - like sites of cell - matrix interaction . moreover , the α v β 3 integrin was found in the mineralization nodules in osteoblasts derived from bone marrow stromal cells , as determined by anti - α v β 3 antibody / calcein staining . applicants also demonstrated that mechanical strain leads to an increased formation of mineralized nodules ( fig9 ). osteopontin and other extracellular proteins ( including fibronectin and bone sialoprotein ) were previously shown to initiate and / or direct the spatial deposition of mineral in the extracellular matrix ( 49 - 51 ). therefore , applicants &# 39 ; results indicate that mechanical strain - enhanced interaction between osteopontin and α v β 3 integrin is indicated to lead to enhanced matrix mineralization . osteopontin , a known substrate of tissue transglutaminase , may be covalently cross - linked by this enzyme ( 52 ). tissue transglutaminases are a widely distributed class of enzymes found intra - as well as extracellularly ( 53 ). tissue transglutaminase participates in the assembly and organization of extracellular matrix via its activity as an externalized enzyme , and thus has an impact on cell spreading and adhesion ( 54 ). tissue transglutaminase catalyzes a calcium - dependent formation of a specific isopeptide bond , therefore promoting intra - and / or intermolecular cross - linking in proteins ( 55 ). tissue transglutaminase is a highly selective enzyme , with only a few native proteins identified as its substrates ( 53 , 56 ). the high specificity of this enzyme renders the tissue transglutaminase - catalyzed cross - linking of proteins , including ostcopontin , physiologically significant . our study indicates that the α v β 3 integrin colocalizes with tissue transglutaminase in bone cells ( fig7 ), and suggests that osteopontin colocalizes with tissue transglutaminase in the large , blotchy sites of matrix organization , thus suggesting that other matrix proteins may be cross - linked with osteopontin in these complexes . finally , burger and veldhujzen have reported that production of extracellular matrix decreases at high strain levels , while cellular proliferation increases ( 57 ). applicants observed the enhancement of mineralization of extracellular matrix in osteoblasts grown in osteogenic medium for 20 days , resulting from an average mechanical strain level of approximately 70 , 000 μe , as determined on the basis of the strain curve described by gilbert et al ( 33 ). thus , applicants suggest that the majority of the cells in our studies were exposed to a strain level at which no inhibition of bone matrix production occurred . since applicants were limited to a non - uniform strain distribution by our apparatus , applicants were unable to determine the applied strain that lead to increased mineralization . our data indicates that the sequential process of matrix production , deposition and mineralization was enhanced by the level of mechanical strain employed . in view of the above , it will be seen that the several objects of the invention are achieved . as various changes could be made in the above compositions and processes without departing from the scope of the invention , it is intended that all matter contained in the above description be interpreted as illustrative and not in a limiting sense . 1 . wozney , j . j ., rosen , v ., celeste , a . j ., mitsock , l . m ., whiters , m . j ., kriz , r . w ., hewick , r . m ., and wang , e . a . 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