microvessel endothelial cells and pericytes increase proliferation and repress osteoblast phenotypic...

Jortmnl of Orthopaedic Research 13553-561 The Journal of Bone and Joint Surgery. Inc 0 1995 Orthopaedic Research Socicty

Microvessel Endothelial Cells and Pericytes Increase Proliferation and Repress Osteoblast Phenotypic Markers

in Rat Calvarial Bone Cell Cultures

A. R. Jones, C. C. Clark, and C. T. Brighton

Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

Summary: To investigate the influence of microvessel cells on osteoblasts, we exposed osteoblast-enriched cultures of rat calvarial cells to cultured endothelial cells and pericytes using feeder-layer co-cultures, co-culture dish inserts, and conditioned media experiments. When co-cultured with growth-arrested feeder- layers of endothelial cells or pericytes for 10 days, bone cell cultures showed an increase in cell number and reduction in alkaline phosphatase activity. The response of bone cells to endothelial cells was nearly twice their response to pericytes. A similar response was demonstrated by exposure to microvessel cells in co-culture dish inserts and by exposure to media conditioned by microvessel cells. In long-term cultures of bone cells, the levels of osteocalcin and the number of mineralized nodules both were reduced by exposure to media conditioned by the microvessel cells. Transient exposure to conditioned media from the microvessel cell cultures for 3 days, during the period from initial plating to cell confluence, produced nearly the same effect on the cultures of bone cells as did continuous exposure to these conditioned media. The influence of isolated microvessel cells on osteoblast-enriched calvarial cells was found to be primarily mitogenic, mediated by soluble factors, independent of cell contact, and a cause of prolonged reduction in the expression of early and late markers of the osteoblast phenotype.

Angiogenesis long has been recognized as an essential part of osteogenesis. Microvessel ingrowth oc- curs concurrent with the onset of mineralization in fracture callus (31,48), developing bones of the limb (16,34) and growth plate (4,15), and at extraskeletal sites of endochondral ossification (35). Beyond their role of maintaining a vascular conduit, microvessel cells are suspected of having a direct relationship to bone formation because of their proximity to osteoblasts and osteoprogenitor cells at sites of new bone formation. The appearance of microvessel cells during angiogenesis seems to accelerate bone formation early, before microvessel flow appears to have been established (47), and in an environment known to be hypoxic (2,19). In our laboratory’s studies of bone induction either by electricity (3) or after fracture ( 5 ) , microvessels appear to contribute cells to the pool of large, polymorphic mesenchymal cells that align along rows of osteoblasts. These studies have suggested that microvessel-derived cells participate in early pro- cesses of bone repair, either as osteoprogenitor cells

Received October 21,1993; accepted September 15,1994. Address correspondence and reprint requests to A. R. Jones

at Division of Orthopaedic Surgery, Burnett-Womack 242, CB# 7055, University of North Carolina, Chapel Hill, NC 27599-7055, U.S.A.

or as regulatory cells that promote osteogenesis. Microvessels largely are composed of two types of

cells-endothelial cells and pericytes (for reviews, see references 21 and 45). Endothelial cells maintain a nonthrombogenic microvessel lumen and modu- late the delivery and activity of blood-borne agents, whereas pericytes are believed to support the struc- tural integrity of the microvessel, regulate microvessel flow, and control the growth of microvessel cells during angiogenesis.

Several studies have supported the hypothesis that osteoprogenitor cells are derived from endothelial cells (7,24,29,48) or pericytes (6,14,26,30,37); however, none has been conclusive. Many have implicated microvessel cells as putative osteoprogenitor cells because of their presence at sites of increasing numbers of osteoblasts.

Whether microvessel cells promote the growth or the differentiation of osteoblastic cells, or both, has not been investigated in detail. Two studies used in vitro methods to evaluate the influence of vascular cells on osteoblasts. Guenther et al. (18) found that medium conditioned by cultured bovine large-vessel endothelial cells was mitogenic to cultures of rat calvarial bone cells, and Villanueva and Nimni (49) found that more alkaline phosphatase activity and mineral content developed in diffusion chambers containing

553

5.54 A . R. JONES ET AL.

both bone cells and hepatic endothelial cells than in chambers containing bone cells alone. To expand on these findings, we used in vitro methods to examine the intluence of microvessel endothelial cells and pericytes on osteoblastic cells isolated from the same species of rat.

Several methods have been established for the study of cellular interactions in vitro, and they differ in complexity and in capacity for experimental manip- ulation. Culture of different cell types together in direct contact is likely to support maximum interaction of cells but fails to isolate the response of the cell type of interest. The present study relied on three in vitro methods: (a) bone cells were cultured in direct contact with growth-arrested endothelial cells or pericytes, (b) bone cells were grown in the same plate in medium shared with microvessel cells, but contact between the two cell types was prevented by the use of co-culture dish inserts, and (c) media were conditioned by microvessel cells and added to cultures of bone cells.

MATERIALS AND METHODS Cell Culture

Bone cell culrure: Osteoblast-enriched rat calvarial bone cell culturcs were grown by the method of Cohn and Wong (11). Rat calvaria. obtained from newborn Sprague-Dawley rat pups, were digested in 0.2% bacterial collagenase for four 30-minute periods. Cells I-eleased from the final three digests were plated at a density of 35.000 cells/cm2 on uncoatcd 3.5 mm plastic dishes (Falcon: Bec- ton Dickinson. Mountainview, CA, U.S.A.) in a mixture of 45% (volivol) NCTC 135 (Gibco, Grand Island, NY, U.S.A.), 45% Dul- beccok modified Eagle's mcdiuni (Gibco). and 10% newborn calf serum at 37°C in 21 % Oz and 5 % C 0 2 (balance N2). Following cell conflucnce, the medium also contained SO @ml sodium ascorbate and 10 mM P-glycerophosphate.

Microvessel endodielid (,el/ culture: Microvessel endothelial cells were cultured by the method of Madri and Williams (28). Rat cpididymal fat pads were digcsted in 0.2% collagenase for 20 minutes. and the digest was centrifuged at 500 g for 5 minutes. The pellcted cells were resuspended in a solution of 40% (vol/vol) Pcrcoll (Pharmacia Hiosystems, Piscataway, NJ. LJSA.) in Hanks' solution and centrifuged at 14,000 g for 20 minutes. Endothelial cells were collected at a density of 1.025 g/ml and were washed and plated at 25.000 cclls/cmz on 35 mm plastic dishes precoated with 1% gelatin in phosphate buffered saline for 2 hours at 4°C. Plates were washed briefly 20 minutes after plating to remove less adher- ent cells. Dulbecco's modified Eagle's medium with 10% serum and SO pgiml cndotheiial cell growth supplement (ECCS: Sigma Chemical. St. Louis, MO, U.S.A.) was used. Following cell conflucnce. the cndothelial cell growth supplement was omitted from the medium.

Pericyte culture: Pericytes were allowed to outgrow from microvessels isolated from newborn rats by an established technique (13). Briefly. rat forebrains were gently homogenized in cation-free Hanks' solution with 1 % bovine serum albumin and were passed by gravity through successive nylon sieves of 750,500, 250,100, and 80 pm. Microvessels collected on the 100 and 80 pm sieves were harvested by backwashing, plated o n uncoated plastic dishes at a density of approximately SO0 microvessel fragments/cm*. and grown in medium composed of 45Y0 NCTC, 45% minimum essential medium. and 10% newborn calf serum. Primary cultures were grown

3 days beyond cell confluence before cells were replated by passage at a density of 20,000 cells/cm*; under these conditions, pericytes inhibited contaminating endothelial cell growth (33). Stock cultures of pericytes and endothelial cells were discarded after the third passage. Rat pericytes cultured by this method were identical in morphology to the bovine pericytes studied previously (6) and sim- ilarly formed alkaline phosphatase-rich focal.areas of high cell density, or nodules, that mineralized beginning 3 weeks after confluence in cultures supplemented with P-glycerophosphate (10 mM) and sodium ascorbate (50 pg/ml).

Charucterization of microvessel endothelial cell and pericyte crcl- lures: Endothelial cells grew to a cobblestone morphology that characteristically was less uniform than the cobblestone pattern of large vessel endothelial cells (52). Pericytes displayed a typical spreading morphology and quickly grew into layers of cells with nodular areas of increased cell density. To further characterize the cell populations within the endorhelial cell and pericyte cultures. experiments were conducted with several cell-specific stains, rc- ported previously (1,20,32,42,50), which compared endothelial cell and pericyte cultures with cultures of pulmonary artery endothelial cells, smooth muscle cells, microglial cells, and dermal fibroblasts (gifts of Dr. E. Macarak, Dr. P. Howard, Dr. J. Buchalter, and Dr. J. Fine, University of Pennsylvania). Frozen sections of newborn rat brain, kidney, and heart also were used as positive staining controls.

Receptor-facilitated pinocytosis of low-density lipoprotein in endothelial cells was tested by incubation of cultures with fluorescent-labeled low-density lipoproteins (Di-LDL; Biomedi- cal Technologies, Stoughton, MA, U.S.A.). The following immunocytochemical stains also were used to identify endothelial cells: antibodies against angiotensin converting enzyme (ACE; Infinity Reagents, Neshanic Station, NJ, U.S.A.) and factor VIII-relatcd antigen (Accurate Chemical and Scientific, Westbury, NY. U.S.A.).

To detect pericytes, immunocytochemical studies were per- formed using a polyclonal affinity-fractionated anti-smooth muscle actin antibody (ASMA; gift of Dr. Ira Herman. Tufts University). a monoclonal anti-a smooth muscle actin antibody (aSMA; Sigma Chemical), and a monoclonal anti-y smooth muscle actin antibody (HUC-1; gift of Dr. J. Lessard, University of Cincinnati). To detect contamination by microglial cells from brain. cultures also were stained with polyclonal antibodies against glial fibrillar acidic protein (GFAP gift of Dr. N. Gonadas, University of Pennsylvania).

Cultured cells were passaged to eight-well culture slides (Tissue Tek; Miles, Pittsburgh, PA, U.S.A.), precoated with gelatin in the case of the endothelial cells, and allowed to attach overnight. Fro- zen sections were obtained from the brain, heart. and kidney of newborn rat for use as tissue controls. Culture slides and sections were fixed in acetone, rinsed in phosphate buffered saline, and pretreated to reduce background staining by a 30-minute exposure to a 1:10 dilution of non-immune serum from the host species of the secondary antibody. After rinsing, each of the primary antibodies listed in Table 1 was applied to the slides at a 1:200 working dilution (except the monoclonal anti-a smooth muscle actin antibody, at 1500) and incubated for 60 minutes at room temper- ature. Negative staining controls were exposed to a 1 :20 dilution of non-immune serum from the primary antibody host species in lieu of primary antibody. Secondary antibodies were fluoroscein isothiocyanate-conjugated, and fluorescence microscopy was used for manual counting of the cells showing positive or negative staining patterns.

In V i m Cell Interaction Study Methods Direct contact co-culture: Microvessel cells (endothelial cells or

pericytes) wcre plated at a density of 15,000 cells/cm2 in 35 mm plastic dishes (Falcon) and allowed to attach overnight. Further growth then was arrested by exposure to 10 pg/mi mitomycin-C (Sigma Chemical) for 1 hour. Monolayers were washed with me-

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BONE C E L L RESPONSE TO MICROVESSEL CELLS 555

TABLE 1. Biochemical daiu from cnlvarial bone cell ciiltirres following exposiire IO rnediiini coriditionerl by endothrlinl cells or pericytes for either 3 d ~ y s foilowing plrrting or continuoilsly

control Endothelial cell-conditioned medium Pericvte-conditioned medium medium 3 days Continuous 3 days Continuous

DNA (pglplate) 16.5 2 2.1 31.5 i 4.3" 28.7 i 6.6'' 24.4 2 5.1" 22.2 t 4.4" Alkaline phosphatase activity 1.62 2 0.17 0.52 ? 0.12" 0.41 t 0.15" 0.93 t 0.17,' 0.85 2 0NPi

Incorporation of [ "Clproline 54.2 2 4.1 49.1 2 5.7 46.8 2 6.3 55.4 2 9.1 48.2 i 3.5 (nmoles p-nitrophenollminlpg DNA)

(dpm x lO'/pg DNA)

Osteocalcin (nglpg DNA) 3.06 3 0.48 0.98 t 0.09".h 0.51 t 0.10" 2.21 i 0.42"." 1.34 5 0.22"

The proportion of conditioned medium was 50%. DNA level. alkaline phosphatase activity. and [ "Clproline incorporation were

Statistically different from the controls (p < 0.05). Transiently exposed groups and continuously exposed groups were each compared measured after 10 days. and osteocalcin concentrations were measured after 4 weeks. Values represent mean ? SD (n = 6 ) .

with controls by two-way analysis of variance. "Statistically different from the group exposed continuously to the same conditioned medium (Student r test, p < 0.05).

dium. and bone cells were passaged to these dishes at a density of 15.000 cells/cm* to establish the co-cultures. As controls, separate cultures of growth-arrested microvessel cells and bone cells were plated and maintained under identical conditions. At harvest. quantitative data for the control cultures were compared with the co-cultures by the Student t test with the Neuman-Keuls multiple comparisons procedure.

Co-culture in shured medium, wirholct cell contact: Microvessel cells (endothelial cells or pericytes) were grown to confluence on gelatin-precoated tissue culture dish inserts containing membranes of 0.45 pm pore size (Millicell-CM: Millipore. Marlborough, MA. U.S.A.). Bone cells were plated into extra-deep 35 mm six-well plates (Falcon) at a density of 20.000 cellslcm' and allowed to attach overnight before inserts containing microvessel cells were moved into dishes containing bone cells. These inserts suspended the microvessel cells 2 ml above the monolayers of bone cells. so that the two different cell layers shared medium but had no direct contact. At harvest, the dish inserts were removed and the under- lying bone cell layers were analyzed. Control cultures of bone cells were grown under the same conditions in plates with gelatin- coated culture dish inserts devoid of cells. Data were compared with use of the Student r test with the Neuman-Keuls multiple comparisons procedure.

Conditioned medio exprrimenrs: Media were collected after 48 hours of exposure to immediately postconfluent cultures of either endothelial cells or pericytes. The conditioned media were passed through filters to remove floating cells and were combined with fresh bone cell culture medium in pre-established proportions. Bone cells were plated on uncoated plasticware at a density of 20,000 cells/cm2 and were allowed to attach overnight before being fed medium supplemented with endothelial cell-conditioned medium. pericyte-conditioned medium. or control medium. Control media were incubated in empty culture plates under conditions identical to those used to generate conditioned media from the microvessel cell cultures. The data from control and experimental cultures were compared by means of analysis of variance with Fisher's protected least significant difference multiple comparison test.

Culture Analysis At 10 days following the initiation of co-cultures. and at sev-

eral time points over 2 weeks in dose-response conditioned mc- dia experiments. cultures were harvested by scraping cell layers into phosphate buffered saline. DNA content was determined by a lluorometric assay (25). and alkaline phosphatase activity was determined by a colorimetric assay (27). Activity was expressed as nanomoles of p-nitrophenol produced per minute per microgram of DNA. Culture histology was reviewed at the light micro- scopic level after histochemical staining for alkaline phosphatase

activity (Sigma Chemical) and counterstaining with hematoxylin. In experiments with microvessel cell-conditioned media, bone

cell cultures were radiolabeled with [''C]proline, 7.5 pCiiml. for 48 hours. The culture media were removcd and monolayers were washed. harvested by scraping into phosphate buffered saline. and homogenized. Equal proportions of each sample's medium and cell layer were mixed and, after dialysis to remove unincorporated counts, total incorporation was quantified by scintillation counting and was normalized to the DNA content of the culture.

Synthesis of osteocalcin was measured by radioimmunoassay (Biomedical Technologies) in the medium of 4-week-old bone cell cultures. Thirty-six hours prior to sampling. the cell layers were washed and serum was removed from the medium to exclude serum osteocalcin from these measurements. Levels of osteocalcin were normalized to the DNA content o l the cell layer and were expressed as nanograms per microgram of DNA. Mineralization was assessed in 2. 3. and 4-week-old bone cell cultures by histochemical staining with the von Kossa method (40). The formation of mineralized nodules was assessed by light microscopy. and a grade of mineralization was assigned to each plate by a single observcr. Grades of mineralization were averaged for cach experimental group, with n = 5.

RESULTS Characterization of Cell Populations in Endothelial Cell and Pericyte Cultures

Endothelial cell cultures exposed to Di-LDL showed uniform staining of cell cytoplasm consistent with the pattern of staining previously reported for this cell type (SO), and all of the cells stained positively (data not shown). Concentration of fluorescence in these cells could be seen after exposure to 10 pg/ml of Di-LDL at 37°C for as brief a period as 30 minutes. Positive control cultures of large vessel endothelial cells showed identical staining charac- teristics. Cultures of rat pericytes, bovine pericytes. and rat skin fibroblasts failed to concentrate stain within any of the cultured cells after a labeling period as long as 24 hours.

Immunocytochemical staining for endothclial cell- specific markers showed that with anti-angiotensin converting enzyme antibodies there was positive staining of the capillaries in frozen sections of the rat brain. the vascular endolhelium in sections of rat heart

556 A . R. J O N E S ET AL.

bone cells EC's bone cell : EC pericytes bone cell / pericyte co-cultures cocuitures

FIG. 1. Calvarial bone cells were plated onto preconfluent monolayers of growth-arrested endothelial cells (ECs) or pericytes and grown for 10 days. DNA and alkaline phosphatase activity were compared with that in control cultures of the individual cell types grown under identical conditions. Asterisks indicate that values differ statistically from the summed values of the respective control cultures (p < 0.05). Error bars represent 1 SD.

and kidney, and within 95% of the cells by count in control cultures of pulmonary artery endothelial cells and microvessel endothelial cell cultures from rat epididymus. Cells from three different isolations of microvessel endothelial cells showed positive staining for angiotensin converting enzyme in more than 95% of the cells, whereas rat pericyte cultures and control cultures of smooth muscle cells or fibroblasts showed uniformly negative staining for angiotensin converting enzyme.

Factor-VIII staining studies revealed that endothelial cells from three different isolations showed staining patterns identical to those of control cultures of pulmonary artery endothelial cells, whereas the microvessel endothelial cells from two other isolations displayed reduced intensity of staining. For the isolations that stained intensely, 90% of the cells showed the perinuclear staining typical for factor VIII, whereas in the other two isolations 40 and 50% of the cells stained positively. Cells from the isolations with reduced staining for factor VIII stained positively with Di-LDL and anti-angiotensin converting enzyme. Cells from late passage cultures of microvessel endothelial cells showed weak staining for factor VIII in 25% of the cells, although they stained intensely for angiotensin converting enzyme and took up Di-LDL avidly. Cul- tures bf pericytes and smooth muscle cells were uniformly negative for staining for factor VIII.

Pericyte cultures showed positive immunocytochemical staining with antibodies against the polyclonal anti-smooth muscle actin antibody and the monoclonal anti-a smooth-muscle actin antibody in 95% of cells, whereas control cultures of smooth muscle cells showed of the cells labeled by each of these stains. Ten percent of the cells in the pericyte cultures showed positive staining with the monoclonal anti-y smooth muscle actin antibody, whereas 90% of the cells in the smooth muscle cell cultures stained

positively. Endothelial cell cultures were uniformly negative for staining with these anti-smooth muscle stains.

Immunocytochemical stains with glial fibrillar acidic protein antibody revealed intensely positive staining in microglial colonies of frozen sections of newborn rat brain and also labeled 85% of the cells in control cultures of microglial cells. However, no positively labeled cells in the pericyte cultures were obtained from three different cell isolations from rat brain microvessels.

Co-cultures with Cell Contact The initial experiments employed direct contact co-

cultures to support maximum cellular interaction. Feeder-layers of pericytes and endothelial cells, gen- erated by exposure to mitomycin-C, maintained normal morphology but did not increase cell number, as verified by levels of total DNA. Notably, alkaline phosphatase activity increased significantly in endothelial cell and pericyte cultures following growth arrest. Compared with control cultures at 10 days following cell confluence, alkaline phosphatase activity increased 66% for the microvessel endothelial cells and 35% for the pericytes (n = 6, p < 0.05).

Biochemical data for the experiments with feeder- layer co-cultures are shown in Fig. 1. Both the bone cell/pericyte and bone celllendothelial cell co-cultures showed significantly higher levels of DNA and lower alkaline phosphatase activity than would have been predicted from the values measured in the control cultures of these cells grown alone. The difference between the values for a co-culture and the sum of the values for the respective control cultures reflected the effects of the interaction between the two cell types. This difference was proportionally larger for the bone cell/endothelial cell co-cultures; it was nearly twice that seen in the bone cell/pericyte co-cultures. Histo-

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BONE C E L L RESPONSE TO MICROVESSEL CELLS

* 1 -

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-.' Q

3 30 a I-

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bone cells

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d z .- = a Gtj--3

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FIG. 2. Bone cell cultures were exposed to 50% endothelial cell (EC)-conditioned medium or to endothelial cells within culture dish inserts. After 10 days, DNA and alkaline phosphatase activity were measured in the bone cell cultures and were compared with the activity in control cultures that had been given sham-conditioned medium or had been exposed to empty culture dish inserts. Error bars represent 1 SD (p < 0.05).

chemical staining for alkaline phosphatase in these experiments showed that roughly 20% of the cells by count stained heavily in focal areas of the bone cell control cultures. Cultures of growth-arrested endothelial cells or pericytes stained homogeneously for alkaline phosphatase at a level of intensity only slightly greater than the background staining seen in cultures used as negative staining cultures. The pattern of staining in the co-cultures was the same whether the bone cells were grown with growth-arrested endothelial cells or pericytes. In these co-cultures, staining was homogeneous, without the focal pattern of heavy staining seen in the cultures of bone cells grown alone (data not shown).

Co-cultures without Cell Contact In methodological pilot experiments with co-culture

dish inserts, endothelial cells and pericytes both at- tached and grew on the inserts with the same morphology seen in control cultures. To assess the effects of the inserts on culture monolayers, bone cells cultured with and without the presence of empty inserts were compared after 10 days in culture. DNA content and alkaline phosphatase activity were decreased in the cultures with inserts, and in two of three experiments these differences reached statistical significance at p < 0.05 (Student t test, n = 6). Another pilot finding was that the inserts were fragile when wet, which made handling and changes of the medium difficult.

The next experiments examined the response of bone cells to endothelial cells in culture dish inserts and to endothelial cell-conditioned medium. In the bone cell cultures exposed to endothelial cell-conditioned medium, culture dish inserts were not used. Therefore, in these experiments, two groups of control bone cell cultures were established that either did or

did not contain inserts. After 10 days, DNA levels and alkaline phosphatase activity in the bone cell cultures were measured (Fig. 2). Comparison of the first two groups indicated that exposure to endothelial cell- conditioned medium increased DNA in layers of bone cells and decreased alkaline phosphatase activity. Bone cell cultures that included endothelial cells in inserts also showed an increase in DNA and a decrease in alkaline phosphatase when compared with cultures that contained empty inserts. Expressed as a percentage of the values of their respective control groups, DNA levels in bone cell cultures were increased 144% in the group exposed to endothelial cell-conditioned medium and 134% in the group exposed to endothelial cells in inserts, whereas alkaline phosphatase activity decreased 76% in the endothelial cell-conditioned medium group and 59% in the group with endothelial cells in inserts. In summary, this influence of endothelial cells on bone cell cultures was expressed either through noncontact co-culture or through conditioned medium, and the effect of the endothelial cell-conditioned medium was greater. These changes in bone cell cultures were proportionally similar to those seen previously after growth in direct contact with the endothelial cells (Fig. 1).

Similar experiments measured levels of DNA in bone cell cultures and alkaline phosphatase activity after 10 days of exposure to either pericyte-conditioned medium or to pericytes in co-culture dish inserts. DNA (micrograms per plate) increased 131% in response to pericyte-conditioned medium (stimulated, 24.2 2 4.4; control, 18.5 5 3.7; n = 6) and increased 122% in the group that contained pericytes in inserts (stimulated, 16.6 2 3.4; control, 13.6 t 3.1). Alkaline phosphatase activity (nanomoles of p-nitrophenol per minute per microgram of DNA) decreased 44% in the

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558 A . H. J O N E S E T AL.

TABLE 2. Mean grades o,f mineralized nodule formcrtion in groups of calvarial hone cell c u h r e s following exposure to medium conditioned by enrtothelial cells or pericykv either f i)r 3 rlczys

following plating or continuously

Endothelial cell-conditioned medium Pericyte-conditioned medium Control medium 3 days Continuous 3 days Continuous

2 weeks + 0 0 0 0 3 weeks +++ + + + + 4 weeks ++++ + + +++ +

The proportion of conditioned medium was 50%. The cultures were graded, and the scores were averaged with n = 5.0 = no nodules, + = small sparse nodules, ++ = scattered nodules of moderatc size. +++ = many large nodules, and ++++ = dense plate coverage with large coalescing nodules (see Materials and Methods section).

group with pericyte-conditioned medium (stimulated, 1.12 +- 0.16; control, 0.74 5 0.10) and decreased 37% in the group with pericytes in inserts (stimulated, 0.75 f- 0.11; control, 0.47 2 0.06). These changes in DNA and alkaline phosphatase were statistically significant at p < 0.05 for groups with pericytes in inserts and pericyte-conditioned medium.

A dose-response study of the effects of endothelial cell and pericyte-conditioned media on the bone cell cultures is shown in Fig. 3. In control cultures of calvarial bone cells, alkaline phosphatase activity peaked at 7 days in culture at a level six times that measured 2 days following plating. In contrast, bone cell cultures exposed to endothelial cell-conditioned medium as either 20 or 50% the proportion of total volume of medium failed to show any statistically significant change in alkaline phosphatase activity over the culture period. Bone cell cultures exposed to pericyte- conditioned medium as 5 , 20, or SO% of the culture medium also showed significant reductions in alkaline phosphatase activity. However, pericyte-conditioned medium failed to completely blunt the normal rise in alkaline phosphatase in bone cell culture as did the endothelial cell-conditioned medium.

The effect of transient exposure to conditioned me-

EC-conditioned media; % I I pericyte-conditioned media; % I

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dia from the microvessel cells was compared with the effect of continuous exposure. Conditioned media were added to the bone cell cultures for 3 days from initial cell plating to cell confluence. The total DNA in the bone cell cultures, alkaline phosphatase activity, and incorporation of radiolabeled proline were measured after 10 days, and the levels of osteocalcin and mineralization were assessed after 4 weeks (Tables 1 and 2). A significant increase in DNA and decrease in alkaline phosphatase activity occurred after both continuous and transient treatment with conditioned media from both cell types; no statistically significant difference was seen in these values when the continuously exposed and transiently exposed groups were compared. Incorporation of proline by bone cell cultures, however, was not significantly affected by the microvessel cell-conditioned media under either regi- men. Levels of osteocalcin were reduced by both continuous and transient exposurc to conditioned medium from both microvessel cell types; continuous exposure led to the greater reduction.

The mineralization of bone cell cultures was mark- edly impaired by continuous exposure to medium conditioned by either endothelial cells or pericytes (Table 2). Mineralized nodules developed in control cultures

50 20

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FIG. 3. Beginning on the day following primary plating, endothelial cell (EC) or pericyte-conditioned medium was added lo the bone cell cultures. in different proportions. DNA and alkaline phosphatase levels were assayed every third day thereafter. Error hars represent 1 SD.

of bone cells after 2 weeks, whereas the conditioned media-treated cultures failed to show any until 3 weeks. By the fourth week of culture, the mineralized nodules in the control cultures were large and had coalesced, while those of the conditioned media- treated cultures remained small. Transient treatment with microvessel cell-conditioned media led to re- pressed formation of mineralized nodules, similar to that seen in cultures exposed to these media continuously: nodules were not seen until 3 weeks, and at 4 weeks the pattern of the nodules was less dense and the nodules were small to moderate in size, There was a trend for the transiently treated cultures to have larger nodules at a higher density than the continuously treated cultures; this was more evident in the groups exposed to the pericyte-conditioned medium. However. these differences in mineralization between groups of bone cells exposed to microvessel cell- conditioned media in different regimens were rela- tively minor in comparison with the major difference seen between all of these groups and the control bone cell cultures.

DISCUSSION This in vitro study of the influence of microvessel

cells on osteoblastic bone cells revealed that exposure to either microvessel endothelial cells or pericytes produced a proliferative response in bone cell cultures and that this response was associated with repression of the normal expression of osteoblastic phenotypic markers by these cells. The mitogenic influence was demonstrated in co-cultures with cell contact, in co- cultures that permitted only the sharing of medium, and in experiments using media conditioned by the microvessel cells. The findings suggest that the medi- ator of the observed effect is a stable soluble factor whose production by microvessel cells is not affected by the presence of bone cells.

Much has been learned about microvessel cells re- cently, due in large part to advances in the methods by which these cells can be maintained in culture. The culture of microvessel endothelial cells once relied on technically difficult and time-consuming methods of cloning (17). Less demanding methods of isolation and optimum culture conditions have been established whereby populations of microvessel endothelial cells can now be cultured with little contamination by other cell types, as determined by testing with endothelial cell-specific stains (10,28). We found that the uptake of low-density lipoproteins and the presence of angiotensin converting enzyme were reliable markers for microvessel endothelial cells, whereas factor VIII-related antigen was not expressed in some of the isolations in amounts as large as those in control cultures of large vessel endothelial cells. Reduced staining for factor VIII in microvessel endothelial cells has

been reported previously (22,46) and may be related to the variability in expression of this antigen in the animals from which the cells are isolated. The overall pattern of staining of our cultures of microvessel endothelial cells suggested a high level of purity for cells with endothelial cell-specific markers. with very little contamination by other cell types.

Pericytes can be cultured without unusual culture substrates or medium additives. so much of the work to develop the methods of cell isolation and culture has been to identify the conditions that select for and optimize the expression of phenotypic markers known to be associated with pericytes in vivo (8,20,38). To date, identification of pericytes in culture has relied on identification of a typical morphology and the presence of selected smooth muscle antigens along with the absence of endothelial cell-specific markers. We found that polyclonaI anti-smooth muscle actin antibody and monoclonal anti-a smooth muscle actin antibody yielded staining patterns in the pericyte cultures similar to those of smooth muscle cells and anti-y smooth muscle actin antibody labeled antigen present in smooth muscle cells not widely expressed by pericyte cultures. Pericyte cultures failed to stain with any of the endothelial cell-specific stains or with glial fibrillar acidic protein, which detected microglial cells of brain, and failed to phagocytose Congo red stain. which was avidly taken up by cultured macrophages. On the basis of these criteria, the predominant type of cell in these cultures was the pericyte, and there was no indication of significant contamination by cells likely to be found in the tissues used for isolation. A new antibody against a small proteoglycan, HMW- MAA (36), has been shown to label pericytes in vivo and may become another pericyte-specific marker in vitro, but it was not tested in this study.

We employed well established methods for the culture of microvessel endothelial cells from adipose tissue and pericytes from brain. Microvessel cells isolated from a variety of tissues have been shown to share phenotypic features (21,41,52). To our knowl- edge, no techniques have been established for the isolation of useful quantities of microvessel endothelial cells or pericytes from bone. A promising approach to the further study of the interaction between bone cells and microvessel cells would be to develop methods for the culture of pericytes or endothelial cells from sites of active bone formation, so that the physiology of those cells could be studied and their effects on bone cells could be compared with the effects seen in this study with microvessel cells from tissues that were not from bone.

We believed it important that all the cells for the study be isolated from the same animal species, so that celluiar interactions that were species-specific would not be overlooked. Guenther et al. (18) found that

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560 A . R. JONES ET AL.

medium conditioned with endothelial cells from bovine pulmonary artery contained mitogens for rat calvarial bone cells. Our study demonstrated a similar mitogenic influence in conditioned medium from both microvessel endothelial cells and pericytes isolated from the rat. Comparison of these studies does not indicate how much of this influence is species-specific but does suggest that release of bone cell mitogens is a common feature of cells isolated from different vascular tissues. In our study, the mitogenic effect from microvessel endothelial cells was greater than that from pericytes. Likely mediators of this vascular mitogenic influence on bone cells are those growth factors and cytokines that are known to be both produced by vascular cells and mitogenic to bone cells. They in- clude basic fibroblast growth factor (FGF), acidic FGF, insulin growth factor-I (IGF-I), IGF-11, trans- forming growth factor-p (TGF-P), and endothelin

Cultures of rat calvarial osteoblast-enriched bone cells have been studied extensively (12,4331) and ex- hibit a typical sequence of gene expression. Soon after plating, genes express cellular products associated with proliferation such as histones, and once cell confluence is reached, the cultures reduce synthesis of proliferative products and increase synthesis of their more differentiated cell products-those typical of the osteoblastic phenotype. In this study, early transient exposure to microvessel cell-conditioned media following plating led to a significant reduction of osteoblastic markers later in culture. This finding suggests that the microvessel cell factors interfere with the normal early transitions in bone cell cultures and that this interference causes a reduction in the subsequent expression of the osteoblastic phenotype.

Our study’s findings appear to conflict with those of Villanueva and Nimni (49), who found an increase in alkaline phosphatase activity and mineral content in diffusion chambers containing bone cells and hepatic vascular endothelial cells, as compared with those containing bone cells alone. The two studies differ significantly. Our experiments were conducted in vitro and were controlled for all substances except those produced by, or in response to, the microvessel cells. Their study relied on diffusion chambers, which exposed the cells to many local substances, some of which may be important to osteogenesis and were omitted from our system.

The complexity of the environment in vivo makes it difficult to correlate the findings of this in vitro study with physiologically significant events in the biology of bone cells. If one assumes that the mitogenic in- fluences of microvessel cells on bone cells shown in this study are active in vivo, then a model whereby microvessel cells cause an increase in the local cell population at sites of bone formation and repair may

(9944).

be proposed for further investigation. Whether or not the cells of this enlarged population respond to osteoinductive stimuli is an important question. There is increasing evidence that mesenchymal cells at many stages of differentiation can respond to osteoinductive stimuli under the proper conditions. For example, TGF-P induces chondrogenic markers in cultured muscle cells (39), and bone morphogenetic protein-2 (BMP-2) induces osteoblastic expression in a non- osteogenic cell line (23). Further experiments to test this model are warranted in order to determine whether calvarial bone cell cultures, expanded in response to microvessel cell-derived mitogens, retain their capacity to respond to osteoinductors. If so, the mitogenic effect of the influence of microvessel cells shown in this study might be expected to provide a net increase in the bone-forming ability of local cells at sites of bone formation.

Another noteworthy finding of this study was the increase in alkaline phosphatase activity that occurred after pharmacologic growth arrest of the pericytes and microvessel endothelial cells. Because both types of cell have been implicated as potential osteoprogenitor cells (6,14,24,30,48), we are studying their capacity to express other osteoblast phenotypic markers. If the microvasculature is directly involved in the regulation of bone formation, it may offer new methods by which therapeutic control of bone formation may be tar- geted to the local level. This goal may be approached through further studies to clarify the dynamics and scope of the influence of the microvessel cell on bone metabolism.

Acknowledgment: The authors gratefully acknowledge the as- sistance of Dr. Edward Macarak and Dr. Pamela Howard (Univer- sity of Pennsylvania) and of Dr. Antonio Martinez-Hernandez and Dr. Stuart Williams (Thomas Jefferson University) and the techni- cal support of Krystyna Knight and Esther Payne. This work was supported by National Institutes of Health Grant AR18033-18 and the Orthopaedic Research and Education Foundation.

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