Study of the growth factor requirements of human bone-derived cells: A comparison with human fibroblasts

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Study of the Growth Factor Requirements of Human Bone-derived Cells: A Comparison with Human Fibroblasts J. E. PICHfi1y2 and D. T. GRAVES2 ‘Department of Periodontics, lDepartment of Periodontics,

Wiiford Hall USAF Medical Center, San Antonio, Texas The University of Texas Health Science Center, San Antonio, Texas, USA

Address for correspondence and reprints: Dr. Dana Graves, Department of Oral Biology, Goldman School of Graduate Dentistry, Boston University Medical Center, 100 East Newton Street, Boston, MA 02118, USA.

processes leading to repair or regeneration of damaged tissue. Three growth factors that are released by platelets following injury are platelet-derived growth factor (PDGF) (Antoniades et al. 1979; Heldin et al. 1979; Deuel et al. 1981; Raines and Ross 1982), transforming growth factor-p (TGF-P) (Assoian et al. 1983; Childs et al. 1982) and an epidermal growth factor (EGF)-like protein (Oka and Orth 1983; Assoian et al. 1984). Platelets sequester these mitogens under normal circumstances and then release them upon degranulation, providing an ideal system for selectively delivering growth factors to injured tissue. The importance of these growth factors is supported by evidence that PDGF, EGF and TGF-P stimulate soft tissue wound healing in vivo (Spom et al. 1983; Grotendorst et al. 1984; Buckley et al. 1985; Roberts et al. 1986; Lynch et al. 1987). Therefore, the in vitro response of bone cells to PDGF, EGF, and TGF-P, which are released at sites of bone injury, ought to provide insight into which factors might locally regulate osseous repair. Recent techniques have been devised for the culture of bone cells derived from bone explants. Several reports have described a technique where adult human cancellous bone fragments are cultured in vitro for 3-4 weeks, allowing bone cells to migrate from the bone fragments onto a tissue culture surface (Beresford, Gallagher et al. 1983; Beresford, MacDonald et al. 1983; Beresford, Gallagher, Poser et al. 1984; Auf’mkolk et al. 1985). Isolating cells by this technique has facilitated in vitro investigation into the cellular control mechanisms of bone-derived cells. These cells respond to PTH with an increase in cyclic AMP content (Auf’mkolk et al. 1985), and have high basal alkaline phosphatase activity, which is increased on exposure to 1,25_dihydroxyvitamin D3 (Skjodt et al. 1985; Beresford et al. 1986) and decreased by PTH (Beresford et al. 1984b; Hesch et al. 1984). These characteristics distinguish these bone-derived cells from skin libroblasts. Since bone cells respond differently than fibroblasts to hormonal control, they may also respond differently to locally generated paracrine factors. While the growth factor response of tibroblasts in vitro has helped identify factors which mediate soft tissue wound healing in vivo, less is known about the paracrine factors which stimulate bone cells. In order to further understand events which occur in osseous wound healing it would be helpful to identify paracrine factors that induce proliferation of bone cells. Thus, normal human

Abstract Recent techniques have been devised for the culture of bone cells derived from human bone explants. These cells, which are thought to represent several stages in the osteoblast lineage, respond to PTH with an increase in cyclic AMP content, and have high basal alkaline phosphatase activity which is increased on exposure to 1,25dihydroxyvitamin D, and decreased by PTH. Such characteristics distinguish these cells from fibroblasts. In this study, we demonstrate that human bone-derived cells also differ from tibroblasts in their growth characteristics. Bone-derived cells proliferated in basal medium supplemented with platelet-poor plasma. The rate of proliferation was enhanced by additional supplementation with platelet-derived growth factor (PDGF), and further increased when a combination of growth factors was added (PDGF, TGF-P and EGF). In contrast, fibroblasts did not proliferate in basal medium supplemented with plateletpoor plasma and the addition of PDGF alone stimulated fibroblast proliferation to the same extent as 10% fetal bovine serum. Supplementation with other growth factors did not further enhance the response of fibroblasts to PDGF. These results emphasize the differences in proliferative responses between human bone-derived cells and human fibroblasts, and indicate that the factors responsible for osseous regeneration in vivo may differ from those factors which regulate repair of soft tissue wounds. Key Words: Human bone-derived cells-Growth Cell culture-Osteoblasts-PDGF.

factors-

Introduction Soft tissue wound healing and osseous regeneration following bone damage or fracture are thought to involve a similar sequence of cellular events. These include chemotaxis, proliferation of cells of mesenchymal origin at the site of injury and production of an extra-cellular matrix (Grotendorst et al. 1984). This cascade of events is largely controlled by locally generated factors that regulate the

The opinions expressed in this paper of the United

do not necessarily represent States Air Force or the Department of Defense.

the views

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J. E. Pichk and D. T. Graves: Bone cell response to growth factors

bone-derived cells isolated by the above technique provide a suitable population to examine the influence of paracrine factors since they represent a population of bone cells at various stages in the osteoblast lineage that are thought to participate in osseous wound healing. The studies presented here sought to investigate the growth factor response of normal human bone-derived cells utilizing growth factors which may contribute to osseous wound healing, and to compare the results with those observed for normal human fibroblasts. The results indicate that the growth factor requirements of normal human bone-derived cells differ from those of normal human fibroblasts.

Materials and Methods Explants

All tissue samples were obtained from healthy adult volunteers and were handled under sterile conditions in a manner similar to recent descriptions in the literature (Beresford, Gallagher et al. 1983; Beresford, Gallagher, Poser et al. 1984; Hanazawa et al. 1987). Adult human bone-derived

cells

Bone-derived cells were obtained from sub-periosteal human trabecular bone. Human bone was obtained from adults with no malignancies or metabolic diseases. Bone particles (l-3 mm9 were placed in a 25 cm2 flask and incubated in culture medium containing Dulbeco Modified Eagles’s Medium (DMEM) (Gibco) supplemented with 10% Fetal Bovine Serum (FBS) (K. C. Biological), penicillin (100 U/ml) and streptomycin (100 U/ml) under standard conditions at 37”C, 5% COZ. The flasks were left for three to four weeks, with media changes every three days, until bone cells had migrated from the particles and the flask was confluent. At confluence, cells were trypsinized (0.25% trypsin, 0.02% EDTA) and transferred to a 75 cm2 flask. Cells were subsequently split at a 1:3 ratio and transferred under standard conditions. The bone-derived cells were studied at passages 2-4.

was also used to confirm the osteoblast-like phenotype. In addition, it was consistently noted that adult human bonederived cells formed multiple layers when confluent while the adult human fibroblasts grew in monolayer cultures. These responses and characteristics of the bone-derived cells are consistent with previous descriptions of cells exhibiting an osteoblast-like phenotype (Luben et al. 1976; Thomas and Ramp 1979; Wergedal and Baylink 1984; Robey and Termine 1985; Beresford et al. 1986). Growth factors

Purified growth factors were used in all studies. Human PDGF from platelets was generously donated by Dr. Harry Antoniades. Human TGF-B from platelets was generously donated by Dr. Michael Sporn. Recombinant human IGF- 1 was purchased from Amgen Biologicals. Mouse EGF was purchased from Collaborative Research. Platelet-poor plasma (PPP) and fetal bovine serum were purchased from K. C. Biologicals. Cellular proliferation

Bone-derived and normal human libroblast cultures were assayed simultaneously for cellular proliferation as described in Graves et al. (1983). Results were confirmed using three different bone explants obtained from different individuals. Briefly, cells were plated at subconfluence in 24-well tissue culture plates and incubated in 0.5 ml DMEM supplemented with 10% FBS for 12 hours at 37”C, 5% COP. On day 0 cells were rinsed and changed to DMEM containing the indicated concentration of plateletpoor plasma. Growth factors were then added singly or in combination to each well. The response to growth factors was compared to a negative control consisting of DMEM supplemented with platelet-poor plasma and to a positive control consisting of cells incubated in DMEM supplemented with 10% FBS. The medium was changed every 3 or 4 days. Cells were trypsinized and counted using a hemocytometer. In all cases there was good agreement between samples for each assay point, with the value of each sample lying within 5% of the mean. DNA synthesis

Adult human fibroblasts

Normal human fibroblasts were established from adults with no malignancies or metabolic diseases. Pieces of dense gingival connective tissue with no epithelium were placed in a 25 cm2 flask and incubated under standard conditions in culture medium containing DMEM supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 U/ml). The flasks were left for three to four weeks, with media changes every three days, until the tibroblasts had migrated from the tissue and the flask was confluent. The cells were then passaged in an identical manner to the bone-derived cells. Normal human tibroblasts were studied at passages two through four. The phenotype of the bone-derived cells and fibroblasts were examined using biochemical tests. Each cell isolate was tested at the same passage that experiments were initiated. In all cases, bone-derived cells had significantly higher basal alkaline phosphatase levels compared to normal human fibroblasts, and the alkaline phosphatase activity was increased following exposure to 1,25(OH)2 vitamin D3 and inhibited following exposure to parathyroid hormone. Parathyroid enhancement of cyclic-AMP levels

DNA synthesis was measured by acid insoluble 3H Thymidine incorporation (Graves et al. 1983). Normal human Iibroblasts and bone-derived cells were plated at approximately 10,000 cells/well in 96-well microtiter plates and left to incubate for 48 hours. They were then depleted for 48 hours in DMEM supplemented with 1% platelet-poor plasma. Depletion medium was removed and growth factors (PDGF, EGF, IGF, and TGF+) were added to DMEM supplemented with 0.1% albumin. Cells were incubated for 24 hours with the addition of 3H-Thymidine (2.5 UCi/ml) for the last two hours. The cells were then fixed with cold 5% tricholracetic acid (TCA) for 20 minutes, rinsed with cold 5% TCA and solubilized in 1% SDS for 30 minutes and transferred to vials containing a scintillation cocktail. Incorporated 3H Thymidine was measured in a Beckman scintillation counter. DNA synthesis was also measured by autoradiography as described by Pledger et al. (1977). Cells were plated in a similar manner to that described for acid insoluble 3H Thymidine experiments and grown to confluence. The cells were then depleted and assayed simultaneously following two slightly different protocols. One group of cells was de-

133

J. E. Picht and D. T. Graves: Bone cell response to growth factors for 72 hours in DMEM supplemented with 3% platelet-poor plasma, rinsed and tested in fresh identical medium. The other group of cells was depleted for 72 hours in DMEM supplemented with 0.1% Bovine Serum Albumin (BSA), insulin (5 Ug/ml) and transferrin (5 Ug/ml), rinsed and tested in fresh identical medium. The concentrations of insulin and transferrin used have been shown to enhance the response of Balb Cl3T3 cells to platelet-derived growth factor while minimally stimulating DNA synthesis (Rockwell 1984; Van Wyk 1984). In addition, this concentration of insulin is sufficiently high to enable insulin to bind to the insulin-like growth factor 1 receptor (Van Wyk 1984). Both groups were compared to a positive control consisting of DMEM supplemented with 10% FBS. 3H-Thymidine (5 @J/ml) was then added to the assay medium for 24 hours followed by methanol fixation and processing for autoradiography. Statistical analysis was performed as indicated using a Statview 512 Software Package on an Apple Macintosh Plus microcomputer. Within each experimental protocol the results were analyzed using an Analysis of Variance (ANOVA). Scheffe’s F-test was used as the post hoc test.

BONE-DERIVED

pleted

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Results The normal human tibroblast and bone-derived cell populations were tested for their proliferative response to growth factors. Basal media, DMEM, was supplemented with platelet-poor plasma because platelet-poor plasma contains essential nutrients including lipids, is minimally stimulatory for fibroblasts, and enhances the response of libroblasts to PDGF. In addition, the concentrations of platelet-poor plasma used contain very little PDGF (Pledger et al. 1977; Antoniades and Owen 1982; Rockwell 1984; Van Wyk 1984). Figure 1A demonstrates the response of bone-derived cells. Bone-derived cells proliferated in 4% platelet-poor plasma so that the cell number increased five-fold over nine days. Low concentrations of PDGF (0.6 &ml) were not stimulatory compared to cells in control medium. At higher concentrations, 6.0 ng/ml of PDGF stimulated a 33% increase in cell number over nine days when compared to cells in 4% PPP alone. This was still less than the increase observed in cells incubated in 10% FBS. Ten percent FBS was chosen for comparison because it provides an enriched supplement of growth factors and nutrients which has been found to be highly stimulatory for cells of mesenchymal origin. In contrast to the bone-derived cells, the tibroblasts did not proliferate in low concentrations of platelet-poor plasma, as has been previously demonstrated (Pledger et al. 1977; Graves et al. 1983). Low concentrations of PDGF (0.6 &ml) were stimulatory, while 6.0 r&ml of PDGF stimulated cellular proliferation to the same extent as cells incubated in 10% FBS. The response we observed for fibroblasts is consistent with previous reports that the mitogenic effect of PDGF for other cells of mesenchymal origin, such as smooth muscle cells, fibroblasts, and glial cells, is maximized between 2-6 rig/ml (Ross et al. 1974; Westermark and Wasteson 1976; Pledger et al. 1977; Raines and Ross 1982). The above results indicate that normal human tibroblasts are more responsive to physiologic concentrations of PDGF than the bone-derived cells. Since bone-derived cells were apparently able to utilize the growth factors present in platelet-poor plasma and proliferate, the response of these cells to platelet-poor plasma

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Fig. 1. Proliferation of bone-derived cells and tibroblasts in response to PDGF. Bone-derived cells (A) and tibroblasts (B) were plated at subconfluence in 10% FBS, incubated overnight and changed to DMEM supplemented with 4% platelet-poor plasma (PPP) and 0, +; 0.6, 0; and 6.0 r&ml PDGF, Cl; or DMEM supplemented with 10% FBS, W. Cells were counted on days 0, 3, 6 and 9.

was further examined. Platelet-poor plasma stimulated a dose dependent increase in the number of bone cells, with 10% platelet-poor plasma demonstrating greater than a 700% increase over six days, which was still less stimulatory than 10% FBS (Table I). Autoradiographic studies were undertaken to assess the relative number of cells responding to PPP (Table II). When cells were incubated in 3% platelet-poor plasma only 1.2% of the normal human tibroblast nuclei were labeled, in contrast to bone-derived cells in which 8.8% of the nuclei were labeled. This indicates that a considerably larger number of bone-derived cells are synthesizing DNA under conditions in which fibroblasts are quiescent. Experiments with cells tested Table I. Growth of bone-derived

Medium DMEM 3% PPP/DMEM 10% PPP/DMEM 10% FBSlDMEM

cells in platelet-poor plasma.

Cell number 4333 27,166 72,500 146,500

Percent change in cell number over 6 days -50 +200 + 723 + 1565

Day 0 cell counts were 8800 cells/well. Following incubation with the indicated culture media the cell number was determined after 6 days. Each number represents the mean of duplicate wells. Duplicates agreed within 10% of the mean.

134

J. E. Piche and D. T. Graves: Bone cell response to growth factors

under defined conditions provide further insight into this response. When incubated in insulin and transferrin in lieu of platelet-poor plasma, all normal human fibroblasts and bone-derived cells were virtually quiescent, with 0.9% and 0.6% respectively having labeled nuclei. This would suggest that there are factors in platelet-poor plasma other than insulin, insulin-like growth factors, or transferrin which support DNA synthesis in bone-derived cells but not normal human fibroblasts. Since cells responding in a wound healing environment are exposed to multiple growth factors, the response of normal human fibroblasts and bone-derived cells to combinations of growth factors was tested (Fig. 2, Table III). The concentrations of growth factors used in this series of experiments were based on concentrations previously reported to be stimulator-y for a variety of cell types of mesenchymal origin (Antoniades et al. 1979; Heldin et al. 1979; Roberts et al. 1981; Raines and Ross 1982; Wrana et al. 1986; Centrella et al. 1987). In addition, the concentrations of TGF-B and EGF-like activity chosen are consistent with the concentrations that may be achieved in clotted serum in vivo (Carpenter and Cohen 1979; O’Connor-McCourt and Wakefeld 1987). In the experiment described in Fig. 2 and Table III, growth factors were added to DMEM supplemented with platelet-poor plasma (2%). The concentrations of EGF and TGF-B selected were shown to only slightly enhance the proliferation of bone-derived cells above the negative control when tested singly (data not shown). EGF and TGF-B were then utilized in multiple combinations to examine their ability to supplement and enhance the response of bone-derived cells and normal human fibroblasts to PDGF. The least stimulatory growth factor combination was EGF (2.5 rig/ml) and TGF-B (7.0 ng/ml). This growth factor combination induced only a slight increase above the negative 2% platelet-poor plasma control, which was not statistically significant (Table IIIB). The combination of PDGF (2.5 ngiml) and either EGF (2.5 ng/ml) or TGF-B (7.0 ng/ml) stimulated an intermediate level of cellular proliferation of bone-derived cells. Only when PDGF, EGF and TGF-l3 were added together did the bone-derived cell population demonstrate a response equal to 10% FBS. For the normal human fibroblast population, Table II. Mitogenic response of bone-derived fibroblasts to platelet-poor plasma. Bone-derived

cells and

Insulin/transfenin PPP FBS

BONE-DERIVED

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any combination of growth factors which included PDGF (2.5 &ml) resulted in a statistically significant increase (p < .Ol) in cellular proliferation equal to that seen in 10% FBS (Fig. 2B, Table IIIB). When EGF (2.5 ng/ml) and TGF-B (7.0 &ml) were incubated in the absence of PDGF no increase in fibroblast proliferation above the 2% platelet-poor plasma negative control was observed. Additional studies were carried out to measure the response of bone-derived cells to growth factors under defined conditions (Table IV). In these experiments growth factors were added to DMEM supplemented with 0.1% crystalline bovine serum albumin (BSA) in the absence of plasma or serum factors. The results were similar to those obtained for cellular proliferation. No single growth factor maximally stimulated DNA synthesis in the bone-derived cells, with TGF-B, IGF and EGF all slightly stimulating DNA synthesis. Of the growth factors tested, PDGF induced the largest increase, but was still less mitogenic than 10% FBS. The addition of either EGF (8 &ml) or IGF (20 &ml) to PDGF (8 ng/ml) did not further increase DNA synthesis in bone-derived cells. The combination of all three (EGF (8 ng/ml), TGF-B (8 ng/ml) and PDGF (8 &ml)) stimulated greater than a 400% increase in DNA synthesis, which was statistically equivalent to the level induced by 10% FBS. DNA synthesis equal to the 10%

Percent labeled nuclei mean 2 S.E.M. 0.5 ? 0.18 8.8 2 0.85 62.0 f 1.45

40000

30000

Fibroblasts Medium

Insuiinltransfenin PPP FBS

Percent labeled nuclei mean 2 S.E.M. 0.9 2 0.44 1.2 2 0.30 71.7 t 5.81

Cells were incubated in DMEM supplemented with insulin (5 UgIml) and transferrin (5 Ug/ml), 3% PPP, or 10% FBS for 72 hours. The culture medium was changed and DNA synthesis was measured by autoradiography. Each number represents the mean of triplicate wells i standard error of the mean. Triplicates agreed within 10% of the mean.

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Fig. 2. The proliferation of bone-derived cells and fibroblasts in response to combinations of growth factors. Bone-derived cells (A) and fibroblasts (B) were plated as described in Fig. I. Cells were incubated in 2% PPP, +; or 2% PPP supplemented with: PDGF alone (2.5 ngiml), A; EGF (2.5 ngiml) and TGF-B (7.0 ng/ml), x ; PDGF (2.5 ng/ml) and EGF (2.5 &ml), + ; PDGF (2.5 ng/ml) and TGF-B (7.0 ngiml). 0; TGF-g (7.0 ngiml), PDGF (2.5 ng/ml) and EGF (2.5 ngiml), q, or in DhIEM supplemented with 10% FBS. n .

J. E. Piche and D. T. Graves: Bone cell response to growth factors

FBS positive control was also achieved when cells were exposed to PDGF, IGF, EGF, and TGF-B simultaneously.

Discussion Cells derived from soft tissue have been extensively studied for their response to paracrine factors @porn and Roberts 1986). The results obtained in these studies have been useful in designing experiments to examine the response to growth factors in vivo @porn et al. 1983; Grotendorst et al. 1984; Buckley et al. 1985; Roberts et al. 1986; Lynch et al. 1987). In contrast, the growth factor response of bone-derived cells is less well characterized. A comparison of in vitro studies measuring the growth factor response of bone cells is complicated by significant differences in the source of bone cells. These studies have frequently utilized immortalized bone cell lines (Graves et al. 1983; Ng et al. 1983; Robey et al. 1987), bone organ cultures (Canalis 1981; Stracke et al. 1984; Tashjian et al. 1985; Pfeilschifter et al. 1987) or embryonic rat cells (Schmid et al. 1983; Hanks et al. 1986; Centrella et al. 1987). In addition, each of the above target cells has specific limitations in applying the results to adult human bone. Immortalized cell lines have been shown to have aberrant expression of oncogenes, and thus, may have altered growth factor responses (Weinberg 1985). In bone organ cultures it is difficult to assess the phenotype of the cell population and it has not been demonstrated that the response of embryonic rat bone cells is similar to adult human bone cells. Recently, non-transformed bone cells explanted from normal human bone tissue have been shown to have a stable osteoblast-like phenotype (Beresford, Gallagher et al. 1983; Beresford, MacDonald et al. 1983; Auf’mkolk et al. 1985) and have been used to study normal bone cells in vitro. These cells have been utilized to Table III. Proliferative response of bone-derived A. Bone-derived

study the response of normal human bone cells to 1,25 dihydroxyvitamin D3 (Skjodt et al. 1985; Beresford et al. 1986), parathyroid hormone (Beresford, Gallagher, Poser et al. 1984; Hesch et al. 1984) and interleukin 1 (Beresford, Gallagher, Gowan et al. 1984), as well as the synthesis and regulation of osteocalcin (Beresford, Gallagher, Gowan et al. 1984; Beresford, Gallagher, Poser et al. 1984; Auf’mkolk et al. 1985; Skjodt et al. 1985), collagen (Beresford, Gallagher, Gower et al. 1984; Auf’mkolk et al. 1985), glucocorticoids (Gallagher et al. 1982, 1984), and prostaglandins (Beresford, Gallagher, Gowan et al. 1984; MacDonald et al. 1984) in vitro. We report here the mitogenic response of similar human bone cells to growth factors when cultured in low concentrations of platelet-poor plasma and under defined conditions. To our knowledge this report represents the first test of the platelet-derived growth factor response of cells obtained from explants of normal human adult bone. These data ought to provide insight into paracrine factors that might interact to stimulate proliferation of bone cells in osseous wound healing. In addition, they suggest growth factors that might be useful for culturing bone cells under defined conditions. Results presented here demonstrate that bone-derived cells proliferate in low concentrations of platelet-poor plasma. Moderate concentrations of PDGF (0.6 ng/ml) did not stimulate proliferation of bone cells, while higher concentrations of PDGF (6.0 ng/ml) stimulated cell proliferation but to a lesser extent than cells incubated in 10% FBS. Canalis has previously reported that high concentrations of PDGF are required to stimulate DNA synthesis in bone organ culture (Canalis 1981). TGF-B and EGF were mildly stimulatory although considerably less so than PDGE Only when a combination of growth factors was tested (PDGF, TGF-P and EGF) did the proliferation of bone cells equal that of cells incubated in 10% FBS. Similar results were observed when growth factors were tested for stimulating

cells and tibroblasts to multiple growth factors.

ceils

Sample 2% PPP TGF-B + TGF-B + PDGF + TGF-B + 10% FBS

135

EGF PDGF EGF PDGF + EGF

Cell number mean i- S.E.M. 39,667 54,333 73,333 75,667 97,000 101,333

+ k 2 ? 2 2

1201 1453 1764 1202 3000 4055

Statistically significant compared to: 2% PPP

10% FBS

NS NS * * 1 *

* * * * NS NS

B. Fibroblasts

Sample 2% PPP TGF-B + TGF-B + PDGF + TGF-B + 10% FBS

EGF PDGF EGF PDGF + EGF

Cell number mean 2 S.E.M. 17,000 20,000 48,333 48,333 52,667 51,667

k 577 ” 577 2 1333 + 882 k 1333 2 882

Statistically significant compared to: 2% PPP

10% FBS

NS NS * * * *

* * NS NS NS NS

Day 0 cell counts were 13,000 cells/well for bone-derived cells and 18,000 cells/well for tibroblasts. Cells were incubated in 10% FBS, 2% PPP or 2% PPP supplemented with the following growth factors in combination: 7.0 &ml TGF-B, 2.5 ng/ml PDGF, 2.5 ng/ml EGE Each value represents the mean of triplicate samples ? the standard error of the mean obtained from the data on day 9 in Figs. 2A and 2B. Statistical significance was determined between groups using Scheffe’s F-test at the p < 0.01 level. Values that are significantly different are denoted by (*). NS indicates not significant

136

J. E. PichC and D. T. Graves: Bone cell response to growth factors

Table IV. The mitogenic response of bone-derived

Treatment 0.1% BSA EGF IGF PDGF TGF-P PDGF + IGF PDGF + EGF PDGF + EGF + TGF-P PDGF + EGF + TGF-P + IGF 10% FBS

cells to growth factors under defined conditions.

CPM ? S.E.M. 757 916 1103 3233 1374 3031 2752 3886 4845 5035

k k + + 2 2 f f + 2

127 131 147 484 104 139 100 304 458 156

Percent increase

Statistically significant compared to: Negative control 10% FBS

-

NS

21 46 327 82 300 263 413 540 565

NS NS *

* * *

NS * * * *

* * *

*

*

NS

NS NS

Bone-derived cells were plated at 10,000 cells/well and tested at confluence. Cells were depleted of serum factors for 48 hours in DMEM and 1% platelet-poor plasma and then incubated in DMEM containing 0.1% BSA with and without the addition of growth factors for 24 hrs. The concentrations of growth factors were as follows; IGF-1, 10 &ml; PDGF, 8 ng/ml; EGF, 10 ng/ml; TGF-P, 8 &ml. DNA synthesis was measured by 3H Thymidine incorporation. Each value represents the mean of triplicate samples 2 the standard error of the mean. Statistical significance was determined using Scheffe’s F-test at the p < 0.01 level. Values that are significantly different are denoted by (*). NSTndicates not statistically significant.

DNA synthesis in these cells under defined conditions. Centrella and co-workers (1987) reported that TGF-P alone is highly mitogenic for cells obtained from embryonic rat calvaria. The difference between our results and Centrella and co-workers probably reflects differences in the source of bone cells and assay conditions. Interestingly, Robey et al. (1987) have recently reported that the mitogenic effect of TGF-P on fetal bovine cells is highly influenced by cell density. The growth factor response that we observed for human bone cells consistently differed from the response that we and others have observed for fibroblasts (Antoniades and Owen 1982; Heldin et al. 1985). These results point out a significant difference in the growth factor requirements of bone-derived cells and fibroblasts. First, normal human fibroblasts do not proliferate in basal medium supplemented with low concentrations of platelet-poor plasma, while bone-derived cells do. This suggests that platelet-poor plasma contains factors which are stimulatory for bone-derived cells, but not fibroblasts. Second, concentrations of PDGF (6.0 ng/ml) which maximally stimulates DNA synthesis in other cells of mesenchymal origin stimulated the proliferation of normal human fibroblasts to the same extent as 10% FBS, while the same concentration of PDGF was less mitogenic for bone-derived cells when tested simultaneously. When PDGF was further supplemented with EGF and TGF-P, the rate of proliferation of bone cells was equal to the rate observed for 10% FBS. This additional supplementation did not enhance the proliferation of normal human fibroblasts. There are several explanations for the difference in response between normal human bone-derived cells and normal human fibroblasts. It is possible that the sub-optimal response of bone-derived cells to PDGF reflects a decreased sensitivity of bone cells to PDGF compared to fibroblasts. Alternatively, there could be a qualitative difference in the growth factor requirement of bone-derived cells and fibroblasts so that growth factors present in low concentrations of platelet-poor plasma enable fibroblasts to respond maximally to 6.0 ng/ml PDGF while they do not for bone-derived cells. This explanation is supported by evidence that supplementation of platelet-poor plasma with IGF, EGF, and TGF-P enables bone-derived cells to respond maximally to PDGE For normal human fibroblasts this supple-

mentation to platelet-poor plasma did not enhance the response to PDGF. Therefore, bone-derived cells may require multiple growth factors for optimal growth. A third interpretation of the above results is that the response observed for bone-derived cells reflects different growth factor requirements for different cell types within a heterogeneous cell population. It is known that in vitro bone cell populations contain a heterogeneous mixture of cells at various stages of the osteoblast lineage (Canalis 1985). If each cell type were to have different growth factor requirements then multiple growth factors may be needed to satisfy the proliferative requirements of each sub-population. If this is the case, the differential response to defined growth factors may be useful in the selection of different bone-derived sub-populations. The examination of a population of bone cells presumed to be at various stages in the osteoblast lineage may be appropriate for studying the response to growth factors, since bone cells at various stages of differentiation may be directly or indirectly involved in osseous wound healing. In fact, Raisz and Kream (1984) have proposed that the proliferation of immature osteoblasts indirectly contributes to bone formation following bone resorption. Knowledge of the in vitro response of normal bone-derived cells to growth factors may be useful in devising strategies for utilizing growth factors to stimulate bone formation in in vivo experiments. Our results would suggest that a combination of growth factors, which include platelet-derived growth factor, would be most stimulatory. This conclusion is not surprising since platelet-derived growth factor is released upon platelet degranulation at sites of injury, along with transforming growth factor beta and a high molecular weight epidermal growth factor-like protein. The above data may also be useful in developing defined culture conditions for bone-derived cells. In order to thoroughly characterize the response of bone cells to systemic and local factors, cultures of normal human bone cells must be developed in vitro. In vitro analysis facilitates quantitation of ligand-receptor interactions, examination of co-operative or inhibitory effects of different cell modulators, investigation of secondary messages, and the study of induced gene expression. Quantitation of the mitogenic effect of growth factors under defined conditions

J. E. Piche and D. T. Graves: Bone cell response to growth factors may assist in developing culture conditions that facilitate extensive and reproducible examination of normal human bone cells.

Acknowledgments: We would like to thank Kazi Begum for providing expert technical assistance and Dr. Gregory Mundy for helpful discussions in preparing this manuscript. These studies were funded by NIH grant DE07559 and the Air Force Institute of Technology.

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Received: February 19, 1988 Revised: December 5, 1988 Accepted: December 27, 1988