Search PubMed⌕ Search

Biomedical subjects

D J Baylink

Publications and source records attributed to D J Baylink.

At least 199 records · Page 11Linked to original sources

Quantitation of growth factors in ossein-mineral-compound.

Study was undertaken to identify polypeptide factors in the commercially available ossein-mineral-compound and to see if they are present in a biologically relevant quantity. Using the guanidine-EDTA extraction, 35.7 +/- 0.1 mg proteins were obtained from 1 g of the ossein-mineral-compound. At concentration 1 micrograms/ml, guanidine-EDTA-extractable proteins stimulated the incorporation of thymidine into DNA by human bone cells to 581 +/- 122% (p less than 0.001) of that by bovine serum albumin-treated control cells, decreasing thereafter. Similarly, it stimulated the activity of alkaline phosphatase in the human bone cells. Growth factors IGF-I, IGF-II, and TGF-beta were identified in the ossein-mineral-compound. This leads to speculation regarding possible role of growth factors in explaining the beneficial effects of the compound in retarding bone loss in patients with osteoporosis.

Animals↗

Contribution of marrow stromal cells to the regulation of osteoblast proliferation in rats: evidence for the involvement of insulin-like growth factors.

Fibroblast-like marrow stromal cells are believed to play a role in the maintenance of osteoblast populations at the marrow-bone interface. We now report that this interaction may be very specific. Stromal cell conditioned medium (SC-CM) stimulated DNA synthesis and proliferation in culture of neonatal rat calvarial osteoblasts at low concentrations (1.25-5%), but was inhibitory at 10%. At growth promoting effective concentrations, the activity of osteoblast alkaline phosphatase was decreased. This action was selective since SC-CM failed to promote the growth of rat calvarial fibroblasts. Characterization of the SC-CM indicated the cells produced IGF-I and -II and a wide range of molecular weight fractions with putative stimulatory action (FPLC analysis using Superose 12 and 6 gel permeation columns). HPAE-PAD analysis showed that some elements were glycosylated, and the composition suggested the presence of N- and O-linked oligosaccharide chains. Because rat marrow stromal fibroblast-like cells produce a number of osteotropic factors which affect calvarial osteoblast growth, these interactions may be important to considerations about the etiology of the osteoporoses.

Alkaline Phosphatase↗

Effects of medroxyprogesterone acetate on some parameters of calcium metabolism in patients with glucocorticoid-induced osteoporosis.

Long-acting medroxyprogesterone acetate (MPA) effect on some important parameters of calcium metabolism in patients with glucocorticoid-induced osteoporosis (GCO) was evaluated. Twelve steroid-dependent asthmatic male patients with GCO were administered 200 mg of MPA (Depo-Provera) intramuscularly, and had fasting serum samples obtained at baseline and at weekly intervals for 5 consecutive weeks. Baseline serum samples were also obtained from 12 control healthy male subjects matched for age. The following measurements were made from each serum sample: osteocalcin (OC), skeletal (SAP) and total alkaline phosphatase (TAP), calcitonin (C), insulin-like growth factor I (IGF1), 1,25-dihydroxyvitamin D and 25-hydroxyvitamin D. Significantly lower baseline serum levels of OC and C were found in the patients with GCO than in controls (P less than 0.001). Following MPA administration in GCO patients statistically significant and sustained increases in OC, SAP and C were noticed during the next 5 weeks. No significant differences in baseline levels for TAP, IGF1, 1,25(OH)2D and 25(OH)D between GCO patients and controls were found, and no significant changes following MPA administration in GCO patients were obtained for these parameters. In conclusion, when administered to patients with GCO, MPA seems to stimulate the osteoblastic activity as suggested by sustained increases in OC and SAP serum levels, and also enhances the C production by the C-cells of the thyroid.

Aged↗

1,25-Dihydroxyvitamin D3 differentially regulates the production of insulin-like growth factor I (IGF-I) and IGF-binding protein-4 in mouse osteoblasts.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces differentiation and inhibits proliferation in many cell types including bone cells. These effects may be mediated by the modulation of the insulin-like growth factor (IGF) regulatory system. Therefore we investigated the effects of 1,25-(OH)2D3 on transcript and protein levels of both IGF-I and IGF binding proteins (IGFBPs) in clonal mouse osteoblasts. Subconfluent cultures were treated in serum-free medium with 1,25-(OH)2D3. Secreted IGF-I was measured using a RIA under conditions eliminating the interference of IGFBPs. 1,25-(OH)2D3 (10(-11)-10(-8) M) inhibited IGF-I release in a dose dependent manner at 24 h (maximally to 30 +/- 5% of control, mean +/- SEM of seven independent experiments). In a time course study IGF-I increased in the media of control cultures over a 48-h period, while IGF-I secretion was completely prevented from 6 h onward in 1,25-(OH)2D3 treated cultures. Northern blot analysis revealed four IGF-I transcripts of 0.9, 1.8, 4.4, and 7.5 kilobases (kb). 1,25-(OH)2D3 decreased levels of the 7.5 kb IGF-I transcript from 4-48 h, with maximal inhibition occurring at 24 h (25% of control). Western ligand blots of the culture medium demonstrated secretion of a 25-kilodalton IGFBP, which comprised greater than or equal to 90% of the secreted IGFBPs. The 25-kilodalton IGFBP had previously been shown to have sequence similarity with IGFBP-4, a binding protein which inhibits the action of IGFs on bone cells. 1,25-(OH)2D3 treatment increased secretion of IGFBP-4 up to 14-fold over 24 h. 1,25-(OH)2D3 also increased IGFBP-4 (2.2 kb) transcript levels within 30 min, with the maximal stimulation of 8-fold occurring after 8 h. [3H]Thymidine incorporation into cells was inhibited by 1,25-(OH)2D3 both under basal and serum-stimulated conditions. Our results are consistent with the hypothesis that the effects of 1,25-(OH)2D3 on osteoblast proliferation may be mediated in part by decreased levels of IGF-I and increased concentrations of inhibitory IGFBP-4. It is proposed that this alteration in the IGF system may be an important functional autocrine or paracrine switch in the transition of osteoblasts from states of proliferation to differentiation.

Animals↗

Insulin-like growth factor II and transforming growth factor beta 1 regulate insulin-like growth factor I secretion in mouse bone cells.

Bone cells in culture produce and respond to growth factors, suggesting that local as well as systemic factors regulate bone volume. Previous studies have shown that IGF-I is the major mitogen produced by mouse bone cells and that its production is regulated by systemic agents such as PTH and estrogen. Because IGF-II and transforming growth factor beta 1 have been shown, respectively, to increase and decrease MC3T3-E1 cell proliferation, we tested the hypothesis that these two growth factors modulate the production of IGF-I in this cell line. In order to eliminate artifacts owing to IGF binding proteins, conditioned media samples were pretreated with IGF-II before measurement of IGF-I by RIA. After 24 h treatment at a density of 2.5 x 10(4) cells/cm2, IGF-II (10 micrograms/l) induced a 2.2-fold increase compared with untreated control (9.5 +/- 1.5 vs 4.2 +/- 0.44 pg/micrograms protein, p less than 0.001), whereas transforming growth factor beta 1 (1 microgram/l) caused a 66% decrease in IGF-I production (1.5 +/- 0.3 vs 4.2 +/- 0.44 pg/micrograms protein, p less than 0.001). Both IGF-II and transforming growth factor beta 1 regulated IGF-I production in a dose-, time- and cell density-dependent manner. The lowest effective doses for IGF-II and transforming growth factor beta 1 were 1 and 0.01 microgram/l, respectively. These results support a role for IGF-II and transforming growth factor beta 1 as potent modulators of IGF-I secretion in mouse bone cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Growth factor-induced proliferation of osteoblasts measured by bromodeoxyuridine immunocytochemistry.

Immuno-localization of BUdr was used to identify DNA synthesis in vitro in chicken embryonic bone cells stained positively or negatively for alkaline phosphatase activity. The results were similar to, but more sensitive than, our standard bioassay which assesses 3H-thymidine incorporation into DNA by liquid scintillation counting, and more rapid than autoradiographic localization of 3H-thymidine. SGF/IGF-II and bFGF stimulated cellular proliferation equally in ALP(+) and ALP(-) cells. In contrast, IGF-I and TGF-beta stimulate proliferation more in the ALP(-) than ALP(+) cells. The greatest increase in DNA replication of ALP(-) cells occurred following incubation with SGF/IGF-II or TGF-beta, and in the ALP(+) cells with SGF/IGF-II or bFGF. TGF-beta stimulated cellular proliferation at the lowest dose (1 ng/ml). The differential effect of the growth factors on each population of cells indicates that all these bone-matrix derived growth factors may play different roles in the local regulation of skeletal metabolism.

Alkaline Phosphatase↗

Evidence that the inhibition of TE85 human bone cell proliferation by agents which stimulate cAMP production may in part be mediated by changes in the IGF-II regulatory system.

Our previous studies have shown that insulin-like growth factor-II (IGF-II) is an important autocrine/paracrine regulator of human bone cell proliferation. In this study, we sought to look at the regulation of IGF-II production by human bone cells since IGF-II synthesis is a key variable that regulates the actions of IGF-II at a local site of bone. For studies of IGF-II regulation, we used TE85 human osteosarcoma cells as a model system since these cells exhibited several characteristics which are similar to that of untransformed normal human bone cells. In this study, we investigated the effects of agents which increase intracellular cAMP (forskolin, isobutylmethyl xanthine and prostaglandin E2) and N6,O2' dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) on the IGF-II regulatory system. It was found that these agents caused an increase in the production of an IGFBP in TE85 cells. Subsequent studies on the purification and characterization of IGFBP from DBcAMP treated TE85 cell conditioned medium revealed that the IGFBP produced by TE85 cells in response to DBcAMP treatment was identical to that of 25 kDa inhibitory IGFBP (now designated as IGFBP-4) purified from TE89 human bone cells based on: 1) N-terminal amino acid sequence, 2) amino acid composition, 3) molecular weight and 4) inhibitory actions on basal and IGF-II induced bone cell proliferation. In addition, forskolin and DBcAMP also caused a dose dependent decrease in the production of IGF-II. Consistent with these results, DBcAMP and agents which increase intracellular cAMP inhibited TE85 cell proliferation in a dose dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗

The effects of estrone (Ogen) on spinal bone density of postmenopausal women.

The effects of cyclical treatment with estrone sulfate (0.3, 0.625, or 1.25 mg), plus calcium carbonate, on spinal trabecular bone density were compared with placebo in 120 postmenopausal women in this 2-year, multicenter, double-blind study. While the placebo and 0.3-mg treatment groups lost bone density (-3.6% and -5.1%), the 0.625- and 1.25-mg treatment groups experienced no significant change from baseline at 24 months (-0.8% and +0.7%). The 1.25-mg treatment group was significantly different from the placebo group at 12, 18, and 24 months. Although the 0.625-mg treatment group was significantly different from the placebo group only at 18 months, the data suggest that 0.625 and 1.25 mg of estrone sulfate had different effects than placebo and 0.3 mg of estrone sulfate and, given with supplemental calcium, are effective doses for the prevention of spinal bone loss.

Bone Density↗

Bone growth factors.

Bone volume is determined by the relative rates of bone formation and bone resorption. Recent research in several laboratories suggests that growth factors may act locally to modulate bone formation by stimulating osteoblast proliferation and activity. A number of bone-derived growth factors have been isolated and characterized from bone matrix extracts and from media conditioned by bone cells and bone organs in culture. The growth factors found in bone matrix include insulinlike growth factors I and II, transforming growth factor-beta, acidic and basic fibroblast growth factor, platelet-derived growth factor, and bone morphogenetic proteins. Conditioned medium from bone cells contains several of these growth factors and also hematopoietic factors. These bone matrix-derived growth factors have different biologic activities, including mitogenic, differentiating, chemotactic, and osteolytic activities. Evidence suggests that bone cells produce substantial quantities of growth factors for extracellular storage in bone matrix. Apart from being produced for extracellular storage, it is possible that growth factors secreted by bone cells have acute effects on their neighboring osteoblastic cells, i.e., paracrine action, or on themselves, i.e., autocrine action. The release of matrix-stored growth factors by bone resorption may mean that growth factors act as delayed paracrine agents, e.g., osteoblasts deposit growth factors in bone and later when these growth factors are released from bone via bone resorption, the growth factors stimulate osteoblast precursors to proliferate. The findings that bone is a storehouse for growth factors and that bone cells in culture produce and respond to bone growth factors suggest bone growth factors may act as potential determinants of local bone formation. This review is focused on the structure, regulation, and biologic actions of the known bone growth factors.

Animals↗

The effects of fluoride on bone.

Fluoride has often been used as a treatment for osteoporosis, a metabolic bone disease of considerable importance in the elderly population. The techniques currently used to monitor a patient's response to fluoride are outlined. New findings concerning (1) a mechanism for interaction of fluoride with osteoblasts (via mitogenic signals or growth factors); (2) toxicity and carcinogenesis; (3) recent clinical trial data; and (4) the importance of dosage, administration regimens, and side effects in an effective fluoride treatment protocol are reviewed. Some recent clinical data challenge the efficacy of fluoride in the treatment of postmenopausal osteoporosis. Because of the implications of these recent studies with respect to fracture incidence during fluoride therapy, fluoride cannot be recommended at this time for general use in the treatment of osteoporosis.

Aged↗

Human prostatic cancer cells, PC3, elaborate mitogenic activity which selectively stimulates human bone cells.

Prostatic cancer typically produces osteoblastic metastases which are not attended by marrow fibrosis (i.e., osteoblast but not stromal fibroblast proliferation). In the present study we sought to test the hypothesis that prostatic cancer cells produce factor(s) which act selectively on human osteoblasts. Such a paracrine mechanism would explain the observed increase in osteoblasts, unaccompanied by an increase in marrow fibroblasts. To test this hypothesis we investigated the mitogenic activity released by the human prostatic tumor cell line, PC3. PC3 cells have been reported previously to produce mitogenic activity for cells that was relatively specific for rat osteoblasts compared to rat fibroblasts. However, the effects of this activity on human cells has not been examined previously. PC3-conditioned medium (CM) (5-50 micrograms CM protein/ml) stimulated human osteoblast proliferation by 200-950% yet did not stimulate human fibroblast proliferation [( 3H]thymidine incorporation). PC3 CM also increased cell numbers in human osteoblast but not fibroblast cell cultures. To determine whether the osteoblast-specific mitogenic activity could be attributed to known bone growth factors, specific assays for these growth factors were performed. PC3 CM contained 10 pg insulin-like growth factor (IGF) I, less than 2 pg IGF II, 54 pg basic fibroblast growth factor, and 16 pg transforming growth factor beta/microgram CM protein. None of these growth factors alone or in combination could account for the observed osteoblast-specific PC3 cell-derived mitogenic activity. Furthermore, when 5 micrograms/ml PC3 CM was tested in combination with maximally effective concentrations of either basic fibroblast growth factor, IGF I, IGF II, or transforming growth factor beta, it produced an additive effect suggesting that PC3 CM stimulates osteoblast proliferation by a mechanism independent of these bone mitogens. Biochemical characterization supported the hypothesis that the PC3 cell growth factor was unique from other growth factors. The PC3 growth factor did not bind to heparin and was resistant to acid as well as the reducing agent, dithiothreitol. Sephadex G-75 and fast protein liquid chromatography Mono S cation-exchange chromatography revealed the PC3-derived mitogen to be an Mr 26,000-30,000 basic protein. Therefore, we conclude that PC3 cells release a mitogen which exhibits higher specificity for human osteoblasts than human fibroblasts and is unique from other growth factors tested. Production of this mitogen by human prostatic carcinoma cells could play an etiological role in the intense osteoblast-specific stimulation that occurs at sites of bone metastases.

Animals↗

Purification and N-terminal amino acid sequence of the tartrate-resistant acid phosphatase from human osteoclastoma: evidence for a single structure.

Tartrate-resistant acid phosphatase type-5 was purified to apparent homogeneity from human osteoclastomas by sequential chromatography on CM-Sepharose, Phenyl-Sepharose, concanavalin A-Sepharose, FPLC Superose-12, and FPLC Mono-S. The purification over the original tissue extract was 1167-fold, with a yield of 16%. An identity in the N-terminal amino acid sequence and Mr was found between this enzyme and two type-5 tartrate-resistant acid phosphatases isolated from hairy cell leukemia spleen. However, they appeared to be different as assessed by amino acid composition. In contrast to a previous report, no evidence was found for two subunits of the tartrate-resistant acid phosphatase.

Acid Phosphatase↗

Skeletal growth factor and other growth factors known to be present in bone matrix stimulate proliferation and protein synthesis in human bone cells.

The purpose of the study was to investigate the effect of skeletal growth factor/insulinlike growth factor II and other growth factors known to be present in bone matrix on the proliferation and differentiation of human bone cells. Cells were isolated by collagenase digestion from femoral heads obtained during hip replacement operations. Cells were cultured in DMEM medium with 10% calf serum. Third to fifth passage cells were plated in multiwell plates and the medium changed to low serum (0.1%) for 2 days. The medium was changed to serum-free medium prior to addition of growth factors. Cell proliferation was measured by the incorporation of [3H]thymidine into DNA and by the percentage of cells that incorporate bromodeoxyuridine. Protein synthesis was measured by the incorporation of [3H]proline into trichloroacetic acid-precipitable material. Skeletal growth factor/insulinlike growth factor II and insulinlike growth factor I stimulated cell proliferation and protein synthesis in a dose-dependent manner. Alkaline phosphatase-specific activity was not increased by these factors. Transforming growth factor beta 1 did not affect cell proliferation but stimulated protein synthesis and increased the specific activity of alkaline phosphatase. Fibroblast growth factor did not affect any of the cell parameters. These studies suggest that skeletal growth factor/insulinlike growth factor II, insulinlike growth factor I, and transforming growth factor beta 1 may play a role in the local control of the proliferation and differentiation of human osteoblasts.

Alkaline Phosphatase↗

Quantitation of growth factors IGF-I, SGF/IGF-II, and TGF-beta in human dentin.

Human bone matrix is known to contain a battery of polypeptide growth factors. Since dentin is a mineralized tissue similar to bone in composition and perhaps in formation, human dentin was assayed for the presence of similar growth factors. Root dentin proteins were extracted by demineralization in 4 M guanidine hydrochloride (Gu) and 30 mM Tris (pH 7.4) containing 20% EDTA and proteinase inhibitors. Gu-EDTA extracts were desalted and used for the following assays: (1) bone cell proliferation in chick calvarial cell mitogenic assay using the incorporation of [3H]thymidine into TCA-insoluble material; (2) osteocalcin by radioimmunoassay (RIA); (3) insulin-like growth factor I (IGF-I) by RIA; (4) skeletal growth factor/insulinlike growth factor II (SGF/IGF-II) by radioreceptor assay; and (5) transforming growth factor beta (TGF-beta) by bioassay. Gu-EDTA extracts stimulated bone cell proliferation. At 10 micrograms/ml, dentin proteins increased the incorporation of [3H]thymidine by calvarial cells to 320% of that by BSA-treated control cells. Consistent with the presence of mitogenic activity, growth factors were found in dentin in the following concentrations (ng/micrograms Gu-EDTA protein): (1) IGF-I, 0.06; (2) SGF/IGF-II, 0.52; and (3) TGF-beta, 0.017. All three growth factors were present in concentrations lower than that found in human bone. Osteocalcin was detected at a concentration of 3.0 mg/g Gu-EDTA protein, also much lower than that in bone.

Calcium↗

The causes of bone scintigram hot spots in fluoride-treated osteoporotic patients.

We previously described new bone formation in fluoride-treated osteoporotic patients. Since then, several investigators have contended that fluoride-induced "hot spots," as seen on bone scintigrams, represent stress fractures. To further evaluate this issue we analyzed scintigrams, radiographs, and quantitative computer tomography (CT) scans of the spine and femoral condyles from 129 patients, obtained before and during therapy. Hot spots, new or of increased intensity, were more than twice as common in the weight-bearing peripheral skeleton than in nonweight-bearing sites (p less than 0.001). The hot spots were usually diffuse, multiple, bilateral, and mostly seen early in therapy, a pattern quite different from that expected of stress fractures. Previously, we postulated that mechanical stress and fluoride act synergistically to stimulate new bone formation. If this hypothesis were correct, we would expect to see a greater increase in femoral condyle bone density in patients with hot spots. Consistent with this hypothesis, patients who developed hot spots in the knees had a greater increase in condylar density (22 +/- 2.5 vs. 9 +/- 3.3 mg/cc) than those without hot spots and stress fractures were not seen in either group. Additionally, patients with more than 5 hot spots in the peripheral skeleton had greater increases in spinal and condylar density than those with fewer than 5 hot spots. Finally, stress fractures were found in less than 2% of our patients. While we do not exclude the possibility that some additional patients may have had stress fractures, our data support the hypothesis that the great majority of hot spots seen on the scintigrams are the sites of new bone formation.

Adult↗

Fluoride therapy for osteoporosis promotes a progressive increase in spinal bone density.

Since osteoporosis is a disease of diminished bone density, and since osteoporotic fractures occur most commonly in the spine, the ideal therapeutic agent for osteoporosis is one which can increase spinal bone density and thereby reduce the risk for vertebral fractures. In the current study we sought to examine the effect of fluoride therapy on spinal bone density utilizing quantitative computed tomography to measure changes in vertebral trabecular bone density during treatment with fluoride. A group of 61 postmenopausal osteoporotic females, aged 70 +/- 9 years, were treated with 34 +/- 7 mg elemental fluoride/day (equivalent to 75 +/- 15 mg NaF/day) and 1500 mg calcium/day for 19 +/- 6 months. Spinal bone density was increased within the first 6 months of fluoride therapy by 42% or 10 +/- 13 mg/cm3 (p less than 0.001) and continued to increased throughout 2 years of observation. The skeletal response to fluoride therapy was also associated with an early increase in serum alkaline phosphatase activity (p less than 0.001), which was related to the increase in spinal bone density (r = .58, p less than 0.001). Large interpatient variation was observed in the spinal bone response to fluoride therapy, which was not explained by variations in the pretreatment spinal bone density (r = .04), age of the patient (r = .15), or dose of fluoride (r = .16). Results from these studies demonstrate (1) the therapeutic value of fluoride to increase trabecular bone density linearly for 2 years in the osteoporotic spine and (2) the clinical value of measuring spinal bone density and/or serum alkaline phosphatase activity as indices of the skeletal response to fluoride.

Aged↗

Fluoride pharmacokinetics in good and poor responders to fluoride therapy.

In this study, the relationship between fluoride pharmacokinetics and the response in spinal bone density to fluoride treatment was studied in 14 patients with primary osteoporosis treated with fluoride for at least 1 year. Serum concentrations and urinary excretion of fluoride were determined after ingestion of 10 mg fluoride as monofluorophosphate. The pharmacokinetic parameters were calculated according to a linear one-compartment open model. The fasting serum fluoride level was 8.8 +/- 0.98 mumol/liter. The peak serum fluoride level was 20.5 +/- 1.4 mumol/liter and was reached within 2 h after ingestion of fluoride. When the patients were divided into good and poor responders, based on whether they did or did not exhibit a change in spinal bone density of 13 mg/cc per year or more, we found that good responders had decreased renal fluoride clearance (-62 +/- 13%, p less than .02), increased maximum change in serum fluoride (+38 +/- 18%, p less than .01), increased extrarenal clearance (+62 +/- 57%, p less than .05) and increased change in serum alkaline phosphatase (ALP) (+241 +/- 169%, p less than 0.02) compared with poor responders. Our data suggest that one factor accounting for a good response is a relatively high serum level of fluoride. However, although the maximum change in serum fluoride was greater in good responders compared with poor responders, variations in fluoride levels could not explain all of the variation in spinal bone density. Therefore, we propose that in addition to differences in serum fluoride, other factors are also responsible for the good response.

Aged↗

Mitogenic action(s) of fluoride on osteoblast line cells: determinants of the response in vitro.

Clinically effective (osteogenic) concentrations of fluoride (5-30 microM) also have direct effects on skeletal tissues in vitro, to increase bone formation and osteoblast line cell proliferation. The effect on cell proliferation was specific for bone cells, modulated by systemic skeletal effectors, and dependent on (a) the [Pi] in the medium, (b) the presence of a bone cell mitogen, and (c) mitogen-responsive osteoprogenitor cells. Together, these data indicate that fluoride increases bone formation in vitro by increasing osteoprogenitor cell proliferation and that fluoride increases osteoprogenitor cell proliferation by enhancing the activity of bone cell mitogens.

Alkaline Phosphatase↗