Effects of serum from rabbits of various ages on cell proliferation

The report of Carrel and Ebeling (J. Exp. Med., 34 (1921) 599-623) generally gives the impression that both serum and blood plasma from old animals inhibit cell proliferation. For confirmation of this, we examined the effects of serum from rabbits of various ages on rabbit fetal skin fibroblasts (RSF cell) and human fetal lung fibroblasts (TIG-1 cell). Serum from young rabbits 8 months of age stimulated proliferation of RSF cells just as did fetal bovine serum, but that from old rabbits 5-7.8 years of age was found to significantly increase proliferation more than serum from the young. This was also the case when using TIG-1 cells. The lesser effect on cell proliferation by young serum apparently does not arise from growth-inhibitory factor(s) in the blood components. An examination showed young serum to possibly contain fewer growth-stimulatory factor(s) than old serum. On the basis of our data, we concluded that old rabbit serum stimulates, not inhibits, the proliferation of RSF and TIG-1 cells.     

Effects of serum from human subjects of different ages on migration in vitro of human fibroblasts

A study was carried out to determine whether human serum from older subjects inhibited cell migration. Sera of both sexes from subjects in their 60s (60-64 years) tended to be more inhibitory (8-14%) to the migration of human fetal lung fibroblasts, TIG-1, than serum from subjects in their 20s (20-29 years). In the case of females, the effects of serum on cell migration were significantly (P less than 0.05) different between the younger and older groups. Next, cell migration-stimulatory activity of serum was measured using human skin fibroblasts from young adult (age 21) and elderly (age 65) donors. The results were similar to those obtained with TIG-1 cells. However, the cell migration-stimulatory activity of serum was not significantly different between the two age groups. A study on the effects of concentration of human serum on the migration of TIG-1 cells showed that cell migration-stimulatory activity of serum declined linearly with increasing concentrations of sera from subjects in their teens (16-19 years) and 50s (50-59 years), and was the same between the two age groups. These results imply that substance(s) inhibitory to cell migration may not have accumulated in serum during the ageing process in humans, although human serum contained substance(s) inhibitory to cell migration.     

Changes in the migratory ability of human lung and skin fibroblasts during in vitro aging and in vivo cellular senescence.

The migration of human lung and skin fibroblasts was determined during in vitro aging and in vivo cellular senescence by measuring their migration from the edge of a denuded area of a monolayer. The migration of human fetal lung fibroblasts (TIG-1 and TIG-3) decreased only very slightly with increasing passage, whereas the migration of human fetal skin fibroblasts (TIG-3S) declined gradually: the difference in cell migratory ability between early and late passages was significant (P less than 0.05). The migratory patterns of skin fibroblasts from adult and elderly donors were also similar to that of fetal skin fibroblasts. Next, the migratory abilities of fibroblast lines from adult and elderly donor groups were compared, using relatively early passaged cells. The migratory ability of the elderly-donor skin fibroblast lines was significantly lower (P less than 0.05) than that of the adult-donor skin fibroblast lines. Addition of suramin and monensin suppressed the migration of fibroblasts from fetal, adult and elderly donors, which implies that fibroblast migration is regulated by growth factors and matrix substances. The relationships between the age-dependent decline of migratory ability, growth factors and the extracellular matrix are discussed.     

Failure in S6 protein phosphorylation by serum stimulation of senescent human diploid fibroblasts, TIG-1

When quiescent young or senescent human diploid cells, TIG-1, were metabolically labeled with 32Pi and stimulated with 10% fetal bovine serum, the phosphorylation of ribosomal S6 protein was enhanced in young cells but not in senescent cells while that of some other proteins were increased in both cells. Inability to stimulate the phosphorylation of S6 protein in senescent cells after serum addition may be the primary cause of the failure of enhancement in protein synthesis followed by the block of prereplicative events dependent on protein synthesis and thus of the failure of cells to enter S phase. However, when the cell-free preparation from serum-stimulated senescent cells was incubated with [gamma-32P]ATP, S6-kinase activity was stimulated and S6 in ribosomal fraction was susceptible to phosphorylation as observed in young cells. Differences in S6 phosphorylation of senescent cells between in vivo and in vitro was discussed.     

Effects of different protein supplements on mitogen responses of human peripheral blood lymphocytes

We evaluated the effects of 6 supplements often used in human lymphocyte cultures, including fetal calf serum, autologous human serum, pooled human AB serum, hypogammaglobulinemic human serum, bovine serum albumin and human serum albumin. Lymphocyte proliferation of unstimulated and mitogen activated peripheral blood mononuclear cells was measured by [³H]thymidine incorporation. The responses of cells stimulated with the T-cell mitogen phytohemagglutinin-P were significantly lower when cultured in bovine serum albumin supplemented media, but were otherwise not supplement dependent. In contrast, responses of cells stimulated with the B-cell mitogens Cowan I strain of S. aureus and antisera against the μ or δ chain of human immunoglobulin were significantly effected by supplement. Cultures containing fetal calf serum and bovine serum albumin had high background responses without a proportional rise in cellular proliferation when B-lymphocyte-specific mitogens were utilized. Autologous human serum and pooled human AB serum contained immunoglobulin which interacted with each of the B cell mitogens, thus limiting their usefulness as in vitro supplements. Cells grown in human serum albumin supplemented media had minimal background and high stimulated responses to B-cell mitogens. These results indicate that human serum albumin is an optimal supplement for in vitro human lymphocyte proliferative assays since it supports high stimulated cell responses with low background activity, is devoid of immunoglobulin and had minimal variability among lots.     

The proliferative activity of myocardial capillary wall cells in variously aged swimming-exercised rats.

Cellular proliferation was studied by incorporation of 3H-thymidine in the hearts of young, adult and old swimming-exercised rats. The proliferative activity was measured by scintillation counting. The swimming exercise induced a significant proliferation of capillary wall cells in the younger age groups, suggesting a neoformation of myocardial capillaries in these rats, whereas no cell proliferation was recorded in the old rats.     

Effects of hydrophobic amino acids and antibodies to nerve growth factor receptors on the development of splenic tissue culture from young and old rats

The effects of hydrophobic L-amino acids alone and in the presence of monoclonal antibodies to nerve growth factor receptors NGFRp75 (apoptosis inductors) were studied on organotypic culture of splenic lymphoid tissue from young (3 months) and old (24 months) rats. Nine amino acids inhibited cell proliferation in splenic explants from young rats. This was paralleled by hyperexpression of p53 proapoptotic protein. Only two amino acids stimulated apoptosis in explants from old rats. The inhibitory effects on the development of splenic explants from young and old rats were abolished in the presence of antibodies to NGFRp75. Hence, the group of hydrophobic amino acids mediates the proapoptotic effect in the lymphoid tissue of old and young rats through nerve growth factor low affinity receptors.     

不同年龄段大鼠血清对大鼠骨髓内皮祖细胞活力的影响

目的：观察不同年龄段大鼠血清对大鼠骨髓内皮祖细胞(endothelial progenitor cells, EPCs) 活力的影响。方法：PBS冲洗出1-2月龄、19-26月龄SD大鼠股骨和胫骨骨髓，Ficoll密度梯度离心法分离单个核细胞(MNCs), 应用含10%FBS的DMEM/F12培养基（含内皮细胞生长添加剂100 mg/L、肝素100 mg/L、青霉素1×10⁵ U/L、链霉素1×10⁵ U/L）差速贴壁法进行体外培养，以Dil-ac-LDL与FITC-UEA-1荧光双染，直接或间接荧光标记CD31 及vWF分别通过流式和免疫组化进行鉴定。制备1-2月龄、19-26月龄大鼠血清，将培养的EPCs分A （老年大鼠EPCs+老年大鼠血清）、B（老年大鼠EPCs+年轻大鼠血清）、C （年轻大鼠EPCs +老年大鼠血清）、D （年轻大鼠EPCs+年轻大鼠血清）4组， EPCs经含10%不同年龄段大鼠血清的DMEM/F12培养基（不含胎牛血清）培养后，激光共聚焦检测EPCs吞噬Dil-ac-LDL后平均荧光强度，改良Boyden 小室和黏附能力测定实验分别观察EPCs迁移和黏附能力，MTT法检测细胞增殖活性。结果：年轻大鼠血清显著促进老年大鼠EPCs吞噬Dil-ac-LDL能力（P<0.01）及增强其迁移（P<0.01）、黏附（P<0.05）和增殖能力（P<0.01），而老年大鼠血清显著抑制年轻大鼠EPCs迁移（P<0.05）和黏附能力（P<0.05）。结论：年轻大鼠血清显著增强老年大鼠EPCs的细胞活力，而老年大鼠血清可部分抑制年轻大鼠EPCs功能活性。     

Loss of responsiveness in senescent human TIG-1 cells to the DNA synthesis-inducing effect of various growth factors

Responses of human diploid cells, TIG-1, were examined with respect to their ability to initiate DNA synthesis under the influence of various growth factors and their combinations. The following agents stimulated DNA synthesis in quiescent TIG-1 cells at 37-49 PDL (population doubling level) (66-79% of lifespan completed): fetal bovine serum; tumor-derived DNA synthesis factors such as those from rat rhodamine fibrosarcoma, human adenoma and from the conditioned medium of cultured human pituitary cells; human and mouse epidermal growth factors; tumor promotors such as 12-O-tetradecanoylphorbol 13-acetate and teleocidin; microtubule-disrupting agents as colchicine, vinblastine, podophyllotoxin and TN-16; melittin; and dexamethasone. Cells at 58-60 PDL (94-97% of lifespan completed) were stimulated to synthesize DNA by fetal bovine serum, tumor-derived DNA synthesis factors and epidermal growth factors, but not by other agents. Finally, in senescent cells at 62 PDL (100% of lifespan completed), any of these growth factors and of their combinations failed to induce DNA synthesis at all. These senescent cells, however, still retained the ability to initiate DNA synthesis following infection with SV40 as reported previously [Exp. Cell Res., 143 (1983) 343-349].     

Effects of in vitro aging and cell growth on the viability and recovery of human diploid fibroblasts,TIG-1, after freezing and thawing

The viability and the recovery (cell attachment to the dish) after thawing of human diploid fibroblasts (TIG-1) frozen by four different methods were studied at different passages. Improved results were observed in a medium of 30% fetal bovine serum plus 15% glycerol, compared with the conventonal medium which contained 10% fetal bovine serum plus 10% glycerol. Centrifugation to remove glycerol immediately after thawing had a negative effect on the viability and recovery of cells. The recovery of cells after freezing and thawing showed a maximal value in the middle of phase II (PD 35) during the finite lifespan of the cell (average PD 67). This result indicates that the cells at early and late passages are sensitive to injury by freezing and thawing. The modified method yielded improved recovery, especially in the cells at early and late passages, except for the extremely senile stage. The recovery was also affected by the state of cell growth after inoculation.     

Growth factor responsiveness declines during adulthood for human skin-derived cells

Keratinocytes and fibroblasts were obtained from upper arm biopsies of young (22-27 years) and old (60-82 years) adult donors. Keratinocytes were grown in serum-free medium containing variable quantities of either epidermal growth factor (EGF) or a bovine hypothalamic extract known to contain keratinocyte growth factor (KGF). Fibroblasts were grown in serum-free medium containing variable quantities of EGF or insulin. Paired keratinocyte cultures were plated in serum-free medium containing 20% newborn keratinocyte-conditioned medium (NM) or 20% control conditioned medium (CM). Newborn foreskin keratinocytes were plated in 20% conditioned media derived from newborn, young adult or old adult keratinocyte cultures. In spite of large inter-donor variability, keratinocyte growth significantly decreased with age (0.05 greater than P greater than 0.01). Cell yield at 7 days showed an 8-fold increase for young adults over the KGF dose range treated, but only a 4-fold increase for old adults. Young adult cells in varying concentrations of EGF achieved 3-fold to 5-fold higher densities than old, although EGF was not stimulatory for either adult age group. Donor age-associated loss of growth factor responsiveness was confirmed with dermal fibroblasts derived from the same biopsies. Newborn but not adult keratinocytes were stimulated by NM, while newborn cells were not stimulated by either young or old adult conditioned media (YM or OM). An epidermal proliferation index, incorporating both donor cell yield and cell yield of newborn cells in donor conditioned medium, was significantly different (P less than 0.01) for newborn vs. young or old adult cells. Our findings confirm that a decreased proliferative capacity is measurable within adulthood, and suggest that this decrease may be due to a reduced ability to synthesize or respond to mitogens, including autocrine factors.     

Cell cycle analysis in bone marrow and kidney tissues of dietary restricted rats

The effect of dietary restriction (DR) on the proportion of cells in various phases of the cell cycle as determined by flow cytometry was investigated in the bone marrow and kidney of young and old Fischer 344 rats. Control rats were fed a standard occurrence of numerous age-associated diseases, including cancer, renal diseases and by the control rats starting at 16 weeks of age until killed at 5 or 20 months old. The relative proportion of cells in the various phases of the cell cycle was independent of tissue type, treatment condition and age, consistently showing an order of G1- greater than S- greater than G2M-phase. In old rats DR did not affect cell cycling in bone marrow of either sex, however, it did cause an increase in the percentage of G1-phase cells in the kidney of male rats. Additionally, DR caused a mathematically significant change in the percentage of cells in all phases of the cell cycle in the bone marrow of young male rats but had no effect in young females. The percentage of S-phase cells in both tissues of both sexes decreased in old rats when compared to young rats regardless of treatment conditions, indicating a parallel decline in cell proliferating activity with aging. To summarize, DR produces a greater cell cycle effect in the young male than the old male rats. Proliferative capacity is enhanced when the young male rats are dietary restricted. This may aid in DNA repair mechanisms and/or immune system response.     

Spontaneous production of thymocyte-activating factor by human gingival fibroblasts and its autoregulatory effect on their proliferation.

The purpose of this study was to examine whether human gingival fibroblasts produce a cytokine which modulates in immune and inflammatory responses including alterations in connective tissue metabolism in periodontal tissue. We found that a cultured human gingival fibroblast cell line (Gin-1) and freshly isolated human gingival fibroblasts produced thymocyte-activating factor(s), so we called the factor(s) fibroblast-derived thymocyte-activating factor (FTAF). Growth of the producing cell was itself modulated by the factor(s). Gin-1 cells spontaneously produced a significant amount of FTAF in a cell growth-dependent manner. Maximum activity was observed in conditioned medium from stationary-phase cells. The activity in conditioned medium of cultures lacking serum was significantly higher than that in those containing serum. Treatment of Gin-1 cell cultures with cycloheximide, an inhibitor of protein synthesis, markedly inhibited FTAF production. When Gin-1 cells were stimulated by triggering with muramyl dipeptide or sonicated extracts of Bacteroides gingivalis, FTAF production was significantly stimulated. Freshly isolated human gingival fibroblasts from gingival biopsies of healthy donors also produced FTAF which enhanced thymocyte proliferation. Peaks of thymocyte proliferation activity in conditioned medium from Gin-1 cells were observed in fractions having molecular weights of 25,000, 35,000, and 45,000, as determined by Sephadex G-75 column chromatography. The peak fractions (partially purified FTAF) significantly suppressed the proliferation of Gin-1 cells themselves as evaluated by [3H]thymidine uptake. The suppressive effect of partially purified FTAF was, at least partially, mediated by endogenous prostaglandin for the following reasons: addition of indomethacin, and inhibitor of prostaglandin synthesis, abrogated the suppressive effect; partially purified FTAF stimulated the production of prostaglandin E2 by the cells; and the suppression of cell proliferation was reinforced by addition of exogenous prostaglandins. These observations suggest that gingival fibroblasts play a significant role in regulation of cell growth of lymphocytes and in their own growth under physiological conditions and in pathological states in periodontal connective tissue.     

Effects of subchronic d-fenfluramine on splenic immune functions in young and old male and female Fischer 344 rats.

The present study was designed to demonstrate age- and sex-related differences in immune functions, and to determine whether subchronic elevations in serotonin (5-HT) availability in vivo would alter immune functions assessed subsequently in vitro. Male and female F344 rats (5 and 21 months of age) were administered the 5-HT releaser and reuptake inhibitor, d-fenfluramine (d-Fen), in their drinking water for 30-38 days then killed. The young animals received a higher dose (1.8 mg/kg/day) of d-Fen than the old rats (0.6 mg/kg/day) in order to compensate for age-related decreases in drug biotransformation and clearance. Brain and spleen d-Fen and metabolite concentrations, however, were considerably higher in the young than in the old rats. d-Fen treatment did not affect body weight or fluid intake. Although substantial sex differences in immune function were not discerned, age-related decreases were observed in absolute splenic cellularity, recombinant interleukin-2 (rIL-2) stimulated natural killer (NK) cytotoxicity, LPS stimulated B-cell mitogenesis, and in the level of Ox19 (CD5) positive cells. d-Fen caused an increase in absolute spleen weight and a decrease in absolute splenic cellularity only in the old rats of both sexes. Spleen cells from young male and old female rats receiving d-Fen had relatively more large granular lymphocytes and enhanced baseline and rIL-2 activated killing of YAC-1 cells than their vehicle matched or opposite sex counterparts. The drug also increased Con A-induced T-cell proliferation in young males and LPS induced B-cell proliferation in old females. d-Fen decreased Ox39 (CD25) levels by 19%, but did not affect any of the other phenotypes examined. The results suggest that 5-HT has a selective stimulatory effect on young male and old female NK activity, and that old female rats are more sensitive to the immunological effects of d-Fen than old male rats.     

Aging and arteriosclerosis. Cell cycle kinetics of young and old arterial smooth muscle cells.

Intimal cell proliferation is a "hallmark" of atherosclerosis. Myointimal hyperplasia in arteries has been shown to be dependent on age after vascular endothelial denudation and injury associated with vascular transplantation. Because myointimal thickening is greater in aged rats than in younger rats, and aortic segments from old rats transplanted into young syngeneic recipients have a greater myointimal proliferative response to injury than its host environment, the authors examined the cell cycle distributions of old and young rat arterial smooth muscle cells (SMCs) by flow-cytometric analysis. They observed that there is an apparent age-dependent variation in the cell cycle distribution. Moreover, old SMCs have a greater percentage of their population in the S phase and not G2/M, compared with young SMCs; and there is a decrease in the percentage of old cells in the G0/G1 phase as compared with young SMCs. These differences may reflect the cellular changes observed during myointimal hyperplasia following vascular injury. It is concluded that our data support the hypothesis that the proliferation of SMCs is dependent, in part, on those processes related to aging as well as to the phenotypic state of the cell.     

供体年龄影响融合生长内皮祖细胞对平滑肌细胞表型转换及增殖迁移的作用

目的:探讨老龄和年轻个体来源融合生长状态内皮祖细胞(EPCs)对血管平滑肌细胞(SMCs)表型转换以及增殖和迁移的调节作用。方法:脱臼处死1~2月龄、19~26月龄SD大鼠,应用含15%FBS的DMEM/F12培养基(含内皮细胞生长添加剂100 mg/L、肝素100 mg/L、青霉素、链霉素各1×105U/L)培养EPCs,取1~2月龄大鼠腹主动脉,组织块法培养血管SMCs,应用Di I-Ac-LDL与FITC-UEA-1荧光双染以及α-SM-actin免疫荧光分别对EPCs和SMCs进行鉴定。建立细胞共培养体系,上室为融合生长状态的EPCs,下室为SMCs,实验分4组:(1)第3代SMCs(P3)组;(2)第4代SMCs(P4)组;(3)第4代SMCs与年轻大鼠来源EPCs共培养(P4YE)组;(4)第4代SMCs与老龄大鼠来源EPCs共培养(P4AE)组。Western blotting检测α-SM-actin和osteopontin蛋白的表达;[3H]-TdR掺入法检测SMCs增殖;细胞划痕实验检测SMCs的迁移能力。结果:与P3组相比,P4组的SMCsα-SM-actin表达显著下调,而osteopontin表达显著增强;P4YE组SMCs的α-SM-actin及osteopontin表达与P3组比较未见有显著差别;与P4组相比,年轻和老龄大鼠来源的EPCs均显著促进第4代SMCs的α-SM-actin和下调osteopontin的表达,抑制第4代SMCs的增殖和迁移;与老龄大鼠来源的EPCs相比,年轻大鼠来源的EPCs更能够显著延迟SMCs表型由收缩型向合成型转换,抑制SMCs增殖和迁移。结论:共培养融合生长状态的EPCs使血管SMCs表型转换延迟、抑制SMCs增殖和迁移,年轻大鼠来源的EPCs较老龄大鼠来源的EPC更显著延迟血管SMCs表型由收缩型向合成型转换,并具有更强的抑制血管SMCs增殖和迁移的能力。     

Filopodia number increases with age and quiescence in populations of normal WI-38 cells, and is correlated with drug-induced changes in proliferation in both normal and transformed populations

Filopodia in log and stationary phase populations of human fetal lung fibroblasts (WI-38) at low and high population doubling levels (PDLs) and of SV40 transformed WI-38 cells (VA13A), were observed and counted under different conditions of in vitro growth by scanning electron microscopy. Cells from old non-vigorously growing WI-38 populations (those at a high PDL) had more filopodia than younger populations (those at a lower PDL) at all times after seeding, and for any given population stationary phase cells (those entering, or in, quiescence), had more than log phase cells. Hydrocortisone (HC, 14 microM), which stimulates proliferation and increases life span of WI-38 cells, was associated with a marked decrease in filopodia. Conversely, retinoic acid (RA, 10 microM), which inhibits growth and decreases life span of WI-38 cells, was associated with an increase in filopodia. Since old cell populations have lower saturation densities than young, it is suggested that cell contact signaling growth cessation in these populations may be mediated by filopodia. The HC-associated decrease in filopodia may thus be possibly interpreted as a decrease in filopodia-mediated "density dependent inhibition," and the increase in filopodia with RA as a possible increase in this "inhibition." Both HC and RA inhibit growth and are associated with an increase in filopodia in VA13A cultures.     

A biologically active VEGF construct in vitro: implications for bioengineering-improved prosthetic vascular grafts.

Prosthetic arterial grafts are unable to develop an intact endothelial lining after implantation, predisposing them to fail. Strategies have been sought to enhance endothelialization using growth factors and cytokines. This study assessed the biologic activity of vascular endothelial growth factor (VEGF) covalently linked to bovine serum albumin (BSA). Native and modified VEGF were assayed for endothelial cell migration and proliferation. Migration assays were performed comparing the effects of 2% fetal bovine serum (FBS), 50 ng/mL, 100 ng/mL, and 200 ng/mL of native VEGF and VEGF-BSA. Proliferation assays were performed by using Alamar Blue comparing cellular growth in 1% FBS, 10% FBS, 100 ng/mL unbound VEGF, and 100 ng/mL VEGF-BSA. VEGF is a potent chemotactic agent for endothelial cells in both unbound and bound states. Native VEGF solutions (50 ng/mL, 100 ng/mL, and 200 ng/mL) stimulated 23.9 cells/high power field (HPF), 35.3 cells/HPF, and 49.1 cells/HPF (p < 0.005). VEGF-BSA solutions stimulated 25.9 cells/HPF, 39.1 cells/HPF, and 69.0 cells/HPF (p < 0.001). VEGF-BSA and native VEGF supported similar increased cellular proliferation compared with 1% FBS media (p < 0.002). Modified VEGF retains its chemotactic and proliferative properties in vitro. These findings suggest that bare prosthetic surfaces lined with VEGF bound to a "basecoat" albumin may support endothelial cell proliferation and migration and thereby offer new strategies to improve graft patency.     

Events blocked in prereplicative phase in senescent human diploid cells, TIG-1, following serum stimulation

When senescent human diploid cells, TIG-1, were stimulated with serum at the end of their proliferative life span, such biochemical events as uptakes of 2-deoxyglucose and uridine, and expression of c-myc, were enhanced. However, RNA synthesis, polyamine accumulation, thymidine uptake and DNA synthesis were not enhanced at all. Protein synthesis increased only moderately as compared with that observed in younger cells. These results indicated that the events in prereplicative phase known to be independent on protein synthesis are induced in senescent cells after the stimulation with serum, whereas those required protein synthesis failed to increase to the same extent as seen in young cells.     

The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro

The effects of chitin [(1 --> 4)-2-acetamido-2-deoxy-beta-D-glucan] and its partially deacetylated derivatives, chitosans, on the proliferation of human dermal fibroblasts and keratinocytes were examined in vitro. Chitosans with relatively high degrees of deacetylation strongly stimulated fibroblast proliferation while samples with lower levels of deacetylation showed less activity. Fraction, CL313A, a shorter chain length, 89% deacetylated chitosan chloride was further evaluated using cultures of fibroblasts derived from a range of human donors. Some fibroblast cultures produced a positive mitogenic response to CL313A treatment with proliferation rates being increased by approximately 50% over the control level at an initial concentration of 50 microg/ml, whilst others showed no stimulation of proliferation or even a slight inhibition (< 10%). The stimulatory effect on fibroblast proliferation required the presence of serum in the culture medium suggesting that the chitosan may be interacting with growth factors present in the serum and potentiating their effect. In contrast to the stimulatory effects on fibroblasts, fraction CL313A inhibited human keratinocyte mitogenesis with up to 40% inhibition of proliferation being observed at 50 microg/ml. In general highly deacetylated chitosans were more active than those with a lower degree of deacetylation. These data demonstrate that highly deacetylated chitosans can modulate human skin cell mitogenesis in vitro. Analysis of their effects on cells in culture may be useful as a screen for their potential activity in vivo as wound healing agents, although in the case of fibroblasts it is important to select appropriate strains of cells for use in the screen.