Stimulatory effects of phenytoin on osteoblastic differentiation of fetal rat calvaria cells in culture

Phenytoin (diphenylhydantoin, DPH), an anticonvulsant drug for epileptic patients, has several adverse effects, including calvarial thickening and coarsening of the facial features, which occur with chronic DPH therapy. While previous studies have demonstrated that DPH has an anabolic action on bone cells in vivo and in vitro, the basis of these effects is not fully understood. In this study, the effect of DPH on osteoblastic differentiation of fetal rat calvaria (RC) cells in culture was investigated by measuring bone nodule (BN) formation, cell growth, alkaline phosphatase (ALPase) activity, collagen synthesis, and expression of osteocalcin (OC) and osteopontin (OP) mRNAs. Continuous treatment of RC cells with DPH for 18 days dose-dependently increased the mineralized BN number by 1.2–1.7-fold at concentrations of 12.5–200 μmol/L DPH. Cell growth was not affected at the same concentrations of DPH. ALPase activity was stimulated by DPH (1.1–1.9-fold) dose-dependently and was maintained at higher levels in DPH-treated cells throughout the experimental period. DPH increased mineralized and unmineralized BN formations both in the presence and the absence of 10⁻⁸ mol/L dexamethasone (Dex). Expression of OC and OP mRNAs was markedly augmented by DPH on days 12–24 and on days 12–18, respectively. While control mRNA levels of OC and OP increased with time, the increases in DPH-treated cells were greater than those of the controls and the stimulatory effects were dose-dependent. Type I collagen was also influenced by DPH; mRNA level was enhanced and the percentage of collagen synthesized was increased significantly, by 200 μmol/L DPH. When DPH was added in three different culture stages, days 1–6 (growth), days 7–12 (matrix development), and days 13–18 (mineralization), BN formation was influenced primarily on days 1–6 and secondarily on days 7–12, but not on days 13–18, suggesting that DPH increased BN formation by enhancing not only the proportion of osteoprogenitor cells in the early stage but also the proportion of functional osteoblasts in the middle stage within mixed-cell populations. Moreover, such increases were detected in conditions of both Dex(+) and Dex(−). These findings demonstrate that DPH stimulates osteoblast-associated markers such as BNs, ALPase, OC, OP, and type I collagen by continuously affecting the stages of growth and matrix development in RC cells, and suggests that the stimulatory effects by DPH may possibly be induced independent of those by Dex.     

三参数流式细胞术分析紫外线诱导的MOLT-4细胞旁观者效应

目的 应用三参数流式细胞术探讨紫外线(UV)诱导的MOLT-4细胞的旁观者效应(BSE).方法 以人类T细胞白血病细胞株MOLT-4为对象,以紫外线为诱导剂.将经UV照射的MOLT-4细胞与DiD荧光阳性的MOLT-4细胞联合培养作为试验组,未经任何打击的DiD阳性细胞为对照组.用FITC-Annexiu V/PI法检测细胞凋亡率,用激光扫描共聚焦显微镜(LSCM)观察其荧光及形态学改变,以检测其旁观者效应.结果 实验组中旁观者细胞凋亡及坏死比例在0、2、4、8、12、18、24 h分别为6.84%、8.09%、9.88%、13.50%、22.50%、30.99%、37.93%.而对照组为4.28%、4.93%、5.22%、5.39%、6.54%、6.77%、7.14%.试验组旁观者细胞的凋亡及坏死比例随着时间的延长而逐步增高.LSCM观察可见部分旁观者细胞出现磷脂酰丝氨酸(PS)翻转、核边集、核浓集、核碎裂等形态学改变.结论 在MOLT-4细胞株,用非离子射线--UV诱导的凋亡细胞对联合培养的旁观者细胞具有旁观者效应.     

Single-cell microinjection of cytochrome c can result in gap junction-mediated apoptotic cell death of bystander cells in head and neck cancer

BACKGROUND: Gap junction intercellular channels are required for metabolic cooperation between cells and regulate normal tissue homeostasis by means of the transfer of small molecules between contacting cells. Not surprisingly, the gap junction phenotype is frequently lost during carcinogenesis in human tissues (including those of the upper aerodigestive tract), freeing individual cancer cells from the growth control signals of normal surrounding tissues and less aggressive adjacent cancer cells. We hypothesized that gap junctional intercellular communication (GJIC) could mediate a bystander effect (apoptotic cell death) in squamous cell carcinoma of the head and neck (SCCHN) cells adjacent to individually targeted SCCHN cells. METHODS: Single-cell microinjection of cytochrome c was used to induce apoptosis in target SCCHN cells with endogenous GJIC activity and in an SCCHN cell line with exogenously introduced GJIC activity. Apoptosis was followed in target and surrounding bystander cells through light and time course microscopic characterization. All of the preceding experiments were carried out in the absence and presence of 18-beta-glycerretinic acid, a pharmacologic inhibitor of GJIC. RESULTS: When cytochrome c was introduced into SCCHN cells with endogenous GJIC activity through single-cell microinjection, bystander effects (apoptosis of nontarget cells) were observed. When GJIC activity was blocked with the specific pharmacologic inhibitor of gap junctions, 18-beta-glycerretinic acid, a bystander effect was never seen in GJIC active SCCHN cell lines. CONCLUSIONS: Gap junction intercellular channels can mediate a bystander effect in SCCHN. Inconsistencies in our data will be discussed in the context of recent advances in this field, as well as our future research directions.     

唑来膦酸联合甲氨蝶呤对骨肉瘤细胞生长及化疗敏感性研究

[目的]本试验主要观察第3代二膦酸盐类药物唑来膦酸(zoledronic acid,ZOL)在体外对人骨肉瘤细胞株MG63生长的影响,进一步研究唑来膦酸与甲氨蝶呤(methotrexate,MTX)合用对MG63细胞的生长抑制是否具有协同作用。[方法]采用MTT法分别检测不同剂量的唑来膦酸、甲氨蝶呤对人骨肉瘤细胞株MG63生长作用,检测唑来膦酸联合甲氨蝶呤对人骨肉瘤细胞株MG63的增殖抑制。[结果]唑来膦酸对MG63细胞的抑制效应与药物剂量及作用时间均成正比。单药唑来膦酸组剂量为1、10μmol/L时,MG63细胞凋亡率分别为9.91%、48.95%,单用甲氨蝶呤(1、10、100mg/L)时细胞凋亡率为37.68%、45.93%、52.418%,当唑来膦酸(10μmol/L)联合甲氨蝶呤(1、10、100mg/L)时MG63细胞的凋亡率分别为51.96%、66.77%、69.23%,联合用药组与单药组相比差异有显著性(P<0.01)。唑来膦酸作用MG63细胞72h的IC50值为9.39μmol/L。[结论]唑来膦酸对人骨肉瘤细胞株MG63有生长抑制作用,呈时间、剂量依赖性效应,唑来膦酸与甲氨蝶呤有协同作用。     

2-methoxyestradiol induces apoptosis and cell cycle arrest in human chondrosarcoma cells.

2-Methoxyestradiol (2ME) is an endogenous metabolite with estrogen receptor-independent anti-tumor activity. The current study seeks to determine the mechanism of anti-tumor activity of 2ME on human chondrosarcoma. 2ME caused a time- and dose-dependent cytotoxity in chondrosarcoma cells, while primary chondrocytes were minimally affected. Cells accumulated in G0/G1 phase in response to 2ME and DAPI stain indicated an induction of apoptosis. Bax, Cytochrome C, and Caspase-3 protein expression were increased, while p53 expression was decreased. A higher Bax/Bcl-2 ratio followed 2ME treatment. 2ME has a potentially promising role as a systemic therapy of chondrosarcoma when the mechanism of action is better delineated.     

超生理剂量十一酸睾酮引起生精细胞凋亡的动态研究

目的：以生精细胞凋亡作为指标探讨超生理剂量十一酸睾酮（ＴＵ）抑制精子发生的机制。方法：正常生育史男性２０例肌内注射ＴＵ,应用瑞－吉染色和ＴＵＮＥＬ法对精液中脱落细胞进行凋亡率的动态检测。结果：用药后精子密度和生精细胞数较用药前显著下降（Ｐ＜０．０１）,精母细胞和精子细胞凋亡率则显著上升（Ｐ＜０．０１）。结论：ＴＵ具有显著的抑制精子发生的效应。其作用机制除通过抑制促性腺激素而导致睾丸内睾酮（Ｔ）浓度降低,继之使精子发生停滞外,还可能有促进生精细胞凋亡的作用。     

胞嘧啶脱氨酶基因对胃癌细胞的杀伤作用

目的探讨胞嘧啶脱氨酶基因对胃癌细胞的杀伤作用。方法应用胞嘧啶脱氨酶（ＣＤ）基因,通过逆转录病毒载体转入人胃癌细胞Ｍ８５中,经０．４ｍｇ／ｍｌ的Ｇ４１８筛选阳性克隆扩增后进行前药５ＦＣ敏感试验。结果转ＣＤ自杀基因的Ｍ８５细胞较亲代Ｍ８５细胞对前药的敏感性显著增高（Ｐ＜０．０５）。体外实验中还观察到,前药在对转自杀基因的Ｍ８５细胞产生杀伤作用同时,还对与其共培养的亲代Ｍ８５细胞产生明显的杀伤作用,即“旁观者效应”。进一步将转基因Ｍ８５细胞和未转基因Ｍ８５细胞接种至裸鼠皮下,发现两者成瘤性无明显差异;前药５ＦＣ对未转基因肿瘤的生长无影响,而对转基因肿瘤的生长明显抑制。结论本研究为ＣＤ基因治疗胃癌向临床过渡提供了依据。     

Studies on apoptosis of spermatogenic cells in normal fertile men treated with supraphysiological doses of testosterone undecanoate

Aim: To study the anti-spermatogenic mechanism of supra-physiological doses of testosterone undecanoate (TU).Methods: Twenty fertile adult men received four intramuscular injections of TU at monthly intervals, 1000 mg uponadmission and 500 mg for the subsequent injections. The apoptotic germ cells in the semen were studied under light mi-croscope with tenninal deoxynucleotidyl tmnsferase-mediated dUTP-biotin nick end labeling (TUNEL) and Wright-Giem-sa staining methods. Results: After treatment, the sperm density and the number of spermatogenic cells in the semenwere significantly decreased ( P < 0.01), while the apoptotic ratios of spermatocytes and spermatids increased significantly( P <0.01) as compared with the pretreatment levels. Apoptosis was found to be augmented in the whole series of castoffspennatogenic cells. Conclusion: Besides its suppressive effect on spermatogenesis through a negative feed-backmechanism, TU enhances apoptosis of spermatogenic cells, which may be an additional mechanism o     

肝细胞生长因子对骨髓基质干细胞与纤维连接蛋白涂层黏附行为的影响

目的 观察肝细胞生长因子（HGF）对骨髓基质干细胞与纤维连接蛋白涂层黏附行为的影响。方法 将不同浓度的肝细胞生长因子加入预涂纤维连接蛋白的96孔板，观察细胞的黏附特性、增殖活性及形态学改变。结果 在肝细胞生长因子的诱导下，骨髓基质干细胞在纤维连接蛋白预包被的96孔板底分布均匀，多呈纤维母细胞样外观；肝细胞生长因子诱导的骨髓基质干细胞对纤维连接蛋白的黏附性是一种剂量-依赖性方式，50％的有效剂量（ED50）大约是20μg／L，当达到最大黏附力时剂量为100μg／L；与空白对照组相比，50～200μg／L的肝细胞生长因子能促进骨髓基质干细胞的增殖，其中以50μg／L组细胞增殖活性最强（P〈0．05）。结论 在最适浓度下，肝细胞生长因子能显著提高骨髓基质干细胞与纤维连接蛋白涂层的黏附行为。     

Surface antigen expression on bladder tumor cells induced by bacillus Calmette-Guérin (BCG): A role of BCG internalization into tumor cells

BACKGROUND: The antitumor mechanisms of bacillus Calmette-Guérin (BCG) against bladder cancer is still unclear. We previously reported that BCG was internalized by and survived within murine bladder tumor cells (MBT-2) for at least 40 days. In the present study, we investigated the effect of BCG on the surface antigen expression of bladder tumor cells and the characteristics of these cells as antigen-presenting cells in vitro. METHODS: Surface antigen (major histocompatibility complex (MHC) Class II, CD1, CD80 and intercellular adhesion molecule-1 (ICAM-1)) expression on BCG-treated murine (MBT-2) and human (T-24, J82) bladder tumor cells were analyzed using flow cytometry. The production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) from murine lymphocytes sensitized with BCG or BCG-treated tumor cells were also investigated. RESULTS: The expressions of MHC Class II, CD1, CD80 and ICAM-1 were augmented in all of the bladder tumor cell lines used; however, they were augmented to varying degrees among the cell lines that were treated with live BCG. Heat-killed BCG had little or no effect. When murine lymph node cells sensitized with BCG or BCG-treated MBT-2 cells were cocultured with BCG-treated MBT-2 cells, significant amounts of IL-2 and IFN-gamma were produced in the culture medium. CONCLUSIONS: BCG induced the augmented expression of surface antigens, such as MHC Class II, CD1, CD80 and ICAM-1, of bladder tumor cells. Furthermore, BCG-treated MBT-2 cells could stimulate BCG-sensitized lymphocytes to produce IL-2 and IFN-gamma. These results strongly suggest that bladder tumor cells gained the characteristics and functions of antigen-presenting cells (APC).     

Immunoisolated chromaffin cells implanted into the subarachnoid space of rats reduce cold allodynia in a model of neuropathic pain: a novel application of microencapsulation technology

Intrathecal transplants of adrenal medullary chromaffin cells relieve chronic pain by secreting catecholamines, opioids, and other neuroactive substances. Recently, macrocapsules with semipermeable membranes were used to isolate immunologically xenogenic chromaffin cells, but the poor viability in vivo of the encapsulated chromaffin cells limited the usefulness of this method. In this study, we used a novel method of encapsulation to increase the viability of chromaffin cells. We found that microencapsulated chromaffin cells that were implanted into the subarachnoid space of rats relieved cold allodynia in a model of neuropathic pain. Furthermore, microencapsulated chromaffin cells were morphologically normal and retained their functionality. These findings suggest that the intrathecal placement of microencapsulated chromaffin cells might be a useful method for treating chronic pain.     

脐血源性CIK细胞的体外增殖及其对人胆囊癌细胞株GBC-SD杀伤活性的实验研究

目的探讨脐血CIK细胞的体外增殖活性及其对人胆囊癌细胞株GBC-SD的杀伤作用。方法通过在脐血单个核细胞中加入IFN-γ、IL-2和CD3单克隆抗体进行细胞培养,获得多种细胞因子诱导的杀伤细胞(cytokine inducde killer cells)即脐血CIK细胞。用流式细胞仪对细胞作动态表型分析,用MTT法测定其抗肿瘤活性,并与CD3AK作对比分析。结果脐血CIK细胞在培养2周左右获得大量增殖,CD3~-、CD56~ 双阳性细胞大量增殖达1000倍以上;CIK细胞及其培养上清抗人胆囊癌细胞株GBC-SD活性明显优于CD3AK细胞,其抑瘤率为85%与62.8%(P<0.05),早期培养过程中加入G-CSF可以显著提高CIK细胞的增殖活性。结论脐血CIK细胞是一种新型、高效的免疫杀伤活性细胞,其对人胆囊癌细胞株GBC-SD具有明显的细胞毒作用,研究脐血CIK细胞对胆囊癌的过继性免疫治疗具有重要的临床意义。     

BMMSC输注对脐血CIK/NK细胞在荷瘤小鼠体内抗肿瘤效应的影响

目的 利用荷瘤小鼠来探讨不同的输注途径以及不同时间输注骨髓间充质干细胞( BMMSC)对脐血来源细胞因子诱导的杀伤(CIK)/自然杀伤(NK)细胞在体内抗肿瘤效应的影响。方法 NOD/SCID小鼠共56只,分为7组,每组小鼠经尾静脉输注K562细胞后12 h分别进行以下实验：同时经尾静脉输注人BMMSC+ CIK/NK细胞（A组）;经尾静脉相隔48 h分别输注人BMMSC和CIK/NK细胞（B组）;经小鼠胫骨骨髓腔输注入BMMSC,同时经尾静脉输注CIK/NK细胞(C组);经小鼠胫骨骨髓腔输注人BMMSC,48 h后经尾静脉输注CIK/NK细胞;经小鼠胫骨骨髓腔输注人BMMSC,48 h后经尾静脉输注CIK/NK细胞（E组）：较其他组延迟48 h经尾静脉输注CIK/NK细胞（F组）;除输注K562细胞外,不输注其他细胞（G组）。计算各组小鼠的生存曲线,检测小鼠外周血、骨髓、肝、脾和肺等脏器的肿瘤细胞负荷情况。结果 A、B、G组小鼠的存活时间短于C、D、E、F组(P＜0.05);A、B、G组间以及C、D、E、F组间小鼠的存活时间的差异均无统计学意义(P＞0.05);A、B、G组小鼠外周血、骨髓涂片中肿瘤细胞的比例高于C、D、E、F组(P＜0.05)。A、B、G组小鼠外周血、骨髓及肝、脾、肺组织匀浆中人肿瘤细胞标记CD33的表达率高于C、D、E、F组(P＜0.05);A、G组小鼠肝、脾、肺组织匀浆中CD33表达率高于B组(P＜0.05);C组小鼠肺组织匀浆中CD33表达率高于D组(P＜0.05)。结论 同部位注射BMMSC可明显抑制CIK/NK细胞在荷瘤小鼠体内的抗肿瘤作用,而分部位注射,则BMMS的抑制作用明显减弱。提前输入的BMMSC在体内仍会削弱CIK/NK细胞的抗肿瘤作用。     

Longitudinal study of bone loss in the second metacarpal

Summary This longitudinal study was undertaken to ascertain the rate of bone loss and to identify aging, cohort and/or time effects on bone loss in male participants of the Baltimore Longitudinal Study of Aging. Hand-wrist radiographs were obtained from 1958–1981 and were evaluated for total width, medullary width, and length of the second metacarpal. Data were analyzed using an age-time matrix with 8-year intervals for three epochs and nine age groups. The bone measurements were analyzed in three perspectives (cross-sectional, longitudinal and time-series). The results demonstrate that there is both a cross-sectional and longitudinal loss of cortical bone with age in the second metacarpal. Furthermore, the results show that males lose approximately 14% of their cortical bone, at a rate of about 2% per decade, over the adult lifespan. The majority of this loss occurs between the ages of 45 and 69 and is due primarily to aging and is not an artifact of cohort differences or secular change.     

树突状细胞激活的肿瘤浸润性淋巴细胞体外特异性抗小鼠肝癌研究

目的树突状细胞(DC)是目前已知的功能最强的抗原提呈细胞(APC),可以向包括肿瘤浸润性淋巴细胞(TIL)在内的T淋巴细胞提呈抗原,并诱发细胞毒T淋巴细胞(CTL)反应。本文旨在探讨H22细胞和B16细胞全细胞性抗原致敏的树突状细胞激活的肿瘤浸润性淋巴细胞体外抗小鼠肝癌活性。方法从小鼠四肢长骨骨髓中获取DC,应用粒/巨噬细胞集落刺激因子(GM-CSF)、白介素-4(IL-4)和肿瘤全细胞性抗原致敏DC,然后用DC激活TIL,观察TIL在体外对H22细胞、Hepal-6细胞和B16细胞的杀伤活性。结果经H22细胞全细胞性抗原致敏的DC激活的TIL具有很高的对H22细胞杀伤活性,杀伤率为(71·31±3·11)%,明显高于其对Hepal-6和B16细胞的杀伤活性[杀伤率分别为(50·11±3·03)%,(30·31±2·89)%];也明显高于未经H22细胞全细胞性抗原致敏的DC激活的TIL、DC激活的脾淋巴细胞和未经DC激活的脾淋巴细胞对H22细胞杀伤活性[杀伤率分别为(49·80±3·21)%,(48·76±3·60)%和(19·23±2·71)%]和对Hepal-6细胞杀伤活性[杀伤率分别为(39·4±3·21)%,(38·62±2·87)%和(18·73±2·40)%]以及对B16细胞杀伤活性[杀伤率分别为(26·38±2·51)%,(25·82±2·70)%和(18·34±3·01)%],同时经B16细胞全细胞性抗原致敏的DC激活的TIL(来源于H22瘤体)也可诱导相对较低的对B16细胞的特异性细胞杀伤活性。结论来源于H22瘤体的TIL经H22细胞全细胞性抗原致敏的DC激活后可产生很强的针对H22细胞的特异性杀伤活性,明显高于其他各组,说明DC能诱导TIL产生高效而特异的体外抗小鼠肝癌免疫。     

HSV-TK/GCV系统对前列腺癌细胞的杀伤作用和旁观者效应

目的 :研究胸苷激酶 (TK)自杀基因疗法对人前列腺癌细胞的杀伤作用和旁观者效应 ,探讨其治疗前列腺癌的可行性。方法 :采用形态学观察、活细胞拒染法及MTT法检测单纯疱疹病毒胸苷激酶 (HSV TK) /羟基无环鸟苷 (GCV)系统对前列腺癌细胞的杀伤作用及旁观者效应。结果 :GCV对转染TK基因前列腺癌细胞的杀伤作用呈剂量及时间依赖关系 (P <0 .0 5～ 0 .0 1) ;但其旁观者效应较弱 ,TK基因阳性细胞比例约为 10 %的混合细胞 ,经 10 μmol/L和 10 0 μmol/L的GCV处理 72h后 ,仅有16.15 %± 1.64 %和 2 3 .46%± 3 .2 1%的细胞被杀灭。结论 :HSV TK/GCV系统对前列腺癌有杀伤作用 ,但由于其旁观者效应不够强大 ,不能期望通过单纯的TK基因治疗达到前列腺癌良好的治疗效果     

Factors affecting the variability of semen analysis results in infertile men

Infertile men who had 3 or more semen analyses performed in one laboratory were placed in 2 groups (I) oligozoospermic group (n = 106), mean sperm concentration between 1 and 20 million/ml (II) asthenozoospermic group (n = 71), mean sperm concentration greater than 20 million/ml, and mean motility less than 60%. With increasing durations of abstinece from ejaculation before the tests there were significant increases in semen volume and sperm concentration. Semen volume increased over the first 4 days to a similar extent in both groups. Sperm concentrations increased over 15 days, but the effect of abstinence was much greater in the asthenozoospermic group than in the oligozoospermic group (14% compared with 1.4% of the within subject variation). Significant changes in results accompanied repeated testing, notably rises in sperm concentration and motility. Sperm motility was lower in winter and higher in summer in both groups and also, but to a lesser extent, in artificial insemination donors who collected semen in the laboratory.     

Antiproliferative effect of heparin on human smooth muscle cells cultured from intimal hyperplastic lesions of vein grafts

The antiproliferative effect of heparin on cultured smooth muscle cells in proliferating human smooth muscle cells derived from clinical lesions of intimal hyperplasia was tested. Smooth muscle cells were obtained from stenotic segments excised from failing in situ saphenous vein bypass grafts in three patients. The nonadventitial portion of the excised tissue was explanted into cell culture using standard techniques without the addition of exogenous growth factors. Under these conditions, rapid cell outgrowth was observed from these explants, in contrast to minimal growth of smooth muscle cells from normal veins from the same patients. Immunohistochemical staining with antiactin antibody confirmed that the cells cultured from the stenotic lesions were smooth muscle cells. Incubation of these cells with porcine mucosal heparin revealed a significant (p<.01) dose-dependent inhibition of cell proliferation as measured by radioactive thymidine incorporation. Mean inhibition of six subcultures tested ranged from 3 to 46%, at heparin concentrations of 1 to 1,000µg/ml. The magnitude of heparin's antiproliferative effect varied among the cell lines from different patients, but 10–30% inhibition was consistently observed at heparin concentrations usually attained in vivo. The maximal inhibition achieved was 65% in one cell line at the highest heparin dose. We conclude that heparin exerts a significant antiproliferative effect on human smooth muscle cells cultured from intimal hyperplastic lesions from in situ saphenous vein bypass grafts.Presented at the 5th Annual Meeting of the Eastern Vascular Society, May 4, 1991, Pittsburgh, Pennsylvania.     

Effect of insulin-like growth factor 1 and basic fibroblast growth factor on DNA synthesis and collagen production in cultured anterior cruciate ligament cells

This study was undertaken to investigate the effects of insulin-like growth factor 1 (IGF-1) and basic fibroblast growth factor (bFGF) on the DNA and matrix synthesis of cells out-grown from the anterior cruciate ligament (ACL). Five batches of ACL cells from five 8-week-old Japanese white rabbits were isolated and maintained in culture until the fifth passage. We analyzed the effects of various concentrations of IGF-1 (1–1000 ng/ml) on [³H]-thymidine uptake in the cells at the first and fifth passages, and collagen content in the cell layer at the third passage, in the presence or absence of bFGF (10ng/ml). In the absence of bFGF, IGF-1 caused a significant increase in the synthesis of DNA and collagen in the ACL cells. IGF-1 and bFGF, in combination, synergistically increased the DNA synthesis of ACL cells, whereas such synergistic enhancement was not observed in their, collagen production. The amounts of [³H]-thymidine incorporated into the cells incubated with IGF-1 (500ng/ml) and bFGF (10ng/ml) combined were 1.3–1.5 times greater at first passage and 1.3–1.9 times greater at fifth passage than the sum of these with the growth factors used individually. Based on this in vitro finding, we consider it clinically relevant that IGF-1 and bFGF, when used together, have the capability to enhance the primary healing of ruptured ACL. Presented at the 11th Annual Meeting for Orthopaedic Research of the Japanese Orthopaedic Association, Kagoshima, Japan, October 17, 1996.