Development In CD-1 Mice Research Articles

Maternal immune activation accelerates pup reflex development and alters immune proteins in pup stomach contents and brain

Prenatal infection increases the risk for neurodevelopmental disorders including autism spectrum disorder and schizophrenia. To better understand this link, a number of maternal immune activation (MIA) rodent models have been studied. However, the majority of these studies focus on adult behavioural outcomes that mirror adult symptoms related to neurodevelopmental disorders. There is little research reporting the effects of MIA on early postnatal development and even fewer using outbred mouse strains. Here, we use a modified version of the Fox scale to assess the effects of two MIA models, a bacterial model (LPS) and a viral model (PolyIC), on overall mouse pup sensorimotor development in CD-1 mice. Surprisingly, both bacterial and viral MIA models resulted in early reflex development when compared with control pups. To better characterize potential factors related to these changes, we examined indicators of sickness/inflammation in the immune-activated dams and in their pups. Sickness behaviour in the dams resulting from immune activation was assessed using a telemetry implant that allowed for continuous recording of temperature and activity in dams exposed to bacterial or viral immune activation. Although MIA dams showed reduced activity on the day immediately following MIA compared to controls, there was no evidence of fever. All dams showed elevated cytokines/chemokines associated with parturition, but this resolved by day 10 post-parturition and was unaffected by previous immune activation. Although circulating cytokines/chemokines in the dams were similar across MIA treatments, there were differences in the amount of interleukin-12p70 and interleukin-13 present in milk taken from milk bands in MIA pups, and interleukin-4 was overall decreased in LPS pup brain. These findings demonstrate that bacterial and viral models of MIA can result in similar precocious development in mice but differing long-term effects on inflammatory markers in both the milk provided to the pups and in their brains.

Tetrabromobisphenol A-bis(2,3-dibromopropyl ether) impairs Postnatal Testis Development in Mice: The Microtubule Cytoskeleton as a Sensitive Target.

Tetrabromobisphenol A-bis(2,3-dibromopropyl ether) (TBBPA-BDBPE), a widely used flame retardant, has been frequently detected in various environmental compartments, but its health hazard remains largely unknown. Here, we investigated the adverse effects of TBBPA-BDBPE (50 and 1000 μg/kg/day) on postnatal testis development in CD-1 mice and the underlying mechanism. Following the first week of maternal exposure, neonatal mice in the high-dose group exhibited reduced seminiferous tubule area, fewer Sertoli cells and germ cells, and damaged microtubules in Sertoli cells; even microtubule damage was also observed in the low-dose group. When exposure extended to adulthood, male offspring in the high-dose group presented more remarkable alterations in reproductive parameters, including reduced sperm count; in the low-dose group, microtubule damage was also observable, along with blood-testis barrier impairment. Further molecular docking analysis and tubulin polymerization assay indicated that TBBPA-BDBPE could interact with tubulin and disrupt its polymerization. Moreover, we observed attenuated microtubules in mouse Sertoli cells in vitro (TM4) following TBBPA-BDBPE treatment, suggesting that TBBPA-BDBPE impaired testis development possibly by interfering with tubulin dynamics. This study not only highlights the male reproductive hazard of TBBPA-BDBPE but also greatly improved the understanding of the molecular mechanism for male reproductive toxicity of chemicals.

4,4′-(9-Fluorenylidene)dianiline (BAFL) is antiestrogenic and has adverse effects on female development in CD-1 mice

Many phenolic compounds have been found to have endocrine disrupting activities, but their arylamine analogs, the phenolic hydroxyl groups substituted by aniline amino groups, have rarely been reported. 4,4′-(9-Fluorenylidene)dianiline (BAFL) is an arylamine analog of fluorene-9-bisphenol (BHPF) and BHPF has been reported to be a strong antiestrogen which could cause endometrial atrophy, ovarian damage and adverse pregnancy outcomes in animals. BAFL has been widely used as material to synthetize polymers, such as polyimides, polyamide, and polyamine, for various uses since the 1970s. Here, we assessed the antiestrogenicity of BAFL using a variety of methods and looked into its impacts on the development of females in CD-1 mice. With the aid of a yeast estrogen screen assay, we found BAFL possessed obviously antiestrogenic activity (IC50 = 8.15 × 10−6 M), which close to that of tamoxifen and BHPF. Using a 10-d mouse uterotrophic assay, we found that BAFL obviously decreased uterine weight in a dose-dependent way. Histological analyses of mouse uteri revealed that BAFL induced marked endometrial atrophy and inhibited the uterine development. Immunohistochemical analyses showed that Sprr2d, an estrogen-responsive gene encoding protein, was mainly expressed in endometrial epithelial cells and BAFL decreased the areas and levels of Sprr2d staining in mouse uteri. It was clear from uterine transcriptome investigations that BAFL significantly downregulated the expressions of multiple genes responding to estrogen. Molecular docking showed that BAFL could effectively occupy the antagonist-binding pocket of hERα, and one of the amino groups of BAFL formed hydrogen bonds with the side chains of Arg394 and Glu353 in the receptor. These results indicated that BAFL exhibited clearly antiestrogenic characteristics and could interfere with normal female development in mice, which should be avoided using in commodities that come into direct contact with humans. Moreover, this study indicated that the arylamine analogs of phenolic endocrine disrupting chemicals might also have endocrine disrupting activities.

Antiestrogenic property of 9,9-bis[4-(2-hydroxyethoxy)phenyl]fluorene (BPEF) and its effects on female development in CD-1 mice

Identifying chemicals with endocrine disrupting properties linked to disease outcomes is a key concern, as stated in the WHO-UNEP 2012 report on endocrine-disrupting chemicals. The chemical 9,9-bis[4-(2-hydroxyethoxy)phenyl]fluorene (BPEF) is widely and increasingly applied in synthesizing fluorene-based cardo polymers with superior optical, thermal and mechanical properties for various uses. However, little toxicological information is available regarding its safety. Here, we studied the endocrine disrupting property of BPEF by multiple toxicological tools and investigated its effects on female development in adolescent mice. Using the yeast two-hybrid bioassay, BPEF showed strong antiestrogenicity which was similar to that of tamoxifen, an effective antiestrogenic drug. In adolescent CD-1 mice, BPEF significantly decreased the uterine weight at relatively low doses and induced marked endometrial atrophy. Immunohistochemical staining and transcriptome analyses of the mice uteri revealed that BPEF could repressed the expressions of estrogen-responsive genes. Molecular simulation indicated that BPEF could be docked into the antagonist pocket of human estrogen receptor α, and the formation of hydrogen bonds and hydrophobic interactions between BPEF and the active site of receptor maintained their strong binding. All of the data demonstrated that BPEF possessed strong antiestrogenic property and might disrupt female development, suggesting it should be avoided in making products that might directly expose to people, particularly immature women.

Tris(4-hydroxyphenyl)ethane (THPE), a trisphenol compound, is antiestrogenic and can retard uterine development in CD-1 mice

Tris (4-hydroxyphenyl)ethane (THPE), a trisphenol compound widely used as a branching agent and raw material in plastics, adhesives, and coatings is rarely regarded with concern. However, inspection of in vitro data suggests that THPE is an antagonist of estrogen receptors (ERs). Accordingly, we aimed to evaluate the antiestrogenicity of THPE in vivo and tested its effect via oral gavage on pubertal development in female CD-1 mice. Using uterotrophic assays, we found that THPE either singly, or combined with 17β-estradiol (E2) (400 μg/kg bw/day) suppressed the uterine weights at low doses (0.1, 0.3, and 1 mg/kg bw/day) in 3-day treatment of weaning mice. When mice were treated with THPE during adolescence (for 10 days beginning on postnatal day 24), their uterine development was significantly retarded at doses of at least 0.1 mg/kg bw/day, manifest as decreased uterine weight, atrophic endometrial stromal cells and thinner columnar epithelial cells. Transcriptome analyses of uteri demonstrated that estrogen-responsive genes were significantly downregulated by THPE. Molecular docking shows that THPE fits well into the antagonist pocket of human ERα. These results indicate that THPE possesses strong antiestrogenicity in vivo and can disrupt normal female development in mice at very low dosages.

Pilot study on the effects of early exposure to hypergravity on the behavioural and cerebellar development of CD-1 mice

Pilot study on the effects of early exposure to hypergravity on the behavioural and cerebellar development of CD-1 mice

Retraction. Prenatal PFOA exposure alters gene expression pathways in murine mammary gland.

Perfluorooctanoic acid (PFOA) is a synthetic surfactant that was previously shown to delay mammary gland development in CD-1 mice. The objective of this study was to elucidate signaling pathways involved in this morphological effect. Timedpregnant CD-1 mice were treated with 0, 0.01, 0.1, or 1.0 mg PFOA/kg/day from gestational days (GD) 10–17 via oral gavage. Mammary glands from postnatal days (PND) 7 and 14 from control and 1.0 mg/kg PFOA treated mice were evaluated via genome-wide microarray analysis to identify PFOA-targeted candidate gene pathways. Selected genes from candidate pathways were evaluated by real-time PCR (RT-PCR) and Western blot analysis for RNA and protein expression changes, respectively, using samples from all treatment groups from PND 7, 14, and 21. Microarray analysis revealed that PFOA altered expression of genes involved in RNA post-transcriptional modification, lipid metabolism, and cholesterol biosynthesis pathways. These transcriptional changes were predicted to be regulated by peroxisome proliferator-activated receptor (Ppar) and endocrine related genes. Selected genes from candidate gene pathways Ppar, estrogen receptor alpha (Era/Esr1), and Wnt were further evaluated. Results from the RT-PCR analysis confirmed that genes in all 3 pathways were altered at varying levels in PFOA-exposed mammary glands. At PND 7, expression of PPARc and ERa protein were increased in a dose-dependent manner. At PND 21, mammary ERa protein expression was downregulated. PPARa protein levels were not affected. These results suggest that PFOA-induced mammary gland delays present on PND 21 may be modulated by changes in PPARc and endocrine-related genes.

Bisphenol A exposure at low dose level affects early placenta development in CD1 mice: Preliminary data of an integrated in vivo/ex vivo study

Bisphenol A exposure at low dose level affects early placenta development in CD1 mice: Preliminary data of an integrated in vivo/ex vivo study

Differential effects of peripubertal exposure to perfluorooctanoic acid on mammary gland development in C57Bl/6 and Balb/c mouse strains

Perfluorooctanoic acid (PFOA), a common and persistent industrial byproduct detected in human sera, has raised health concerns. PFOA is detrimental to lactational function and postnatal mammary gland development in CD-1 mice after gestational exposure. We have examined the peripubertal period (21 through 50 days of age) as an important window of mammary gland susceptibility to environmental exposures that may affect breast cancer risk later in life. The effects of PFOA (0.1–10mg/kg BW) were examined in Balb/c and C57BL/6 mice. PFOA treatment caused hepatocellular hypertrophy and delayed vaginal opening in both mouse strains. While Balb/c mice exhibited only inhibition of mammary gland and uterine development (5, 10mg/kg), C57BL/6 mice exhibited stimulatory effects in both organs at low dose (5mg/kg) and inhibition at higher dose (10mg/kg). This underscores the need for caution when drawing conclusions about the effects of PFOA and possibly other environmental pollutants on the basis of studies in a single mouse strain.

The effects of gestational and lactational exposure to chromium picolinate or picolinic acid on neurological development of CD-1 mice

The effects of gestational and lactational exposure to chromium picolinate or picolinic acid on neurological development of CD-1 mice

Effects of Pre- and Postnatal Exposure to Chromium Picolinate or Picolinic Acid on Neurological Development in CD-1 Mice

Chromium picolinate, Cr(pic)3, a popular dietary supplement marketed as an aid in fat loss and lean muscle gain, has also been suggested as a therapy for women with gestational diabetes. The current study investigated the effects of maternal exposure to Cr(pic)3 and picolinic acid during gestation and lactation on neurological development of the offspring. Mated female CD-1 mice were fed diets from implantation through weaning that were either untreated or that contained Cr(pic)3 (200 mg kg(-1) day(-1)) or picolinic acid (174 mg kg(-1) day(-1)). A comprehensive battery of postnatal tests was administered, including a modified Fox battery, straight-channel swim, open-field activity, and odor-discrimination tests. Pups exposed to picolinic acid tended to weigh less than either control or Cr(pic)3-exposed pups, although the differences were not significant. Offspring of picolinic acid-treated dams also appeared to display impaired learning ability, diminished olfactory orientation ability, and decreased forelimb grip strength, although the differences among the treatment groups were not significant. The results indicate that there were no significant effects on the offspring with regard to neurological development from supplementation of the dams with either Cr(pic)3 or picolinic acid.

Chemopreventive effects of sarcotriol on ultraviolet B-induced skin tumor development in SKH-1 hairless mice.

Sarcotriol (ST) has been shown to be chemopreventive on 7,12-dimethyl-benz(a)anthracene (DMBA) initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin tumor development in CD-1 mice in recent studies from our laboratory. The objective of this study was to determine the chemopreventive effects of ST on ultraviolet B (UVB)-induced skin tumor development in female SKH-1 hairless mice, an experimental model relevant to human skin cancer development, and its possible mechanisms of action. Female SKH-1 mice were divided into two groups: Control and ST treated. Control was topically treated with 100 microliter acetone and ST treated group administered with 30 microgram ST in 100 microliter acetone one hour before UVB exposure. For UVB-induced tumorigenesis, carcinogenesis was initiated and promoted by UVB (180 mJ/cm(2)). Group weights and tumor counts were taken once every week. After 30 weeks, mice were sacrificed and dorsal skin samples were collected. The proteins from the skin sample were further used for SDS-PAGE and Western blotting using specific antibodies against caspase-3, caspase-8, caspase-9 and p53. Tumor multiplicity was found 19.6, 5.2 in the control and ST treated groups respectively. Caspase-3, -8, -9 and p53 were significantly (P < 0.05) upregulated in ST treated group compared to Control group. Together, this study for the first time identifies the chemopreventive effects of ST in UVB-induced carcinogenesis possibly by inducing apoptosis and upregulating p53.

Chemopreventive effects of sarcophine-diol on skin tumor development in CD-1 mice

The objective of this study was to determine the chemopreventive effects of sarcophine-diol (SD) on 7,12-dimethylbenz(a)anthracene initiated and 12- O-tetradecanoylphorbol-13-acetate promoted skin tumor development in mice and its possible mechanisms of action. SD pretreatment significantly ( P < 0.05) decreased skin papilloma development during promotion phase. SD significantly ( P < 0.05) increased caspase-3 and decreased cyclooxygenase-2 during initiation phase or promotion phase. SD significantly ( P < 0.05) increased caspase-8 during promotion phase. SD resulted in a 95% reduction in 12- O-tetradecanoylphorbol-13-acetate-induced DNA synthesis. SD could be an effective chemopreventive agent for skin cancer by enhancing apoptosis and decreasing cell proliferation.

Chemopreventive Effects of Sarcotriol on Ultraviolet B-induced Skin Tumor Development in SKH-1 Hairless Mice

Sarcotriol (ST) has been shown to be chemopreventive on 7,12-dimethylbenz( a)anthracene (DMBA) initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)- promoted skin tumor development in CD-1 mice in recent studies from our laboratory. The objective of this study was to determine the chemopreventive effects of ST on ultraviolet B (UVB)-induced skin tumor development in female SKH-1 hairless mice, an experimental model relevant to human skin cancer development, and its possible mechanisms of action. Female SKH-1 mice were divided into two groups: Control and ST treated. Control was topically treated with 100 μL acetone and ST treated group administered with 30 μg ST in 100 μL acetone one hour before UVB exposure. For UVB-induced tumorigenesis, carcinogenesis was initiated and promoted by UVB (180 mJ/cm2). Group weights and tumor counts were taken once every week. After 30 weeks, mice were sacrificed and dorsal skin samples were collected. The proteins from the skin sample were further used for SDSPAGE and Western blotting using specific antibodies against caspase-3, caspase-8, caspase-9 and p53. Tumor multiplicity was found 19.6, 5.2 in the control and ST treated groups respectively. Caspase-3, -8, -9 and p53 were significantly (P < 0.05) upregulated in ST treated group compared to Control group. Together, this study for the first time identifies the chemopreventive effects of ST in UVB-induced carcinogenesis possibly by inducing apoptosis and upregulating p53.

A Possible Mechanism of Action of the Chemopreventive Effects of Sarcotriol on Skin Tumor Development in CD-1 Mice

Sarcophine derivatives have been suggested to be chemopreventive in nature. One of its derivatives, Sarcotriol (ST), was investigated to study the skin cancer chemopreventive effects in female CD-1 mice. Three groups (control, promotion, initiation) of 30 female CD-1 mice each were taken. Carcinogenesis was initiated with 7, 12-dimethylbenz (a) anthracene (DMBA) and promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). One hour before treating with DMBA (200 nmol/100 μl acetone), control and promotion groups were treated with acetone (100 μl) and initiation group with ST (30μg/100μl of acetone). Beginning one week after initiation with DMBA, control and initiation groups were treated with acetone and promotion group with ST (30μg/100μl of acetone), one hour before treating with TPA (5 nmol/100 μl acetone). This was carried out twice a week for the next 20 weeks. The effects of ST on 3H-thymidine incorporation in epidermal DNA, the possible role of apoptotic proteins and COX-2 involved in the prevention of skin tumor development of CD-1 mice were investigated. Tumor incidence and multiplicity was found to be 100%, 73%, 100% and 8.2, 4.8, 9.7 in control, promotion and initiation groups respectively. ST treatment resulted in a significant (P &lt; 0.05) inhibition in the incorporation of 3H-thymidine in epidermal DNA. The promotion group showed higher levels of caspase-3, -8 and –9 compared to the control. COX-2 expression was significantly lower (P &lt; 0.05) in the promotion group as compared to the control. No significant difference in caspase-3, -8, -9 and COX-2 levels were observed in the initiation group compared to control. Together, this study confirms the chemopreventive effects of ST, and for the first time identifies the stage of carcinogenesis at which ST exerts its chemopreventive effect, and elucidates the mechanism possibly by inducing apoptosis and decreasing the COX-2 levels, contributing to its overall cancer chemopreventive effects in the mouse skin cancer model.

Chemopreventive effects of various concentrations of α-santalol on skin cancer development in CD-1 mice

Previous studies from this laboratory have indicated that alpha-santalol (5%) provides chemopreventive effects in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin cancer in CD-1 and SENCAR mice. Skin cancer development is associated with increased ornithine decarboxylase (ODC) activity, DNA synthesis and rapid proliferation of epidermal cells. The purpose of this investigation was to determine the effects of various concentrations (1.25% and 2.5%) of alpha-santalol on DMBA-initiated and TPA-promoted skin cancer development, TPA-induced ODC activity, and DNA synthesis in CD-1 mice. alpha-Santalol treatment at both concentrations (1.25% and 2.5%) prevented the skin cancer development. alpha-Santalol treatment (1.25% and 2.5%) resulted in a significant decrease in the TPA-induced ODC activity and incorporation of [3H]thymidine in DNA in the epidermis of CD-1 mice. There was no significant difference in the effects of 1.25% and 2.5% alpha-santalol on tumour incidence, multiplicity, epidermal TPA-induced ODC activity, or DNA synthesis in CD-1 mice.

Chemopreventive effects of pomegranate seed oil on skin tumor development in CD1 mice.

Pomegranate seed oil was investigated for possible skin cancer chemopreventive efficacy in mice. In the main experiment, two groups consisting each of 30, 4-5-week-old, female CD(1) mice were used. Both groups had skin cancer initiated with an initial topical exposure of 7,12-dimethylbenzanthracene and with biweekly promotion using 12-O-tetradecanoylphorbol 13-acetate (TPA). The experimental group was pretreated with 5% pomegranate seed oil prior to each TPA application. Tumor incidence, the number of mice containing at least one tumor, was 100% and 93%, and multiplicity, the average number of tumors per mouse, was 20.8 and 16.3 per mouse after 20 weeks of promotion in the control and pomegranate seed oil-treated groups, respectively (P <.05). In a second experiment, two groups each consisting of three CD(1) mice were used to assess the effect of pomegranate seed oil on TPA-stimulated ornithine decarboxylase (ODC) activity, an important event in skin cancer promotion. Each group received a single topical application of TPA, with the experimental group receiving a topical treatment 1 h prior with 5% pomegranate seed oil. The mice were killed 5 h later, and ODC activity was assessed by radiometric method. The experimental group showed a 17% reduction in ODC activity. Pomegranate seed oil (5%) significantly decreased (P <.05) tumor incidence, multiplicity, and TPA-induced ODC activity. Overall, the results highlight the potential of pomegranate seed oil as a safe and effective chemopreventive agent against skin cancer.

Chronic prenatal exposure to paroxetine (Paxil) and cognitive development of mice offspring

This study investigated the impact on cognitive development in CD-1 mice from chronic prenatal exposure to the antidepressant paroxetine. CD-1 mice were given either paroxetine as 30 mg/kg/day or a placebo in food bars for 2 weeks before mating and throughout gestation. One offspring per gender from each litter was tested on each of the following tasks: tube runway, spatial maze, passive avoidance chamber, and water straight runway followed by an unforced decision maze. Learning occurred in both genders in all tasks ( p<0.001) with no significant differences between treatment groups at the final learning session. Juvenile runway was the only task in which the paroxetine-exposed males demonstrated a learning rate that was slower than the placebo-exposed offspring ( p=0.06). Post learning sessions did not show any significant treatment differences during the juvenile and adult periods during the water straight runway, mazes, and avoidance chamber tasks. In conclusion, chronic prenatal exposure in mice of paroxetine did not impact cognition on select tasks.

High Yield Selective Induction of Uterine Endometrial Adenocarcinomas in CD-1 Mice by N-Ethyl-N'-nitro-N-nitrosoguanidine Combined with 17.BETA.-Estradiol.

In order to assess a combined carcinogen and hormone protocol for selective induction of uterine adenocarcinomas in mice, 79 illumination-induced persistent estrous CD-1 mice, divided into four groups, were treated with N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and/or 17β-estradiol (E2). Groups 1 and 3 were given a single intra-uterine administration of polyethylene glycol (PEG) at 10 weeks of age, while Groups 2 and 4 received ENNG (12.5 mg/kg), dissolved in PEG. Mice of Groups 3 and 4 were also implanted with E2 pellets s.c. one week earlier and the pellets were once renewed after 8 weeks. At 15 weeks after the ENNG treatment, the mice were killed for histopathological and endocrinological examination. All groups demonstrated endometrial proliferative lesions, although no severe hyperplasias or adenocarcinomas were found in the control group (Group 1). The incidences of adenocarcinomas in Groups 2, 3, and 4 were 6 (1/19), 25 (5/20), and 55% (11/20), respectively, those for Groups 3 and 4 being significant as compared to the lack in Group 1. The E2: progesterone (E2: P) ratios in Groups 3 and 4 were also significantly increased. These results indicate that an increased E2: P ratio is important for endometrial adenocarcinoma development in CD-1 mice, acting with or without chemical carcinogen initiation to cause uterine cancer development. The relatively short duration, and specifically high yield mean that the present protocol has advantages for two-stage uterine carcinogenicity studies in mice.

Expression of bone associated markers by tooth root lining cells, in situ and in vitro

Periodontal disease is marked by inflammation and subsequent loss and/or damage to tooth-supporting tissues including bone, cementum, and periodontal ligament. A key tissue in the initial process of periodontal development as well as regeneration following periodontal disease is cementum. Research efforts aimed toward understanding mechanisms involved in periodontal development and regeneration, and in particular the formation of root cementum, have been hampered by an inability to isolate and culture cells involved in cementum production (i.e., cementoblats). Much has been learned regarding the processes and mechanisms involved in bone formation and function from experiments using bone cell cultures. Therefore, the purpose of this study was to develop a strategy whereby cementoblasts could be isolated, cultured, and characterized. As a first step, using in situ hybridization, we determined the timed and spatial expression of mineral-associated proteins during first molar root development in CD-1 mice. These proteins included dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP), osteocalcin (OCN), and type I collagen. During root development in mice BSP, OPN, and OCN mRNAs were expressed selectively by cells lining the tooth root surface—cementoblasts—with high levels of expression at day 41. Importantly, at this time point BSP, OPN, and OCN mRNAs were not expressed throughout the periodontal ligament. These findings provided us with markers selective to rootlining cells, or cementoblasts, in situ, and established the time (day 41) for isolating cells for in vitro studies. To isolate cells from tissues adherent to the root surface, enzymatic digestion was used, similar to what are now considered classical techniques for isolation of osteoblasts. To determine whether cells in vitro contained root-lining cells and cementoblasts, cultured cells were analyzed for expression of mineral-associated proteins. Cells within this heterogeneous primary population expressed type I collagen, BSP, OPN, and OCN as determined by in situ hybridization. In contrast, cells within this population did not express dentin sialoprotein, an odontoblast-specific protein. These procedures have provided a means to obtain root-lining cells in vitro that can now be cloned and used for studies directed at determining the properties of root-lining cells, or cementoblasts, in vitro.