Analysis of lead in circumpulpal dentin of deciduous teeth

Absorbed lead continuously adds to the lead deposited in the vascularized circumpulpal dentin of the teeth. Thus, this dental tissue is expected to contain a lead concentration which reflects the integrated lead exposure during the time from completion of tooth formation to tooth extraction or shedding. A method has been developed to assess the lead level in the dentin surrounding the pulp chamber in deciduous teeth. Variation within the tooth is minimal, but upper medial incisors show a slightly lower lead level than do other incisors, as assessed in 714 teeth from first-grade Danish school children. This tendency was not confirmed, however, in a small number of paired teeth from the same children. The new method appears advantageous for epidemiological studies of lead neurotoxicity in children.     

Cementum and periodontal ligament-like tissue formation induced using bioengineered dentin

Stem cell-mediated root regeneration offers opportunities to regenerate a bio-root and its associated periodontal tissues to restore tooth loss. Periodontal ligament (PDL) and cementum complex and dentin pulp complex have been tissue engineered using human dental pulp stem cells and PDL stem cells, respectively. The aim of this study was to explore whether dentin formation could be induced using an inductive substrate and whether bioengineered dentin could induce cementum and PDL formation. First, dentin was bioengineered from tooth papillae of Sprague-Dawley (SD) rats with an inductive substrate, and its phenotype was characterized; then primarily cultured human PDL cells were seeded on the surface of dentin and transplanted under the skin of immunocompromised mice. Histological, immunohistochemical, and scanning electronic microscopy examinations results showed that bioengineered dentin could induce cementogenesis and PDL formation, and condense PDL arranged perpendicularly on the dentin surface via a layer of cementum-like tissue. The results indicated that tissue-engineered dentin could be induced using an inductive substrate and could be used as a further substrate for cementum and PDL tissue engineering.     

Distribution of BMP6 in the alveolar bone during mouse mandibular molar eruption

Eruption requires synchrony of the tooth with the surrounding tissues, particularly the bone. One important step during eruption is remodelling of the alveolar bone at the base of the tooth and along the roots. Expression of BMP6 was reported to be increased in the basal half of the dental follicle prior to eruption and inhibition of BMP6 affected bone formation at the base of the alveolar crypt. The aim of this study was to further investigate BMP6 protein in relation to tooth eruption and the corresponding bone remodelling using temporospatial correlations of BMP6 localization with morphogenetic events (proliferation, differentiation, apoptosis and bone apposition/resorption), other BMPs (BMP2 and BMP7) and three-dimensional images of tooth–bone development. BMP6 expression pattern was mapped in the mandibular molar teeth and related structures around eruption. Localization of BMP6 dominated in osteoblasts, in regions of bone formation within the alveolar crypt. These findings positively correlated with proliferation at the tooth base region, osteocalcin expression in the osteoblasts/osteocytes and BMP2 and BMP7 presence in the alveolar bone surrounding the tooth. Osteoclast activity and apoptotic elimination in the root region gradually decreased before eruption and totally ceased at eruption stages. Generally, BMP6 positively correlated with BMP2, BMP7 and osteocalcin-positive osteoblasts, and areas of bone remodelling. Moreover, BMP6 was found in the periodontium and cementoblasts. BMP6 expression in the alveolar bone accompanied tooth eruption. Notably, the expression pattern of BMP6 in the bone did not differ around individual molar teeth at the same stage of development. The expression of BMP6 in periodontal ligaments may contribute to interaction between the tooth and bone during the eruption and anchoring process.     

The expression patterns of gremlin 1 and noggin in normal adult and tumor tissues

Gremlin 1 and noggin are inhibitors of bone morphogenetic protein (BMP) signaling. They are vital during early development but their role in adult tissues has remained largely unresolved. The BMP signaling pathway has also been implicated in tumorigenesis, however with emphasis on the role of the ligands and receptors. We performed a concurrent survey of gremlin 1 and noggin protein expression in multiple normal and cancer samples, using immunohistochemistry on tissue microarrays containing 96 samples from 34 different normal organs/tissue sites and 208 samples of 34 different tumor types. In majority of both normal and tumor samples, gremlin 1 and noggin expression was negative or weak. However, normal stomach and skin demonstrated distinct gremlin 1 and noggin expression indicating a role in adult tissues. Likewise, strong expression of both antagonists was detected in Leydig cells of testis. In the tumor panel, the expression patterns were more variable but elevated BMP antagonist expression was detected for the first time in few cases, such as glioblastoma, hepatocellular carcinoma and diffuse B-cell lymphoma for gremlin 1 and renal granular cell tumor and thyroid papillary carcinoma for noggin. Even though gremlin 1 and noggin were not widely expressed in adult tissues, in a subset of organs their expression pattern indicated a potential role in normal tissue homeostasis as well as in malignancies.     

Autophagy in SDF-1α-mediated DPSC migration and pulp regeneration

Critical morphological requirements for pulp regeneration are tissues replete with vascularisation, neuron formation, and dentin deposition. Autophagy was recently shown to be related to angiogenesis, neural differentiation, and osteogenesis. The present study aimed to investigate the involvement of autophagy in stromal cell-derived factor-1α (SDF-1α)-mediated dental pulp stem cell (DPSC) migration and pulp regeneration, and identify its presence during pulp revascularisation of pulpectomised dog teeth with complete apical closure. In vitro studies showed that SDF-1α enhanced DPSCs migration and optimised focal adhesion formation and stress fibre assembly, which were accompanied by autophagy. Moreover, autophagy inhibitors significantly suppressed, whereas autophagy activator substantially augmented SDF-1α-stimulated DPSCs migration. Furthermore, after ectopic transplantation of tooth fragment/silk fibroin scaffold with DPSCs into nude mice, pulp-like tissues with vascularity, well-organised fibrous matrix formation, and new dentin deposition along the dentinal wall were generated in SDF-1α-loaded samples accompanied by autophagy. More importantly, in a pulp revascularisation model in situ, SDF-1α-loaded silk fibroin scaffolds improved the de novo ingrowth of pulp-like tissues in pulpectomised mature dog teeth, which correlated with the punctuated LC3 and Atg5 expressions, indicating autophagy. Our findings provide novel insights into the pulp regeneration mechanism, and SDF-1α shows promise for future clinical application in pulp revascularisation.     

Forensic study of sex determination using PCR on teeth samples

In this study, sex determination using polymerase chain reaction (PCR) on tooth material was evaluated from the viewpoint of forensic medicine. The sensitivity of PCR for detection of the Y chromosome-specific alphoid repeat sequence and the X chromosome-specific alphoid repeat sequence was 0.5 pg of genomic DNA. Sex could be determined by PCR of DNA extracted from the pulp of 16 freshly extracted permanent teeth and dentine including the surface of the pulp cavity of 6 freshly extracted milk teeth. Sex could be determined using the pulp in all 20 teeth (10 male and 10 female) preserved at room temperature for 22 years. For the pulp of teeth stored in sea water, the sex could be determined in all 8 teeth immersed for 1 week and in 5 of 6 teeth immersed for 4 weeks. In the remaining 1 tooth, in which sex determination based on the pulp failed, the sex could be determined correctly when DNA extracted from the tooth hard tissue was examined. For teeth stored in soil, the sex could be determined accurately in all 8 teeth buried for 1 week, 7 of 8 teeth buried for 4 weeks, and in all 6 teeth buried for 8 weeks. When teeth were heated for 30 min, sex determination from the pulp was possible in all teeth heated to 100, 150, and 200 degrees C, and even in some teeth heated to 250 degrees C. When this method was applied to actual forensic cases, the sex of a mummified body estimated to have been discovered half a year to 1 year after death could be determined readily by examination of the dental pulp. In the skeletons of 2 bodies placed under water for approximately 1 year and approximately 11 years and 7 months, pulp tissues had been dissolved and lost, but sex determination was possible using DNA extracted from hard dental tissues. These results indicate that this method is useful in forensic practices for sex determination based on teeth samples.     

Mutation in SSUH2 Causes Autosomal‐Dominant Dentin Dysplasia Type I

Dentin dysplasia type I (DDI) is an autosomal‐dominant genetic disorder resulting from dentin defects. The molecular basis of DDI remains unclear. DDI exhibits unique characteristics with phenotypes featuring obliteration of pulp chambers and diminutive root, thus providing a useful model for understanding the genetics of tooth formation. Using a large Chinese family with 14 DDI patients, we mapped the gene locus responsible for DDI to 3p26.1–3p24.3 and further identified a missense mutation, c.353C>A (p.P118Q) in the SSUH2 gene on 3p26.1, which co‐segregated with DDI. We showed that SSUH2 (p.P118Q) perturbed the structure and significantly reduced levels of mutant (MT) protein and mRNA compared with wild‐type SSUH2 . Furthermore, MT P141Q knock‐in mice (+/? and ?/?) had a unique partial obliteration of the pulp cavity and upregulation or downregulation of six major genes involved in odontogenesis: Dspp , Dmp1 , Runx2 , Pax9 , Bmp2 , and Dlx2 . The phenotype of missing teeth was determined in zebrafish with morpholino gene knockdowns and rescued by injection of normal human mRNA. Taken together, our observations demonstrate that SSUH2 disrupts dental formation and that this novel gene, together with other odontogenesis genes, is involved in tooth development.     

Canonical Wnt signaling acts synergistically on BMP9-induced osteo/odontoblastic differentiation of stem cells of dental apical papilla (SCAPs)

Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. Here, we investigate if BMP9 and Wnt/β-catenin act synergistically on odontogenic differentiation. Using the immortalized SCAPs (iSCAPs) isolated from mouse apical papilla tissue, we demonstrate that Wnt3A effectively induces early osteogenic marker alkaline phosphatase (ALP) in iSCAPs, which is reduced by β-catenin knockdown. While Wnt3A and BMP9 enhance each other's ability to induce ALP activity in iSCAPs, silencing β-catenin significantly diminishes BMP9-induced osteo/odontogenic differentiation. Furthermore, silencing β-catenin reduces BMP9-induced expression of osteocalcin and osteopontin and in vitro matrix mineralization of iSCAPs. In vivo stem cell implantation assay reveals that while BMP9-transduced iSCAPs induce robust ectopic bone formation, iSCAPs stimulated with both BMP9 and Wnt3A exhibit more mature and highly mineralized trabecular bone formation. However, knockdown of β-catenin in iSCAPs significantly diminishes BMP9 or BMP9/Wnt3A-induced ectopic bone formation in vivo. Thus, our results strongly suggest that β-catenin may play an important role in BMP9-induced osteo/ondontogenic signaling and that BMP9 and Wnt3A may act synergistically to induce osteo/odontoblastic differentiation of iSCAPs. It's conceivable that BMP9 and/or Wnt3A may be explored as efficacious biofactors for odontogenic regeneration and tooth engineering.     

The blood-vessel thrust theory of tooth eruption and migration

The Blood-Vessel Thrust Theory is a new hypothesis regarding the forces which produce the normal eruption of teeth, and the movement of 'nonerupted' teeth through bone away from their normal position in the jaws. It points out that the flow of blood through the vessels of the dental pulp, and of the tissues surrounding the tooth, must produce hydrodynamic and hydrostatic forces within the blood vessels, and that these forces have a resultant towards the tooth crown, thus causing the tooth to move, crown first, through the bone during normal eruption or abnormal tooth migration.     

Combination of aligned PLGA/Gelatin electrospun sheets,native dental pulp extracellular matrix and treated dentin matrix as substrates for tooth root regeneration

In tissue engineering, scaffold materials provide effective structural support to promote the repair of damaged tissues or organs through simulating the extracellular matrix (ECM) microenvironments for stem cells. This study hypothesized that simulating the ECM microenvironments of periodontium and dental pulp/dentin complexes would contribute to the regeneration of tooth root. Here, aligned PLGA/Gelatin electrospun sheet (APES), treated dentin matrix (TDM) and native dental pulp extracellular matrix (DPEM) were fabricated and combined into APES/TDM and DPEM/TDM for periodontium and dental pulp regeneration, respectively. This study firstly examined the physicochemical properties and biocompatibilities of both APES and DPEM in vitro, and further investigated the degradation of APES and revascularization of DPEM in vivo. Then, the potency of APES/TDM and DPEM/TDM in odontogenic induction was evaluated via co-culture with dental stem cells. Finally, we verified the periodontium and dental pulp/dentin complex regeneration in the jaw of miniature swine. Results showed that APES possessed aligned fiber orientation which guided cell proliferation while DPEM preserved the intrinsic fiber structure and ECM proteins. Importantly, both APES/TDM and DPEM/TDM facilitated the odontogenic differentiation of dental stem cells in vitro. Seeded with stem cells, the sandwich composites (APES/TDM/DPEM) generated tooth root-like tissues after being transplanted in porcine jaws for 12 w. In dental pulp/dentin complex-like tissues, columnar odontoblasts-like layer arranged along the interface between newly-formed predentin matrix and dental pulp-like tissues in which blood vessels could be found; in periodontium complex-like tissues, cellular cementum and periodontal ligament (PDL)-like tissues were generated on the TDM surface. Thus, above results suggest that APES and DPEM exhibiting appropriate physicochemical properties and well biocompatibilities, in accompany with TDM, could make up an ECM microenvironment for tooth root regeneration, which also offers a strategy for complex tissue or organ regeneration.     

A transmission electron microscopy study of mineralization in age-induced transparent dentin

It is known that fractures are more likely to occur in altered teeth, particularly following restoration or endodontic repair; consequently, it is important to understand the structure of altered forms of dentin, the most abundant tissue in the human tooth, in order to better define the increased propensity for such fractures. Transparent (or sclerotic) dentin, wherein the dentinal tubules become occluded with mineral as a natural progressive consequence of aging, is one such altered form. In the present study, high-resolution transmission electron microscopy is used to investigate the effect of aging on the mineral phase of dentin. Such studies revealed that the intertubular mineral crystallites were smaller in transparent dentin, and that the intratubular mineral (larger crystals deposited within the tubules) was chemically similar to the surrounding intertubular mineral. Exit-wave reconstructed lattice-plane images suggested that the intratubular mineral had nanometer-size grains. These observations support a “dissolution and reprecipitation” mechanism for the formation of transparent dentin.     

E- and N-cadherin distribution in developing and functional human teeth under normal and pathological conditions

Cadherins are calcium-dependent cell adhesion molecules involved in the regulation of various biological processes such as cell recognition, intercellular communication, cell fate, cell polarity, boundary formation, and morphogenesis. Although previous studies have shown E-cadherin expression during rodent or human odontogenesis, there is no equivalent study available on N-cadherin expression in dental tissues. Here we examined and compared the expression patterns of E- and N-cadherins in both embryonic and adult (healthy, injured, carious) human teeth. Both proteins were expressed in the developing teeth during the cap and bell stages. E-cadherin expression in dental epithelium followed an apical-coronal gradient that was opposite to that observed for N-cadherin. E-cadherin was distributed in proliferating cells of the inner and outer enamel epithelia but not in differentiated cells such as ameloblasts, whereas N-cadherin expression was up-regulated in differentiated epithelial cells. By contrast to E-cadherin, N-cadherin was also expressed in mesenchymal cells that differentiate into odontoblasts and produce the hard tissue matrix of dentin. Although N-cadherin was not detected in permanent intact teeth, it was re-expressed during dentin repair processes in odontoblasts surrounding carious or traumatic sites. Similarly, N-cadherin re-expression was seen in vitro, in cultured primary pulp cells that differentiate into odontoblast-like cells. Taken together these results suggest that E- and N-cadherins may play a role during human tooth development and, moreover, indicate that N-cadherin is important for odontoblast function in normal development and under pathological conditions.     

A Journey from Dental Pulp Stem Cells to a Bio-tooth

The ultimate goal of tooth regeneration is to replace the lost teeth. Stem cell-based tooth engineering is deemed as a promising approach to the making of a biological tooth (bio-tooth). Dental pulp stem cells (DPSCs) represent a kind of adult cell colony which has the potent capacity of self-renewing and multilineage differentiation. The exact origin of DPSCs has not been fully determined and these stem cells seem to be the source of odontoblasts that contribute to the formation of dentin-pulp complex. Recently, achievements obtained from stem cell biology and tooth regeneration have enabled us to contemplate the potential applications of DPSCs. Some studies have proved that DPSCs are capable of producing dental tissues in vivo including dentin, pulp, and crown-like structures. Whereas other investigations have shown that these stem cells can bring about the formation of bone-like tissues. Theoretically, a bio-tooth made from autogenous DPSCs should be the best choice for clinical tooth reconstruction. This review will focus on the location, origin, and current isolation approaches of these stem cells. Their odontoblastic differentiation and potential utilizations in the reconstruction of dentin-pulp complex and bio-tooth will be extensively discussed.     

Differential effects of fibromodulin deficiency on mouse mandibular bones and teeth: a micro-CT time course study

Fibromodulin (Fmod) is a keratan sulfate small leucine-rich proteoglycan which is enriched in bones and teeth. In order to determine its functions on bone and tooth mineralization we characterized the phenotype of Fmod-deficient (Fmod-KO) mice using a new-generation microfocus computerized tomography system (micro-CT) and software allowing advanced visualization of 3-D data. Three-week-old and 10- week-old Fmod-KO mandibles and teeth were compared with those of age-matched wild-type (WT) mice. In both young and mature mice the Fmod-KO mandibles were hypomineralized, especially the posterior (proximal) part of the mandible as it appeared to be the main target of the molecule deficiency whereas less extensive alterations were found in the alveolar bone. In transverse sections, larger marrow spaces were observed in the Fmod-KO mice compared with age-matched young or mature WT mice. Quantitative evaluation of the pulp volume of the first molar and 3-D reconstructions suggested that dentinogenesis was diminished in 3-week-old Fmod-KO teeth. In contrast, increased dentin formation was found in 10-week-old Fmod-KO mice and it was accompanied by a reduced pulp volume. Thus, the differential effects of Fmod deficiency on bones and teeth appear to diverge in adult mice. This may result from the previously reported differences in the molecular weight of Fmod in the 2 tissues or from compensatory mechanisms due to the overexpression of DSP and DMP-1 in the dental pulp of Fmod-KO. It is also possible that a single molecule plays diverging roles in a tissue-specific or region-specific manner.     

Comparative Qualitative and Quantitative Assessment of Biomineralization of Tooth Development in Man and Zebrafish (Danio rerio)

It was the aim of this study to investigate the distribution of Ca, P, and C in predentin, mineralizing dentin, and mature dentin of human tooth buds and compare these results with those of zebrafish (Danio rerio) teeth using energy dispersive X‐ray analysis (EDX) element analysis. The mandible of a 16‐week‐old human fetus containing 6 mineralizing tooth buds and three complete heads of zebrafish were fixed in formaldehyde and embedded in Technovit 9100. Serial sections of 80‐μm thickness were cut in frontal‐dorsal direction, and from these sections, polarized light micrographs were taken. The sections with tooth buds were then investigated with scanning electron microscopy, and EDX element analysis was performed with a Philips XL 30 FEG scanning microscope and an EDAX energy‐dispersive X‐ray system using spot measurements, EDX line‐scans and element mapping. Quantitative measurements were made in predentin, mineralizing dentin adjacent to predentin, and mature dentin. The Ca and P content increased rapidly from outer predentin toward mineralizing dentin in human tooth buds and in zebrafish teeth. The Ca/P ratio was different for predentin and dentin areas, reflecting different calcium phosphate compositions in predentin and fully mineralized dentin. Because of the similarities between human tooth buds and zebrafish teeth, it can be concluded that the zebrafish tooth development may be an excellent model for studying biomineralization processes during odontogenesis. Anat Rec, 291:571–576, 2008. © 2008 Wiley‐Liss, Inc.     

Side population cells isolated from porcine dental pulp tissue with self-renewal and multipotency for dentinogenesis, chondrogenesis, adipogenesis, and neurogenesis

Dental pulp has the potential to form dentin as a regenerative response to caries. This regeneration is mediated by stem/progenitor cells. Thus, stem cell therapy might be of potential utility in induction of reparative dentin. We isolated side population (SP) cells from dental pulp based on the exclusion of the DNA binding dye Hoechst 33342 by flow cytometry and compared its self-renewal capacities and multipotency with non-SP cells and primary pulp cells. The cumulative cell number of the SP cells was greater than the non-SP cells and primary pulp cells. Bmi1 was continuously expressed in SP cells, suggesting longer proliferative lifespan and self-renewal capacity of SP cells. Next, the maintenance of the multilineage differentiation potential of pulp SP cells was investigated. Expression of type II collagen and aggrecan confirmed chondrogenic conversion (30%) of SP cells. SP cells expressed peroxisome proliferator-activated receptor gamma and adaptor protein 2, showing adipogenic conversion. Expression of mRNA and proteins of neurofilament and neuromodulin confirmed neurogenic conversion (90%). These results demonstrate that pulp SP cells maintain multilineage differentiation potential. We further examined whether bone morphogenetic protein 2 (BMP2) could induce differentiation of pulp SP cells into odontoblasts. BMP2 stimulated the expression of dentin sialophosphoprotein (Dspp) and enamelysin in three-dimensional pellet cultures. Autogenous transplantation of the Bmp2-supplemented SP cells on the amputated pulp stimulated the reparative dentin formation. Thus, adult pulp contains SP cells, which are enriched for stem cell properties and useful for cell therapy with BMP2 for dentin regeneration.     

Ultimate tensile strength of dentin: Evidence for a damage mechanics approach to dentin failure

Dentin structure and properties are known to vary with orientation and location. The present study explored the variation in the ultimate tensile strength (UTS) of dentin with location in the tooth. Hourglass specimens were prepared from dentin located in the center, under cusps, and in the cervical regions of human molar teeth. These were tested in tension at various distances from the pulp. Median tensile strengths ranged from 44.4 MPa in the inner dentin near the pulp, to 97.8 MPa near the dentino-enamel junction (DEJ). This increase in the median UTS with distance from the pulp to the DEJ was statistically significant (P <.001). Of particular importance was the observation that the UTS measurements followed a Weibull probability distribution, with a Weibull modulus of about 4.5. The Weibull behavior of the UTS data strongly suggests that the large variances in fracture strength data result from a distribution of preexisting defects in the dentin. These findings justify a damage-mechanics approach to studies of dentin failure.     

Delayed tooth eruption in membrane type-1 matrix metalloproteinase deficient mice

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is expressed highly in mineralizing tissues including bones and teeth. Mice deficient in MT1-MMP (-/-) display severe defects in skeletal development including dwarfism, osteopenia, and craniofacial abnormalities. Death occurs in these mice by about 3 weeks of age. Since MT1-MMP is expressed by the ameloblasts of the enamel organ and by the odontoblasts of the dental papilla, we asked if the developing teeth were adversely affected in the knockout animals. Molars from MT1-MMP -/- mice and controls were examined by histological, X-ray, and SEM analysis at 4, 18-20, and 25 days of postnatal development. At 4 days of development the molars from the -/- mice appeared histologically normal. At 18-20 days of development, the first molars of the -/- mice had apparently normal tooth crowns with normal dentin and enamel; however, the roots were truncated and the teeth had not yet erupted. In contrast to the -/- mice, the first molars of the 18-20-day control animals had erupted. SEM analysis of a -/- first molar and incisor revealed a normal enamel prism pattern. However, X-ray analysis demonstrated that tooth eruption was delayed by approximately 5 days and that the tooth roots were abnormally short in the knockout animals. Since MT1-MMP-deficient mice have been demonstrated to display a generalized increase in bone resorption, these data suggest that inefficient growth of bone surrounding the tooth root complex causes a delay in tooth eruption.     

Biological evaluation of a new pulp capping material developed from Portland cement

This study evaluates the biological properties of a new pulp capping material developed from Portland cement. This study was conducted on 48 teeth in 4 dogs (12 teeth/dog). The dogs were classified into two equal groups (n = 24 teeth) according to the evaluation period including: group A (3 weeks) and group B (3 months). Each group was further subdivided into three equal subgroups (n = 8 teeth) according to the capping material including: subgroup 1: mineral trioxide aggregate (MTA), subgroup2: Portland cement + 10% calcium hydroxide + 20% bismuth oxide (Port Cal) and subgroup 3: Portland cement + bismuth oxide. After general anesthesia, a class V buccal cavity was prepared coronal to the gingival margin. After pulp exposure and hemostasis,the capping materials and glass ionomer filling were placed on the exposure sites. All histopathological findings, inflammatory cell count and dentin bridge formation were recorded. Data were analyzed statistically. After 3 months, the histopathological picture of the pulp in subgroup 1 showed normal pulp, continuous odontoblastic layer and complete dentin bridge formation while subgroup 2 showed partial and complete dentin bridge over a normal and necrotic pulps. Subgroup 3 showed loss of normal architecture, areas of necrosis, complete, or incomplete dentin bridge formation, attached and detached pulp stones and fatty degeneration in group B. For group A, MTA subgroup showed the least number of inflammatory cell infiltrate followed by Port Cal subgroup. While subgroup 3 showed the highest number of inflammatory cell infiltrate. For group B, the mean inflammatory cell count increased with the three tested materials with no statistical difference. Regarding dentin bridge formation at group A, no significant differences was found between subgroups, while at group B, MTA subgroup exhibited significantly higher scores than other subgroups. In conclusion, addition of calcium hydroxide to Portland cement improves the dentin bridge formation qualitatively and quantitatively.