Effects of an experimentally developed adhesive resin system and CO2 laser irradiation on direct pulp capping

This study examined the wound healing process of rat pulp directly capped with various experimentally developed adhesive resin systems and treated with CO2 laser irradiation. The experimental adhesive resins used in this study were made from Clearfil Mega Bond (MB). The adhesive resin groups were capped with a combination of the following primers and bonding agents: commercially available MB primer (MBP), experimental MB primer containing 2wt% N-methacryloyl 5-aminosalicylic acid (5-NMSA: MP3) and 5wt% 12-methacryloyloxydodecylpyridinium bromide (MDPB: ABP); and commercially available MB bonding agent (MBB), experimental MB bonding agent containing 5wt% and 10wt% hydroxyl-calcium phosphate (hydroxyapatite: MB1, MB2) and 5wt% dicalcium phosphate dihydrate (brushite: MB3) as a reparative dentin-promoter. The combination of the three primers and four bonding agents yielded the 12 adhesive resin groups used in this study. The CO2 laser group was irradiated with a laser and directly capped with MB. The CO2 laser used was an Opelaser 03S II SP, and irradiation conditions were as follows: a power output of 0.5 W, superpulse mode 1, repeat pulse mode (a cycle of 10 msec irradiation and 10 msec interval), defocused beam (approximately distance 20 mm from pulp exposure surface) and an irradiation time of three seconds, with air cooling. The control group was capped with Dycal (DY) and MB. After the direct pulp capping procedures were undertaken, all cavities were restored with Clearfil AP-X resin composite. The rats were sacrificed on the 14th post-operative day. The specimens were alternately stained with Mayer's H & E, Hucker-Conn bacterial stain and the ABC method on TGF-beta1. These stained sections were observed using light microscopy and the following parameters were evaluated: pulp tissue disorganization, inflammatory cell infiltration, reparative dentin formation and bacterial penetration. The results of this study include the following: all experimentally developed bonding agent groups showed reparative dentin formation; whereas, the MBB-capped groups showed very little reparative dentin formation. The descending order regarding the amount of reparative dentin formation was MB2 > MB3 > MB1 > MBB, which tended to be dependent on the concentration of the blended reparative dentin-promoter. In terms of the quality of the formed dentin, it was observed that MB1-capped teeth tended to form tubular dentin; whereas, MB2- and MB3-capped teeth formed irregular and osteodentin types of dentin. Among the primers used, the descending order regarding the amount of reparative dentin and tubular type dentin formation was MP3 > MBP > ABP. The descending order of migration of macrophages and leukocytes was ABP > MBP > MP3. The CO2 laser group showed a very irregular fibrous dentin matrix in the vicinity of the denatured and carbonized tissue but definite reparative dentin formation was not observed. The control group showed reparative dentin, which was very thick, compared with the other groups. In all the groups, pulp tissue showed almost normal morphology. Positive staining of TGF-beta1 was only observed slightly in some specimens of all groups. There was no difference in the staining of each group. Based on the results of this study, it was concluded that the combination of MP3 (containing 2wt% 5-NMSA) and MB1 (containing 5wt% hydroxyapatite) was effective in initiating an early repair process after direct pulp capping. CO2 laser irradiation is effective for field control, but a longer observation time will be required to determine findings concerning dentin bridge formation.     

Dentonin, a MEPE fragment, initiates pulp-healing response to injury

Phosphorylated extracellular matrix proteins, including matrix extracellular phosphoprotein (MEPE), are involved in the formation and mineralization of dental tissues. In this study, we evaluated the potential of Dentonin, a synthetic peptide derived from MEPE, to promote the formation of reparative dentin. Agarose beads, either soaked with Dentonin or unloaded, were implanted into the pulps of rat molars, and examined 8, 15, and 30 days after treatment. At day 8, Dentonin promoted the proliferation of pulp cells, as visualized by PCNA-labeling. RP59-positive osteoblast progenitors were located around the Dentonin-soaked beads. PCNA- and RP59-labeling were decreased at day 15, while osteopontin, weakly labeled at day 8, was increased at 15 days, but dentin sialoprotein was undetectable at any time. At 8 days, precocious reparative dentin formation occurred in pulps containing Dentonin-soaked beads, with formation slowing after 15 days. These results suggest that Dentonin affects primarily the initial cascade of events leading to pulp healing.     

Modulation of dental inflammation by the sympathetic nervous system

Recent findings have indicated that immune responses are subjected to modulation by the sympathetic nervous system (SNS). Moreover, the findings show that the SNS inhibits the production of pro-inflammatory cytokines, while stimulating the production of anti-inflammatory cytokines. The present review is an attempt to summarize the current results on how the SNS affects inflammation in dental tissues. In dental tissues, it has been found that the SNS is significant for recruitment of inflammatory cells such as CD 43+ granulocytes. Sympathetic nerves appear to have an inhibitory effect on osteoclasts, odontoclasts, and on IL-1alpha production. The SNS stimulates reparative dentin production, since reparative dentin formation was reduced after sympathectomy. Sprouting of sympathetic nerve fibers occurs in chronically inflamed dental pulp, and neural imbalance caused by unilateral sympathectomy recruits immunoglobulin-producing cells to the dental pulp. In conclusion, this article presents evidence in support of interactions between the sympathetic nervous system and dental inflammation.     

表皮生长因子与胶原蛋白复合物对小型猪修复性牙本质形成作用的实验研究

目的：观察表皮生长因子(Epidermal growth factor,EGF)与胶原蛋白海绵(col1agen sponge,CS)复合物对小型猪修复性牙本质形成的影响。方法：选用实验用小型猪牙齿36颗,采用EGF／CS复合物为材料进行直接盖髓术,通过组织病理学方法常规制备牙-颌骨联合切片,光学显微镜下观察修复性牙本质形成情况,并与氢氧化钙糊剂直接盖髓对照。结果：小型猪牙用EGF／CS直接盖髓术后2周,穿髓孔周边可见修复性牙本质团块形成,团块周围有成牙本质细胞样细胞围绕;术后4周有完整的修复性牙本质桥形成;术后12周修复性牙本质桥增厚且结构致密,严密封闭穿髓孔,桥体近髓腔面形成明显的管样牙本质,下方为整齐排列的典型成牙本质细胞。用氢氧化钙糊剂直接盖髓,术后2周可见穿髓孔处有炎症坏死区,只有少量钙化团块形成,术后4～12周逐渐形成完整的修复性牙本质桥,但形成速度较慢且牙本质桥厚度较薄。结论：EGF与胶原蛋白复合物显著促进牙髓细胞分化和修复性牙本质形成,是一种有临床应用价值的生物活性盖髓剂。     

Effects of enamel matrix derivative and transforming growth factor-beta1 on human periodontal ligament fibroblasts

AIM: The objective of this study was to evaluate the effects of enamel matrix derivative (EMD), transforming growth factor-beta1 (TGF-beta1), and a combination of both factors (EMD+TGF-beta1) on periodontal ligament (PDL) fibroblasts. MATERIAL AND METHODS: Human PDL fibroblasts were obtained from three adult patients with a clinically healthy periodontium, using the explant technique. The effects of EMD, TGF-beta1, or a combination of both were analysed on PDL cell proliferation, adhesion, wound healing, and total protein synthesis, and on alkaline phosphatase (ALP) activity and bone-like nodule formation. RESULTS: Treatment with EMD for 4, 7, and 10 days increased cell proliferation significantly compared with the negative control (p<0.05). At day 10, EMD and EMD+TGF-beta1 showed a higher cell proliferation compared with TGF-beta1 (p<0.01). Cell adhesion was significantly up-regulated by TGF-beta1 compared with EMD and EMD+TGF-beta1 (p<0.01). EMD enhanced in vitro wound healing of PDL cells compared with the other treatments. Total protein synthesis was significantly increased in PDL cells cultured with EMD compared with PDL cells treated with TGF-beta1 or EMD+TGF-beta1 (p<0.05). EMD induced ALP activity in PDL fibroblasts, which was associated with an increase of bone-like nodules. CONCLUSION: These findings support the hypothesis that EMD and TGF-beta1 may play an important role in periodontal regeneration. EMD induced PDL fibroblast proliferation and migration, total protein synthesis, ALP activity, and mineralization, while TGF-beta1 increased cellular adhesion. However, the combination of both factors did not positively alter PDL fibroblast behaviour.     

Adenovirus-mediated recombinant human bone morphogenetic protein-7 expression promotes differentiation of human dental pulp cells

Recombinant human bone morphogenetic protein-7 (BMP-7) has been shown to stimulate new reparative dentin formation in animal models. However, little is known about whether BMP-7 could promote the odontoblast-like differentiation and the formation of mineralized nodules in human dental pulp cells. Here, we reported that the infection with adenovirus-BMP-7 (Ad-BMP-7), a BMP-7-expressing adenoviral vector, induced the expression of BMP-7 in primarily cultured human dental pulp cells in the long term with little effect on their proliferation and viability. Importantly, BMP-7 expression significantly increased alkaline phosphatase activity and induced the dentin sialophosphoprotein expression in a dose- and time-dependent manner, suggesting that BMP-7 promoted the odontoblast differentiation. Furthermore, BMP-7 expression stimulated the formation of many mineralized dentin-like calcified nodules. Our data suggest that Ad-BMP-7-mediated BMP-7 expression can promote the differentiation of human pulp cells into odontoblast-like cells and mineralization in vitro, which may provide insight for the design of new gene therapy for the pulp capping in the clinic.     

Temporospatial Expression of Neuropeptide Substance P in Dental Pulp Stem Cells During Odontoblastic Differentiation in Vitro and Reparative Dentinogenesis in Vivo

IntroductionSubstance P (SP) is a neuropeptide released from the nervous fibers in response to injury. In addition to its association with pain and reactions to anxiety and stress, SP exerts various physiological functions by binding to the neurokinin-1 receptor (NK1R). However, the expression and role of SP in reparative dentinogenesis remain elusive. Here, we explored whether SP is involved in odontoblastic differentiation during reparative dentinogenesis.MethodsDental pulp stem cells (DPSCs) were isolated from healthy human dental pulp tissues and subjected to odontoblastic differentiation. The expression of SP and NK1R during odontoblastic differentiation was investigated in vitro. The effects of SP on odontoblastic differentiation of DPSCs were evaluated using alizarin red staining, alkaline phosphatase staining, and real-time polymerase chain reaction. After direct pulp capping with mineral trioxide aggregate, the expression of SP and NK1R during reparative dentin formation in rats were identified using histological and immunohistochemical staining.ResultsSP and NK1R expression increased during the odontoblastic differentiation of DPSCs. SP translocated to the nucleus when DPSCs were exposed to differentiation medium. NK1R was always present in the nuclei of DPSCs and odontoblast-like cells. Additionally, we discovered that 10^?8 M SP marginally enhanced the odontoblastic differentiation of DPSCs, and that these effects could be impaired by the NK1R antagonist. Furthermore, SP and NK1R were expressed in odontoblast-like and dental pulp cells during reparative dentin formation in vivo.ConclusionsSP contributes to odontoblastic differentiation during reparative dentin formation by binding to the NK1R.     

The role of canonical Wnt signaling in dentin bridge formation

ObjectiveWnt signaling has been reported to be involved in dentin bridge formation. However, the detailed mechanism has not yet been clarified. We elucidated the localization of canonical Wnt signaling molecules during dentin bridge formation.MethodsPulp of the maxillary first molar in mice was exposed and directly capped with MTA cement. Maxillae were collected on the 1st, 4th, 7th, 14th, and 28th days after treatment. After μCT analysis, immunohistochemistry for Wnt3a, Wnt10a, β-catenin, F4/80, and osterix was performed in paraffin-embedded sections.ResultsOn the 4th and 7th days after pulp capping, odontoblasts and dental pulp cells expressed Wnt3a, Wnt10a, and β-catenin. On the 14th day, reactionary dentin was formed around the pulp exposure area. Odontoblasts and dental pulp cells express Wnt3a, Wnt10a, and β-catenin. Additionally, F4/80- and Wnt10a-positive macrophages were observed at the center of the dental pulp. When the dentin bridge was formed on the 28th day, reparative odontoblasts expressed Wnt3a, β-catenin and osterix.ConclusionWnt ligands derived from odontoblasts and dental pulp cells are important for the activation of odontoblasts and the differentiation of reparative odontoblasts during dentin bridge formation. Macrophage-derived Wnts are also involved in reparative odontoblast differentiation.     

Formation of Dentinal Bridge on Surface of Regenerated Dental Pulp in Dentin Defects by Controlled Release of Fibroblast Growth Factor–2 From Gelatin Hydrogels

IntroductionPulp regeneration therapy is important to overcome the limitations of conventional therapy to induce reparative dentinogenesis. In the present study, we examined the effects of controlled release of different dosages of fibroblast growth factor–2 (FGF-2) from gelatin hydrogels to regenerate the dentin-pulp complex.MethodsAfter the amputation of dental pulp of rat molars, gelatin hydrogels incorporating various dosages of FGF-2 were individually implanted into dentin defects above the sites of the amputated pulps. Histologic changes as well as the expression of dentin matrix protein–1 (DMP-1) and nestin in the dentin defect area above the amputated pulp were analyzed.ResultsWe found that controlled release of high doses of FGF-2 from gelatin hydrogels induced DMP-1–positive calcified particles in the proliferating pulp, whereas a moderate dose of FGF-2 induced DMP-1–positive dentinal bridge on the surface of the proliferating pulp. These findings indicate that the dosage of released FGF-2 has an influence on the structure of calcified tissue regenerated in dentin defects. In addition, pulp cells near calcified tissues regenerated in dentin defects were nestin-negative, suggesting that the calcified tissues might be osteodentin.ConclusionsOur results showed that the dentin regeneration on amputated pulp, not reparative dentin formation toward amputated pulp, can be regulated by adjusting the dosage of FGF-2 incorporated in biodegradable gelatin hydrogels.     

Different roles of dexamethasone on transforming growth factor-beta1-induced fibronectin and nerve growth factor expression in dental pulp cells

During pulp injury, it has been hypothesized that transforming growth factor-beta1 (TGF-beta1) is released from dentin into pulp tissue and promotes pulp tissue healing. Dexamethasone is a glucocorticoid that has been used to treat pulp injury and shown to induce differentiation of hard tissue forming cells. However, the interaction between dexamethasone and TGF-beta1 is still unknown. This study aimed to examine the effects of dexamethasone on human pulp cells in the presence of TGF-beta1. TGF-beta1 increased expression and synthesis of both fibronectin and nerve growth factor (NGF), whereas dexamethasone stimulated fibronectin synthesis but inhibited NGF expression. The application of both TGF-beta1 and dexamethasone resulted in an additional effect on fibronectin; however, dexamethasone inhibited the TGF-beta1-induced NGF expression. Dexamethasone promotes fibronectin synthesis and suppresses NGF secretion, suggesting that this reagent could be used clinically to reduce pain and promote dental pulp tissue healing.     

An immunohistochemical study of the behavior of cells expressing interleukin-1 alpha and interleukin-1 beta within experimentally induced periapical lesions in rats

Cells expressing interleukin-1 (IL-1) were examined in rat periapical lesions using immunohistochemical methods. During the acute phase, both IL-1 alpha- and IL-1 beta-expressing cells were observed adjacent to many osteoclasts that contained large cytoplasms, and consequently the periapical lesions tended to expand. However, the number of IL-1 beta-expressing cells was about twice that of IL-1 alpha-expressing cells during the same period. On the other hand, there were significantly less IL-1 beta-expressing cells during the chronic phase and consequently a significant expansion of the periapical lesions was not seen. Furthermore, IL-1 alpha- and IL-1 beta-expressing cells were located far from the bone surfaces, whereas a fibroblastic cell layer lay between the IL-1-expressing cells and the bone surfaces. Both IL-1 alpha and IL-1 beta were mainly expressed by macrophages. In addition, after the percolated application of formocresol to experimentally induced periapical lesions, numerous macrophages expressing IL-1 beta became conspicuous within the vicinity of the extensive bone resorption sites. These results suggest that macrophages expressing IL-1 beta may play an important role in the activation and recrudescence of osteoclastic bone resorption, and this may be due to their localization close to the bone resorption sites noted within experimental periapical lesions.     

Octacalcium phosphate-based cement as a pulp-capping agent in rats

OBJECTIVE: The purpose of this study was to evaluate the pulpal response to an octacalcium phosphate (OCP)-based cement used as a pulp-capping material. STUDY DESIGN: The pulps of 60 maxillary first molars of male Sprague-Dawley rats were exposed and then capped directly by using either OCP-based cement or a calcium hydroxide slurry (control). Histologic examinations were performed at 1, 2, and 5 weeks after the surgical procedure, and the results were analyzed statistically by using the Mann-Whitney U test (P<.05). RESULTS: One week after pulp capping, the initial formation of reparative dentin in the exposed areas was more notable in the calcium hydroxide group than in the OCP-based cement group. At 2 weeks, reparative dentin covered by a layer of odontoblast-like cells was observed in both groups. However, at 5 weeks, reparative dentin consisting of regular dentinal tubules was observed more frequently in the OCP-based cement group. CONCLUSION: OCP-based cement allowed favorable healing processes to occur in the dental pulp.     

Contribution of Bone Marrow–derived Cells to Reparative Dentinogenesis Using Bone Marrow Transplantation Model

IntroductionThe aim of this study was to analyze the contribution of bone marrow–derived cells (BMDCs) to reparative dentinogenesis using bone marrow transplantation (BMT) and pulp capping as an in vivo model.MethodsA chimeric mouse model was created through the injection of BMDCs expressing green fluorescent protein (GFP+ BMDCs) from C57BL/6 GFP+ transgenic donor mice into irradiated C57BL/6 wild-type recipient mice (GFP− mice). These GFP− chimeric mice (containing transplanted GFP+ BMDCs) were subjected to microscopic pulp exposure and capping with white mineral trioxide aggregate (n = 18) or Biodentine (Septodont, St Maur-des-Fossés, France) (n = 18) in the maxillary first molar. Maxillary arches from GFP− chimeric mice (with the capped tooth) were isolated and histologically processed 5 (n = 9) and 7 (n = 9) weeks after BMT. Confocal laser microscopy and immunohistochemical analysis were performed to assess the presence of GFP+ BMDCs and the expression of dentin sialoprotein, an odontoblast marker, for those cells contributing to reparative dentinogenesis in the dental pulp.ResultsConfocal laser microscopic analyses evidenced the presence of GFP+ BMDCs in close association with reparative dentin synthesized at the site of pulp exposure in GFP− mice 5 and 7 weeks after BMT. Immunohistochemical analysis revealed that GFP+ BMDCs in close association with reparative dentin expressed DSP, suggesting the contribution of nonresident GFP+ BMDCs to reparative dentinogenesis.ConclusionsThese data suggest the presence of nonresident BMDCs in reparative dentinogenesis and its contribution to dental pulp regeneration in the pulp healing process.     

Enamel Matrix Derivative Promotes Healing of a Surgical Wound in the Rat Oral Mucosa

Background: Enamel matrix proteins (EMPs) play a role in enamel formation and the development of the periodontium. Sporadic clinical observations of periodontal regeneration treatments with enamel matrix derivative (EMD), a commercial formulation of EMPs, suggest that it also promotes post‐surgical healing of soft tissues. In vitro studies showed that EMD stimulates various cellular effects, which could potentially enhance wound healing. This study examines the in vivo effects of EMD on healing of an oral mucosa surgical wound in rats. Methods: A bilateral oral mucosa wound was created via a crestal incision in the anterior edentulous maxilla of Sprague‐Dawley rats. Full‐thickness flaps were raised, and, after suturing, EMD was injected underneath the soft tissues on one side, whereas the EMD vehicle was injected in the contralateral side. Animals were sacrificed after 5 or 9 days, and the wound area was subjected to histologic and immunohistochemical analysis of the epithelial gap, number of macrophages, blood vessels, proliferating cells, and collagen content in the connective tissue (CT). Gene expression analysis was also conducted 2 days post‐surgery. Results: EMD had no effect on the epithelial gap of the wound. On both days 5 and 9, EMD treatment increased significantly the number of blood vessels and the collagen content. EMD also enhanced (by 20% to 40%) the expression of transforming growth factors β1 and β2, vascular endothelial growth factor, interleukin‐1β, matrix metalloproteinase‐1, versican, and fibronectin. Conclusion: EMD improves oral mucosa incisional wound healing by promoting formation of blood vessels and collagen fibers in CT.     

Autocrine growth factors in human periodontal ligament cells cultured on enamel matrix derivative

OBJECTIVE: Enamel extracellular matrix proteins in the form of the enamel matrix derivative EMDOGAIN (EMD) have been successfully employed to mimic natural cementogenesis to restore fully functional periodontal ligament, cementum and alveolar bone in patients with severe periodontitis. When applied to denuded root surfaces EMD forms a matrix that locally facilitates regenerative responses in the adjacent periodontal tissues. The cellular mechanism(s), e.g. autocrine growth factors, extracellular matrix synthesis and cell growth, underlying PDL regeneration with EMD is however poorly investigated. MATERIAL AND METHODS: Human periodontal ligament (PDL) cells were cultured on EMD and monitored for cellular attachment rate, proliferation, DNA replication and metabolism. Furthermore, intracellular cyclic-AMP levels and autocrine production of selected growth factors were monitored by immunological assays. Controls included PDL and epithelial cells in parallel cultures. RESULTS: PDL cell attachment rate, growth and metabolism were all significantly increased when EMD was present in cultures. Also, cells exposed to EMD showed increased intracellular cAMP signalling and autocrine production of TGF-beta1, IL-6 and PDGF AB when compared to controls. Epithelial cells increased cAMP and PDGF AB secretion when EMD was present, but proliferation and growth were inhibited. CONCLUSION: Cultured PDL cells exposed to EMD increase attachment rate, growth rate and metabolism, and subsequently release several growth factors into the medium. The cellular interaction with EMD generates an intracellular cAMP signal, after which cells secrete TGF-beta1, IL-6 and PDGF AB. Epithelial cell growth however, is inhibited by the same signal. This suggest that EMD favours mesenchymal cell growth over epithelium, and that autocrine growth factors released by PDL cells exposed to EMD contribute to periodontal healing and regeneration in a process mimicking natural root development.     

Role of human pulp fibroblasts in angiogenesis

After pulp amputation, complete pulp healing requires not only reparative dentin production but also fibroblast proliferation, nerve fiber growth, and neoangiogenesis. This study was designed to investigate the role of pulp fibroblasts in angiogenesis. Human pulp fibroblasts from third molars co-cultured with human umbilical vein endothelial cells induced the organization of endothelial cells and the formation of tubular structures corresponding to capillaries in vivo. The direct contact between both cells was not necessary to induce angiogenesis, and the observed effect was due to soluble factors. This was confirmed with neutralizing antibodies against FGF-2 and VEGF, which decreased the angiogenic effects of these soluble factors. Immunohistochemistry showed that both FGF-2 and VEGF were expressed in human dental pulp fibroblasts, and this expression increased after injury. These results suggest that the pulp fibroblasts secrete angiogenic factors, which are necessary for complete pulp healing, particularly at the pulp injury site.     

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目的观察Dycal和矿物三氧化物凝聚体（MTA）在大鼠磨牙直接盖髓后骨形态发生蛋白（BMP）-2的表达变化。方法本研究于2011年3月至2012年12月在中国医科大学口腔医学院中心实验室及辽宁省口腔医学研究所进行。42只雌性Wistar大鼠双侧上下第一磨牙分别用Dycal和MTA直接盖髓，在各观察时点（盖髓后12h及1、3、7、14、21、28d）处死动物取材，进行BMP-2免疫组织化学染色，用图像分析软件测定各组标本中BMP-2阳性染色的平均光密度值。用SPSS13．0统计软件对数据分别进行配对t检验和Dunnett-t检验。结果在正常牙髓组织中BMP-2表达呈阴性，Dycal和MTA直接盖髓后12h牙髓中BMP-2出现低水平表达并逐渐增强，7d时达到高峰，14d后表达开始下降，至28d时接近正常水平。MTA组修复性牙本质形成较Dycal组连续且致密，7、14d时MTA组BMP-2表达强度高于Dycal组。结论MTA组形成修复性牙本质较Dycal组连续且致密，BMP-2的表达高于Dycal组；MTA可能通过调节BMP-2的表达参与诱导修复性牙本质的形成，BMP-2的表达强度可能影响了牙髓损伤修复。     

Formation of dentin-like particles in dentin defects above exposed pulp by controlled release of fibroblast growth factor 2 from gelatin hydrogels

The induction of dentin formation on exposed dental pulp is a major challenge in research on the regeneration of the dentin-pulp complex. We examined the effects of fibroblast growth factor 2 (FGF2), which was delivered in either a collagen sponge (noncontrolled release) or incorporated into gelatin hydrogels (controlled release), on the formation of dentin in exposed rat molar pulps. During the early phase of pulp wound healing, pulp cell proliferation and invasion of vessels into dentin defects above exposed pulp were induced in both groups. In the late phase, the induction of dentin formation was distinctly different between the 2 types of FGF2 release. The noncontrolled release of free FGF2 from collagen sponge induced excessive reparative dentin formation in the residual dental pulp, although dentin defects were not noted. In contrast, controlled release of FGF2 from gelatin hydrogels induced the formation of dentin-like particles with dentin defects above exposed pulp. These results suggest the possibility of a novel therapeutic approach for dentin-pulp complex by controlled release of bioactive FGF2.