Repair of facial nerve crush injury in rabbits using collagen plus basic fibroblast growth factor.

Facial nerves are frequently crushed or cut during facial surgery. In this study, the feasibility of repairing facial nerves in rabbits after crush or cut off injury was evaluated using collagen conduits with A collagen-binding domain (CBD)-human basic fibroblast growth factor (bFGF). A total of 39 six-month-old New Zealand White rabbits were randomly divided into four groups of nine rabbits, and bilateral crush or cut off injuries were made on each animal's face. Three rabbits were classified as the healthy control. The facial nerves were cut or crushed and then were either untreated or wrapped with a collagen conduit plus bFGF. At the 15, 30, and 90 days after the injury, three rabbits in each group were sacrificed. Regeneration of the injured facial nerve was evaluated using electrophysiological examination (compound muscle action potentials, CAMPs), scanning electron microscopy, and histological observation. The results suggested that using collagen conduits with recombinant proteins CBD-bFGF to repair facial nerves with crush or cut off injuries promoted functional facial nerve recovery. This treatment, as a possible therapeutic for patients with facial nerve injury, requires further investigation.

Collagen/nano-sized ß-tricalcium phosphate conduits combined with collagen filaments and nerve growth factor promote facial nerve regeneration in miniature swine: an in vivo study.

OBJECTIVE: The aim of this study was to investigate the efficiency of a novel biomedical system that repairs facial nerve gaps in a miniature swine model. STUDY DESIGN: A collagen (COL)/nano-sized ß-tricalcium phosphate (nß-TCP) conduit combined with COL filaments and nerve growth factor (NGF) was prepared and used to bridge a 35-mm-long facial nerve gap in miniature swine. The functional recovery and axonal regeneration were evaluated by electrophysiologic and histologic assessments in the different groups at 6 months postoperatively. RESULTS: Morphologic analysis revealed that the COL+NGF, COL/nß-TCP+NGF, and autograft groups exhibited a superior recovery compared with the COL and COL/nß-TCP groups. The compound muscle action potential ratios were significantly greater in the COL/nß-TCP+NGF group compared to the COL+NGF and COL/nß-TCP groups (P < .01). Moreover, transmission electron microscopy demonstrated significantly larger axon diameters and myelin sheath thicknesses in the COL/nß-TCP+NGF group compared with the COL, COL+NGF, and COL/nß-TCP groups (P < .05). The expression of S-100 was significantly greater in the COL/nß-TCP+NGF group than in the COL+NGF and COL/nß-TCP groups (P < .05). CONCLUSIONS: The functional nerve biomedical system containing the COL/nß-TCP conduit combined with COL filaments and NGF could promote facial nerve regeneration, thus offering promising potential for clinical applications.

Collagen/ß-TCP nerve guidance conduits promote facial nerve regeneration in mini-swine and the underlying biological mechanism: A pilot in vivo study.

This study aimed to evaluate the efficiency of new nerve guidance conduits (NGCs) in bridging facial nerve gaps, and investigate the underlying biological mechanisms implicated in the regeneration process. A collagen/ß-TCP conduit was prepared and applied to a facial nerve gap in a mini-swine model. Functional recovery and axonal regeneration were further evaluated by electrophysiological and histological examinations at 3 months after surgery. Furthermore, the global transcriptomic profiles of regenerated and normal tissues were analyzed by gene microarray to identify the differentially expressed genes at day three and seven, postoperatively. Subsequently, associated biological processes were analyzed by gene ontology (GO) enrichment analysis. The electrophysiological examination and morphological analysis revealed that significant nerve regeneration effects were achieved in the Col/ß-TCP group (p < 0.05). Transcriptional analysis revealed that at day three post-surgery, the majority of overexpressed genes were associated with inflammatory, immune and stimuli response, accompanied by angiogenesis, while at day seven, the majority of overexpressed genes were associated with cell, tissue and organ regeneration and development, synaptic transmission, neurogenesis, and neuronal differentiation, as well as the WNT, MAPK/ERK, and JAK/STAT signaling pathways. In conclusion, the present results suggest that collagen/ß-TCP NGCs provide a promising tubular micro-environment for nerve regeneration. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1122-1131, 2019.

Restoration of mandibular bone defects with demineralized bone matrix combined with three-dimensional cultured bone marrow-derived mesenchymal stem cells in minipig models.

Mandibular defects, caused by congenital, pathological or iatrogenic insults, can significantly affect patient quality of life. The reconstruction of mandible has recently gained the interest of clinical and tissue engineering researchers. The purpose of this study was to evaluate the effectiveness of three-dimensional (3-D) cultured autologous grafts prepared using bone marrow-derived mesenchymal stem cells (BMSCs) combined with demineralized bone matrix (DBM) scaffolds for the restoration of mandibular defects. Cylindrical defects were created in the mandibular body of minipigs and filled with 3D-cultured BMSCs/DBM autografts, 2D-cultured BMSCs/DBM autografts, DBM material (without cells), or were left unfilled (blank). Using computed tomographic (CT) imaging and histological staining, we found that treatment of mandibular defects using 3-D cultured BMSCs/DBM autografts offered improvements in bone formation over both 2-D cultured autografts and cell-free DBM scaffolds. We found increased osteoid formation in 3D and 2D cultures, with more osteogenic cells present in the 3D constructs. We suggest that 3-D cultured homograft BMSCs combined with DBM scaffolds represents a new strategy for bone reconstruction, with potential future clinical applicability.

Functional collagen conduits combined with human mesenchymal stem cells promote regeneration after sciatic nerve transection in dogs.

Numerous studies have focused on the development of novel and innovative approaches for the treatment of peripheral nerve injury using artificial nerve guide conduits. In this study, we attempted to bridge 3.5-cm defects of the sciatic nerve with a longitudinally oriented collagen conduit (LOCC) loaded with human umbilical cord mesenchymal stem cells (hUC-MSCs). The LOCC contains a bundle of longitudinally aligned collagenous fibres enclosed in a hollow collagen tube. Our previous studies showed that an LOCC combined with neurotrophic factors enhances peripheral nerve regeneration. However, it remained unknown whether an LOCC seeded with hUC-MSCs could also promote regeneration. In this study, using various histological and electrophysiological analyses, we found that an LOCC provides mechanical support to newly growing nerves and functions as a structural scaffold for cells, thereby stimulating sciatic nerve regeneration. The LOCC and hUC-MSCs synergistically promoted regeneration and improved the functional recovery in a dog model of sciatic nerve injury. Therefore, the combined use of an LOCC and hUC-MSCs might have therapeutic potential for the treatment of peripheral nerve injury.

Efect of longitudinally oriented collagen conduit combined with nerve growth factor on nerve regeneration after dog sciatic nerve injury.

The research on artificial nerve conduits has become a focus of study in peripheral nerve reconstruction so as a possible replacement for the treatment of autologous nerve grafts in clinics. In this study, we used longitudinally oriented collagen conduit (LOCC) combined with nerve growth factor (NGF) to reconstruct long distance of sciatic nerve defects (35 mm) in adult dog model. The long term follow-up evaluation demonstrated that the LOCC/NGF conduit allowed functional and morphological nerve regeneration at the transection site of the injured sciatic nerve. Furthermore, the functional study confirmed that when NGF was loaded onto LOCC it promoted a better recovery of regenerated axons than LOCC alone. The gastrocnemius muscle mass in the LOCC/NGF group was significantly greater than in the LOCC alone group. The results indicated that when LOCC conduit combined with NGF it would provide a preferential environment for sciatic nerve regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2131-2139, 2018.

Microarray gene expression of periosteum in spontaneous bone regeneration of mandibular segmental defects.

Spontaneous bone regeneration could occur to reestablish mandibular bony continuity in patients who underwent partial or total mandibulectomy for tumors with periosteum-preserving. However, scarce data is available related to the precise role of periosteum in this bone regeneration. Therefore we aimed to investigate the gene expression of periosteum that were involved in the mandibular bone regeneration. Mandibular segmental defects were created in six mini-pigs with periosteum preserved. The periosteum of defects and control site were harvested at 1 and 2 weeks. Gene ontology (GO) analysis showed that the mechanisms concerning immature wound healing were clearly up-regulated at week 1. In contrast, by week-2, the GO categories of skeletal development, ossification and bone mineralization were significantly over-represented at week-2 with several genes encoding cell differentiation, extracellular matrix formation, and anatomical structure development. Furthermore, Tgfß/Bmp, Wnt and Notch signaling were all related to the osteogenic process in this study. Besides osteogenesis, genes related to angiogenesis and neurogenesis were also prominent at week-2. These findings revealed that the gene expression profile of the periosteum's cells participating in bone regeneration varied in different time points, and numbers of candidate genes that differentially expressed during early healing stages of intramembranous bone regeneration were suggested.

Individualized treatment for the mandibular segmental defect: a case report.

Ameloblastomas are slowly growing, locally invasive tumors with high recurrence rate and more common in the mandible, if not treated they can grow to enormous size. Radical resection is the only predictable form of treatment for ameloblastomas. However, mandibular resection can lead to dysfunctions in appearance, speech, mastication, and deglutition, which severely impair the patients' quality of life. The reconstruction of extensive bone defects in the maxillofacial area is still challenging. To meet the demands of functional reconstruction, minimizing the negative influence of mandibular malformation, and disability on patients, the individualized systematic treatment plans highlight denture prosthodontics and require much consideration of multidisciplinary cooperation, with such related fields as maxillofacial surgery, oral implantology, prosthodontics, and radiology taken into account. In this report, we will present a case of reconstructing the mandibular segmental defect after the resection of a rarely giant ameloblastoma. In the case, we took the restoration of the missing teeth and the rehabilitation of the masticatory function as well as restoring bony continuities and facial appearance into consideration, communicated well with prosthodontists and implantologists before surgery, making the individualized systematic treatment plan more effective and efficient.

Collagen scaffolds combined with collagen-binding ciliary neurotrophic factor facilitate facial nerve repair in mini-pigs.

The preclinical studies using animal models play a very important role in the evaluation of facial nerve regeneration. Good models need to recapitulate the distance and time for axons to regenerate in humans. Compared with the most used rodent animals, the structure of facial nerve in mini-pigs shares more similarities with humans in microanatomy. To evaluate the feasibility of repairing facial nerve defects by collagen scaffolds combined with ciliary neurotrophic factor (CNTF), 10-mm-long gaps were made in the buccal branch of mini-pigs' facial nerve. Three months after surgery, electrophysiological assessment and histological examination were performed to evaluate facial nerve regeneration. Immunohistochemistry and transmission electron microscope observation showed that collagen scaffolds with collagen binding (CBD)-CNTF could promote better axon regeneration, Schwann cell migration, and remyelination at the site of implant device than using scaffolds alone. Electrophysiological assessment also showed higher recovery rate in the CNTF group. In summary, combination of collagen scaffolds and CBD-CNTF showed promising effects on facial nerve regeneration in mini-pig models.

Bone forming capacity of cell- and growth factor-based constructs at different ectopic implantation sites.

The aim of this study was to compare the effect of implantation site (i.e., subcutaneous, SQ vs. intramuscular, IM) on bone forming capacity of cell-based and growth factor-based scaffolds in athymic nude rats after an implantation period of 8 weeks. Cell-based scaffolds consisted of porous hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds seeded with either human adipose tissue-derived mesenchymal stem cells (AT-MSCs) only or both AT-MSCs and human umbilical vein endothelial cells (HUVECs), which were precultured in osteogenic medium for 7 days. Growth factor-based scaffolds consisted of porous HA/TCP scaffolds with 20 µg preadsorbed bone morphogenetic protein-2 (BMP-2). Histological and histomorphometrical analysis were used to assess bone formation. A differentiation experiment was performed in parallel to compare the in vitro osteogenic capacity of cell-based scaffolds. The results showed that cell-based scaffolds showed evident osteogenic differentiation in vitro, with only marginal differences between AT-MSCs only and AT-MSCs/HUVECs. In vivo, none of the cell-based scaffolds showed bone formation, irrespective of the site of implantation. In contrast, all growth factor-based scaffolds showed bone formation at both implantation sites without differences in the amount of formed bone. In conclusion, the results of this study demonstrated that the bone forming capacity of HA/TCP scaffolds with pre-adsorbed BMP-2 was equal at different ectopic implantation sites. Further, despite obvious in vitro osteogenic differentiation of AT-MSCs and AT-MSCS/HUVECs on HA/TCP scaffolds, no bone formation of these cell-based scaffolds was observed in vivo. This indicates further investigation on bone formation mechanisms of AT-MSCs is needed before AT-MSCs can be used as a cytotherapeutic treatment in clinics.

Collagen scaffolds modified with CNTF and bFGF promote facial nerve regeneration in minipigs.

Most experiments of peripheral nerve repair after injury have been conducted in the rodent model but the translation of findings from rodent studies to clinical practice is needed partly because the nerve regeneration must occur over much longer distances in humans than in rodents. The reconstruction of long distance nerve injuries still represents a great challenge to surgeons who is engaged in peripheral nerve surgery. Here we used the functional nerve conduit (collagen scaffolds incorporated with neurocytokines CNTF and bFGF) to bridge a 35 mm long facial nerve gap in minipig models. At 6 months after surgery, electrophysiology assessment and histological examination were conducted to evaluate the regeneration of peripheral facial nerves. Based on functional and histological observations, the results indicated that the functional collagen scaffolds promoted nerve reconstruction. The number and arrangement of regenerated nerve fibers, myelination, and nerve function reconstruction was better in the CNTF + bFGF conduit group than the single factor CNTF or bFGF conduit group. The functional composite conduit, which exhibited favorable mechanical properties, may promote facial nerve regeneration in minipigs effectively.

In vitro and in vivo angiogenic capacity of BM-MSCs/HUVECs and AT-MSCs/HUVECs cocultures.

The aim of this study was to comparatively evaluate the angiogenic capacity of cocultures using either human bone marrow- or human adipose tissue-derived mesenchymal stem cells (MSCs) (BM- or AT-MSCs) with human umbilical vein endothelial cells (HUVECs) both in vitro and in vivo at early time points (i.e. days 3 and 7). In vitro, cells were either monocultured (i.e. BM-MSCs, AT-MSCs or HUVECs) or cocultured (i.e. BM-MSCs/HUVECs and AT-MSCs/HUVECs) on Thermanox® (2-dimensional, 2D) or in collagen gels (3-dimensional, 3D). For the in vivo experiment, cells (cocultures) were embedded in collagen gels and implanted subcutaneously in nude mice. For both in vitro and in vivo experiments, samples were collected on days 3 and 7 and histologically processed for hematoxylin-eosin and platelet endothelial cell adhesion molecule (PECAM-1; CD31) staining. For in vivo samples, quantitative parameters for evaluating angiogenesis included CD31-positive staining percentage, total vessel-like structure (VLS) area percentage, VLS density, and average VLS area (i.e. the size of per VLS). In vitro results showed the formation of VLS in both cocultures, while none of the monocultures showed VLS formation, irrespective of 2D or 3D culture condition. Although VLS formation occurred after in vivo implantation, no significant difference in angiogenic capacity was observed between the two cocultures, either on day 3 or on day 7. Further, VLS density decreased and anastomosis of the new human vessels with the murine host vasculature occurred over time. In conclusion, this study demonstrated that AT-MSCs/HUVECs and BM-MSCs/HUVECs have equal angiogenic capacity both in vitro and in vivo, and that vessels from donor origin can anastomose with the host vasculature within seven days of implantation.

Adipose tissue-derived mesenchymal stem cells as monocultures or cocultures with human umbilical vein endothelial cells: performance in vitro and in rat cranial defects.

The aim of this study was to compare the osteogenic capacity between human adipose tissue-derived mesenchymal stem cells (AT-MSCs) and their cocultures with human umbilical vein endothelial cells (HUVECs) in vitro and their biological performance in vivo. First, the optimal cell ratio in cocultures for osteogenic differentiation was determined by seeding AT-MSCs and HUVECs in ratios varying from 100:0 to 0:100 on tissue culture plates. Afterward, AT-MSCs and AT-MSCs/HUVECs (50:50) were seeded on porous titanium fiber mesh scaffolds (Ti) for both in vitro and in vivo osteogenic evaluation. For in vitro evaluation, cell osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity and calcium assay. For in vivo evaluation, the scaffolds were implanted bilaterally into rat cranial defects (5 mm diameter) and bone formation was assessed histologically and histomorphometrically after 8 weeks. The ratio of 50:50 was chosen in the cocultures because this coculture condition retained similar amount of calcium deposition while using the least amount of AT-MSCs. Moreover, AT-MSCs showed higher osteogenic differentiation in comparison to AT-MSCs/HUVECs on Ti in vitro. Furthermore, superior bone formation was observed in AT-MSCs compared to AT-MSCs/HUVECs in rat cranial defects. In conclusion, AT-MSCs showed significantly higher osteogenic potential compared to AT-MSCs/HUVECs both in vitro and in vivo.

Comparison of cell-loading methods in hydrogel systems.

Bone regenerative medicine, based on the combined use of cells and scaffolds, represents a promising strategy in bone regeneration. Hydrogels have attracted huge interests for application as a scaffold for minimally invasive surgery. Collagen and oligo(poly(ethylene glycol)fumarate) (OPF) hydrogels are the representatives of two main categories of hydrogels, that is, natural- and synthetic-based hydrogels. With these the optimal cell-loading (i.e., cell distribution inside the hydrogels) method was assessed. The cell behavior of both bone marrow- and adipose tissue-derived mesenchymal stem cells (BM- and AT-MSCs) in three loading methods, which are dispersed (i.e., homogeneous cell encapsulation, D), sandwich (i.e., cells located in between two hydrogel layers, S), and spheroid (i.e., cell pellets encapsulation, Sp) loading in two hydrogel systems (i.e., collagen and OPF), was compared. The results suggested that the cell behavior was influenced by the hydrogel type, meaning cells cultured in collagen hydrogels had higher proliferation and osteogenic differentiation capacity than in OPF hydrogels. In addition, AT-MSCs exhibited higher proliferation and osteogenic properties compared to BM-MSCs. However, no difference was observed for mineralization among the three loading methods, which did not approve the hypothesis that S and Sp loading would increase osteogenic capacity compared to D loading. In conclusion, D and Sp loading represents two promising cell loading methods for injectable bone substitute materials that allow application of minimally invasive surgery for cell-based regenerative treatment.

Concise review: cell-based strategies in bone tissue engineering and regenerative medicine.

Cellular strategies play an important role in bone tissue engineering and regenerative medicine (BTE/RM). Variability in cell culture procedures (e.g., cell types, cell isolation and expansion, cell seeding methods, and preculture conditions before in vivo implantation) may influence experimental outcome. Meanwhile, outcomes from initial clinical trials are far behind those of animal studies, which is suggested to be related to insufficient nutrient and oxygen supply inside the BTE/RM constructs as some complex clinical implementations require bone regeneration in too large a quantity. Coculture strategies, in which angiogenic cells are introduced into osteogenic cell cultures, might provide a solution for improving vascularization and hence increasing bone formation for cell-based constructs. So far, preclinical studies have demonstrated that cell-based tissue-engineered constructs generally induce more bone formation compared with acellular constructs. Further, cocultures have been shown to enhance vascularization and bone formation compared with monocultures. However, translational efficacy from animal studies to clinical use requires improvement, and the role implanted cells play in clinical bone regeneration needs to be further elucidated. In view of this, the present review provides an overview of the critical procedures during in vitro and in vivo phases for cell-based strategies (both monoculture and coculture) in BTE/RM to achieve more standardized culture conditions for future studies, and hence enhance bone formation.

The in vivo performance of CaP/PLGA composites with varied PLGA microsphere sizes and inorganic compositions.

Enrichment of calcium phosphate (CaP) bone substitutes with poly(lactic-co-glycolic acid) (PLGA) microspheres to create porosity overcomes the problem of poor CaP degradation. The degradation of CaP-PLGA composites can be customized by changing the physical and chemical properties of PLGA and/or CaP. However, the effect of the size of dense (solid rather than hollow) PLGA microspheres in CaP has not previously been described. The present study aimed at determining the effect of different dense (i.e. solid) PLGA microsphere sizes (small (S) ~20µm vs. large (L) ~130µm) and of CaP composition (CaP with either anhydrous dicalcium phosphate (DCP) or calcium sulphate dihydrate (CSD)) on CaP scaffold biodegradability and subsequent bone in-growth. To this end mandibular defects in minipigs were filled with pre-set CaP-PLGA implants, with autologous bone being used as a control. After 4weeks the autologous bone group outperformed all CaP-PLGA groups in terms of the amount of bone present at the defect site. On the other hand, at 12weeks substantial bone formation was observed for all CaP-PLGA groups (ranging from 47±25% to 62±15%), showing equal amounts of bone compared with the autologous bone group (82±9%), except for CaP with DCP and large PLGA microspheres (47±25%). It was concluded that in the current study design the difference in PLGA microsphere size and CaP composition led to similar results with respect to scaffold degradation and subsequent bone in-growth. Further, after 12weeks all CaP-PLGA composites proved to be effective for bone substitution.

Differences in the structure and osteogenesis capacity of the periosteum from different parts of minipig mandibles.

PURPOSE: To compare the structural and cellular differences of the periosteum from different parts of the mandible in minipigs by use of histologic and immunohistochemical methods to confirm the areas in which periosteal osteogenesis in situ can be used to treat mandible defects. MATERIALS AND METHODS: Three minipigs were killed, and the left mandible of each was retrieved with the periosteum remaining and then fixed, decalcified, and embedded. The specimens were cut from the buccal and lingual sides of the ramus, angle, and body of the mandible and the mentum. Sections were stained with hematoxylin-eosin and antibodies for Stro-1 (stem cell marker) and vWF (endothelial cell marker). For each periosteal area, the thickness and number of positive cells for each antibody were measured and analyzed. RESULTS: The mentum and mandibular angle periostea were thicker than those of the body and ramus. In addition, there were more blood vessels in the periostea of the mentum and mandibular body than the angle and ramus. There were more Stro-1-positive cells in the ramus periosteum than the mentum, body, and angle of the mandibles. CONCLUSIONS: The structure and cell populations of the periosteum appear to be site specific. Therefore we suggest periosteal osteogenesis in situ to treat mentum and mandibular body defects. The periosteum should be preserved as much as possible to guarantee a good healing process.

Coculture of osteoblasts and endothelial cells: optimization of culture medium and cell ratio.

Vascularization strategies in cell-based bone tissue engineering depend on optimal culture conditions. The present study aimed to determine optimal cell culture medium and cell ratio for cocultures of human marrow stromal cells (HMSCs) and human umbilical vein endothelial cells (HUVECs) in view of both osteogenic and angiogenic outcome parameters upon two-dimensional and three-dimensional culture conditions. Cultures were performed in four different media: osteoblastic cell proliferation medium, osteogenic medium (OM), endothelial medium, and a 1:1 mixture of the latter two media. Mineralization within the cocultures was observed only in OM. Subsequent experiments in OM showed that alkaline phosphatase activity, mineralization, and CD31(+) staining were highest for cocultures at a 50:50 HMSC/HUVEC ratio. Therefore, the results from the present study show that a HMSC/HUVEC coculture ratio of 50:50 in OM is the best combination to obtain both osteogenic and angiogenic differentiation.

Determination of critical size defect of minipig mandible.

The critical size defect (CSD) of bone can provide a standard for evaluating the usefulness of bone repair materials or methods. The present study aimed to determine the CSDs of the minipig mandible with and without the periosteum. Ten 18 month-old female minipigs were used. First, the premolars and the first molar in the right mandible of each minipig were extracted. Two months later, the animals were randomly divided into two groups, with six animals in group A and four in group B. In group A, bone segments of 4, 5 and 6 cm were removed from the right mandible and the neighbouring periosteum was preserved. In group B, bone segments of 1 and 2 cm were removed from the right mandible and the periosteum was removed. The defects were retained with two reconstructive titanium plates. The mandibles underwent X-ray examinations at 4, 8 and 12 weeks after the operation. The animals were sacrificed at 12 weeks after the operation, and specimens were evaluated by gross and histological examinations. For defects retained by two reconstructive plates, the CSDs in the minipig mandible models with and without periosteum were determined as 6 and 2 cm, respectively.

[Research of immunization with recombinant plasmid pcDNA3-pacA and pcDNA3-pacP against dental caries in gnotobiotic rats. I: Measurement of specific anti-PAc S-IgA, IgG, valence].

OBJECTIVE: In this study, rats were immunized with pcDNA3-pacA and pcDNA3-pacP through submandibular gland, the valence of specific anti-PAc S-IgA,IgG were measured. METHODS: Gnotobiotic rats were vaccinated with pcDNA3-pacA, pcDNA3-pacP and combination use of pcDNA3-pacA and pcDNA3-pacP through submandibular gland. The valence of specific anti-PAc S-IgA in salivary, IgG in serum were measured. RESULTS: The specific anti-PAc S-IgA, IgG could be detected in all of recombinant plasmid groups. The level of salivary specific anti-PAc S-IgA and IgG in pcDNA3-pacA, pcDNA3-pacP, combination use of pcDNA3-pacA and pcDNA3-pacP groups were higher than negative control groups (P < 0.05). CONCLUSION: The recombinant plasmid pcDNA3-pacA and pcDNA3-pacP could provoke specific mucosal immune responses as a novel immunogen against dental caries.