Mapping molar shapes on signaling pathways.

A major challenge in evolutionary developmental biology is to understand how genetic mutations underlie phenotypic changes. In principle, selective pressures on the phenotype screen the gene pool of the population. Teeth are an excellent model for understanding evolutionary changes in the genotype-phenotype relationship since they exist throughout vertebrates. Genetically modified mice (mutants) with abnormalities in teeth have been used to explore tooth development. The relationship between signaling pathways and molar shape, however, remains elusive due to the high intrinsic complexity of tooth crowns. This hampers our understanding of the extent to which developmental factors explored in mutants explain developmental and phenotypic variation in natural species that represent the consequence of natural selection. Here we combine a novel morphometric method with two kinds of data mining techniques to extract data sets from the three-dimensional surface models of lower first molars: i) machine learning to maximize classification accuracy of 22 mutants, and ii) phylogenetic signal for 31 Murinae species. Major shape variation among mutants is explained by the number of cusps and cusp distribution on a tooth crown. The distribution of mutant mice in morphospace suggests a nonlinear relationship between the signaling pathways and molar shape variation. Comparative analysis of mutants and wild murines reveals that mutant variation overlaps naturally occurring diversity, including more ancestral and derived morphologies. However, taxa with transverse lophs are not fully covered by mutant variation, suggesting experimentally unexplored developmental factors in the evolutionary radiation of Murines.

Metameric variation of upper molars in hominoids and its implications for the diversification of molar morphogenesis.

Metameric variation of molar size is in part associated with the dietary adaptations of mammals and results from slight alterations of developmental processes. Humans and great apes exhibit conspicuous variation in tooth morphology both between taxa and across tooth types. However, the manner in which metameric variation in molars emerged among apes and humans via evolutionary alterations in developmental processes remains largely unknown. In this study, we compare the enamel-dentine junction of the upper molars of humans-which closely correlates with morphology of the outer enamel surface and is less affected by wear-with that of the other extant hominoids: chimpanzees, bonobos, gorillas, orangutans, and gibbons. We used the morphometric mapping method to quantify and visualize three-dimensional morphological variation, and applied multivariate statistical analyses. Results revealed the following: 1) extant hominoids other than humans share a common pattern of metameric variation characterized by a largely linear change in morphospace; this indicates a relatively simple graded change in metameric molar shape; 2) intertaxon morphological differences become less distinct from the mesial to distal molars; and 3) humans diverge from the extant ape pattern in exhibiting a distinct metameric shape change trajectory in the morphospace. The graded shape change and lower intertaxon resolution from the mesial to distal molars are consistent with the concept of a 'key' tooth. The common metameric pattern observed among the extant nonhuman hominoids indicates that developmental patterns underlying metameric variation were largely conserved during ape evolution. Furthermore, the human-specific metameric pattern suggests considerable developmental modifications in the human lineage.

Demonstration of d-Octaarginine-Linked Polymers as Promising Adjuvants for Mucosal Vaccination through Influenza Virus Challenge.

Mucosal vaccination is one of the most effective ways to reduce the risk of pandemics as a result of incorrect prediction of epidemic strains of influenza viruses or virus mutation. However, adjuvants and antigen carriers with potent immunostimulatory activities are a prerequisite for significant induction of mucosal immunity because most antigens are poorly immunogenic when solely applied to the mucosa. Our previous studies demonstrated that poly(N-vinylacetamide-co-acrylic acid) bearing d-octaarginine induced the secretion of antigen-specific immunoglobulin A (IgA) on the mucosa when nasally administered with virus antigens and that intranasal IgA reacts to viral strains other than the one used for immunization. Therefore, the present study evaluated capabilities of secreted IgA for protection against virus infection. When mice were inoculated with a mixture of inactivated H1N1 A/Puerto Rico/8/34 influenza viruses and d-octaarginine-linked polymers, antigen-specific secreted IgA was induced on the nasal mucosa. Immunized mice were completely protected from virus infection of the inoculated strain. To the contrary, mice nasally inoculated with inactivated viruses alone were infected with the homologous viruses presumably because of insignificant induction of secreted IgA. Results demonstrated that our polymer would be a promising adjuvant for mucosal vaccination.

Exploring metameric variation in human molars: a morphological study using morphometric mapping.

Human molars exhibit a type of metameric variation, which is the difference in serially repeated morphology within an organism. Various theories have been proposed to explain how this variation is brought about in the molars. Actualistic data that support the theories, however, are still relatively scarce because of methodological limitations. Here we propose new methods to analyse detailed tooth crown morphologies. We applied morphometric mapping to the enamel-dentine junction of human maxillary molars and examined whether odontogenetic models were adaptable to human maxillary molars. Our results showed that the upper first molar is phenotypically distinct among the maxillary molars. The average shape of the upper first molar is characterized by four well-defined cusps and precipitous surface relief of the occlusal table. On the other hand, upper third molar is characterized by smooth surface relief of the occlusal table and shows greater shape variation and distinct distribution patterns in morphospace. The upper second molar represents an intermediate state between first and third molar. Size-related shape variation was investigated by the allometric vector analysis, and it appeared that human maxillary molars tend to converge toward the shape of the upper first molar as the size increases. Differences between the upper first molar and the upper second and third molar can thus be largely explained as an effect of allometry. Collectively, these results indicate that the observed pattern of metameric variation in human molars is consistent with odontogenetic models of molar row structure (inhibitory cascade model) and molar crown morphology (patterning cascade model). This study shows that morphometric mapping is a useful tool to visualize and quantify the morphological features of teeth, which can provide the basis for a better understanding of tooth evolution linking morphology and development.

Potential of D-Octaarginine-Linked Polymers as an in Vitro Transfection Tool for Biomolecules.

We have been investigating the potential use of cell-penetrating peptide-linked polymers as a novel penetration enhancer. Since previous in vivo studies demonstrated that poly(N-vinylacetamide-co-acrylic acid) bearing D-octaarginine, a typical cell-penetrating peptide, enhanced membrane permeation of biomolecules, its potential as an in vitro transfection tool was evaluated in this study. A plasmid DNA encoding green fluorescent protein (pGFP-C1), ß-galactosidase, and bovine serum albumin (BSA) were used as model biomolecules. Anionic pGFP-C1 interacted electrostatically with cationic d-octaarginine-linked polymers. When the ratio of mass concentration of polymers to that of pGFP-C1 reached 2.5, complexes whose size and zeta potential were approximately 200 nm and 15 mV, respectively, were obtained. GFP expression was observed in cells incubated with complexes prepared under conditions in which the polymer/pDNA concentration ratio exceeded 2.5. The expression level elevated with an increase in the concentration ratio, but physicochemical properties of the complexes remained unchanged. Results suggested that free polymers contributed to pGFP-C1 internalization. Another cell study demonstrated that ß-galactosidase premixed with polymers was taken up into cells in its active tetrameric form. Similar electrostatic interaction-driven complex formation was observed for BSA charged negatively in neutral solution. However, it appeared that the internalization processes of BSA differed from those of pGFP-C1. A mass concentration-dependent increase in internalized BSA was observed, irrespective of the polymer/protein concentration ratio. Due to frail interactions, polymers that were released from the complexes and subsequently immobilized on cell membranes might also contribute to membrane permeation of BSA.

Gelatin hydrogel impregnated with platelet-rich plasma releasate promotes angiogenesis and wound healing in murine model.

Platelet-rich plasma (PRP) contains numerous growth factors to promote wound healing and angiogenesis. The objective of this study was to explore the efficacy of biodegradable gelatin hydrogel impregnated with PRP releasate (PRPr) in the wound healing process compared with the single application of PRPr prepared from mouse PRP centrifuged by a double-spin method. Gelatin hydrogel disks with an isoelectric point of 5.0 were used in this study. A total of 180 mice (n = 45/group) were randomly assigned to the following 4 experimental groups: control group, biodegradable gelatin hydrogel group, PRPr group and gelatin hydrogel impregnated with PRPr (PRPrG) group. Wound area and epithelialization were compared on days 1, 5, 7, 14 and 21 post-wounding. After complete epithelialization, wound contraction was also evaluated. Neovascularization using immunohistochemical staining of von Willebrand factor was analyzed on day 14. The wound area of PRPrG on days 5, 7 and 14 was smaller than that in the other groups (p < 0.01). The epithelialization lengths of PRPrG on days 7 and 14 were significantly longer than the others (p < 0.01). The capillary formation of PRPrG was also superior to those in all other groups on day 14. On day 21, all wounds were completely epithelialized and PRPrG prevented wound contraction the most. It is concluded that the sustained-release system of gelatin impregnated with PRPr can stimulate angiogenesis and accelerate wound healing compared with the single application of PRP.

Simultaneous Reconstruction of the Bilateral Maxillae and Nasal Hard Structure Using a Vascularized and Nonvascularized Fibula.

Midfacial reconstruction for extensive defects of the hard nasal structures and bilateral maxillae is challenging. Postoperative radiotherapy causes skin contracture, making secondary reconstruction extremely difficult. A 57-year-old man underwent resection of the nasal bone, nasal cartilage, and hard palate for cancer of the nasal cavity. Postoperative radiotherapy (70 Gy) resulted in bilateral osteoradionecrosis. Severe depression deformity of the midface causes a disorder in closing the mouth, resulting in difficulty in conversation and oral intake. We performed simultaneous reconstruction of the bilateral maxillary and nasal hard structures using double free flaps (fibular osteocutaneous and anterolateral thigh flaps). A 16-cm right fibular osteocutaneous flap was elevated, and an 8-cm proximal bone was resected to obtain the length of the peroneal vessels. The distal 8 cm was cut into three pieces while maintaining the blood flow. The removed nonvascularized fibula was processed into two pieces of cortex: nasal bridge and columella. All areas of the skin island were de-epithelialized to bilaterally fill the maxillary sinuses. Next, the ipsilateral anterolateral thigh flap was elevated with the central 6-cm part for closure of the palate and the proximal area to fill the nasal cavity. The distal area consisted of a fascial flap to cover the reconstructed nasal structure. The chimeric double flap allowed for oral intake, conversation, and nasomaxillary prominence. Computed tomography performed 8 months postoperatively showed maintained bony structures. We used the extra fibula as a nonvascularized cortex piece to prevent infection and exposure, which enabled simultaneous reconstruction of the bilateral maxillae and hard nasal structure.

Lower Lip Reconstruction Using a Sensory Anterolateral Thigh Flap as the First Choice.

Local flaps from the upper lip and cheeks have been the first choice for two-thirds to total resection of the lower lip. However, these local flap techniques involve many clinical problems, including small a mouth, drooling, scarring, and hypesthesia. The improvement of free anterolateral thigh (ALT) flap transfer can solve these problems with expansion of the application of free flaps for lower lip reconstruction. The patient in this case was a 56-year-old man with squamous cell carcinoma of the lower lip (cT3N1M0). Subtotal lower lip resection preserving both corners of the mouth with bilateral neck dissection was performed. Simultaneously, a sensory ALT flap was elevated with an 8 × 6 cm skin island and a lateral femoral cutaneous nerve. The lateral and medial sides of the fascia lata were processed into 1-cm-wide strings, which were tunneled through the orbicularis oris muscle of the upper lip and sutured to the orbicularis oris muscle at the mucosal side of the philtrum. The lateral femoral cutaneous nerve and right mental nerve were sutured. At 3 months, a second surgery was performed to replace the ALT flap on the white labial side with a clavicle full-thickness skin graft. This surgery achieved four important factors: opening and closing of the mouth, sensory function of the lower lip, cosmetic appearance, and minimization of donor-site damage. We believe the worldwide improvement of microsurgery techniques enables lower lip reconstruction using the sensory ALT flap to be selected as the first choice for two-thirds to total lower lip defects.

Purification and characterization of a liquefying α-amylase from alkalophilic thermophilic Bacillus sp. AAH-31.

α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS-PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimum pH and temperature were 8.5 and 70 °C respectively. It was stable in a pH range of 6.4-10.3 and below 60 °C. The calcium ion did not affect its thermostability, unlike typical α-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 °C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards γ-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch.

Stripe and spot selection in cusp patterning of mammalian molar formation.

Tooth development is governed largely by epithelial-mesenchymal interactions and is mediated by numerous signaling pathways. This type of morphogenetic processes has been explained by reaction-diffusion systems, especially in the framework of a Turing model. Here we focus on morphological and developmental differences between upper and lower molars in mice by modeling 2D pattern formation in a Turing system. Stripe vs. spot patterns are the primary types of variation in a Turing model. We show that the complexity of the cusp cross-sections can distinguish between stripe vs. spot patterns, and mice have stripe-like upper and spot-like lower molar morphologies. Additionally, our computational modeling that incorporates empirical data on tooth germ growth traces the order of cusp formation and relative position of the cusps in upper and lower molars in mice. We further propose a hypothetical framework of developmental mechanism that could help us understand the evolution of the highly variable nature of mammalian molars associated with the acquisition of the hypocone and the increase of lophedness.

Double-flap Mandibular Reconstruction around the Condylar Head Using Fibula and Anterolateral Thigh Flaps.

We applied double-flap reconstruction with a vascularized fibula osteo flap and a Y-shaped anterolateral thigh (ALT) flap, which was separated into a fascial flap and a de-epithelialized fat flap to replace the temporomandibular joint capsule and temporal soft tissue volume, respectively. This technique achieved excellent functional and cosmetic results with acceptable operation time and donor site scarring. A 16-year-old girl had a rapidly growing mandibular osteosarcoma. Right mandibulectomy from the right first premolar tooth to right condylar head (including temporalis muscle, masseter muscle, buccal mucosa, and part of the right maxilla) was performed. A left fibula osteo flap and left ALT flap were prepared in a prefabricated manner using a three-dimensional model and a prebent titanium plate. The area of the ALT flap was 23 × 6 cm. A proximal 8 × 4 cm skin flap was positioned to replace the left cheek mucosa, and the distal part was divided into two layers: a de-epithelialized fat flap and a fascial flap. The de-epithelialized fat flap was used to prevent the depression deformation at the temporal area, and the fascial flap was used to cover around the alternate condylar head made by the fibula instead of the resected temporomandibular joint capsule. The peroneal artery and veins were microscopically anastomosed to the distal end of the artery and veins of the descending branch of the ALT flap in a flow-through manner. At 12 months after surgery, the occlusal function and aesthetic balance were excellent.

Central Mandibular Reconstruction by Semiopen Wedge Osteotomy Double-barrel Fibula Flap for a Slim Aesthetic Appearance.

Mandibular reconstruction involving the central segment after malignant tumor resection requires the achievement of both functional and aesthetic quality. Three-dimensional reproduction based on the concept of a double arc composed of a marginal arc and an occlusal arc is important. Most reports of fibula flaps applied three-segmented closed wedge fibula osteotomy (bilateral and central segments); however, the aesthetic outcome sometimes became too stout for female patients because of the large central segment. We performed four-segmented fibula osteotomy for a 78-year-old woman using a semiopen wedge technique characterized by a half-open wedge and half-closed wedge. This procedure obtained a slim mandibular contour and made double-barrel reconstruction easier to apply. We used titanium plates that were prebent according to the shape of a three-dimensional model constructed from CT data. Small bone cortex fragments made from a surplus fibula segment were inserted in a half-opened area. The build-up preparation for central mandibular reconstruction was all performed at the lower leg area before cutting the peroneal pedicle. This prefabricated double-barrel fibula free flap was transferred to the mandibular defect with arterial and venous anastomosis to the right superior thyroid artery, right external jugular vein, and right common facial vein. Although the patient was nearly 80 years of age, the bone segments, including free cortex tips, were fused with smooth remodeling. Semiopen wedge osteotomy can be a key to reproducing an aesthetically slim feminine chin with a functional height of mandibular bone maintained for stability of the dental prosthesis.

Adjustable biodegradability of low-swelling hydrogels prepared from recombinant peptides based on human collagen type 1.

An ideal hydrogel for tissue engineering and regenerative therapy is cytocompatible, biocompatible, and has low-swelling characteristics. Recently, a novel low-swelling hydrogel with a homogenous structure was developed by crosslinking a recombinant peptide, modeled on human collagen type 1 (RCPhC1), with a four-arm polyethylene glycol (tetra-PEG). Here, we hypothesized that the biodegradability of the RCPhC1 hydrogel was adjustable by altering its initial polymer concentration. Three types of RCPhC1 hydrogels were prepared using the initial polymer at different concentrations, and their morphology, swelling ratio, collagenase degradability, cytocompatibility, biocompatibility, and biodegradability were compared. The results revealed a low swelling ratio. The higher the concentration of the initial polymer, the longer it took for it to be degraded by collagenase. The average cell viability ratio was over 92% when using the direct contact method, which suggests that the hydrogels have excellent cytocompatibility. No death, tumorigenesis, exposure of the implants, or skin necrosis associated with the subcutaneous implantation of the hydrogels was found in mice in vivo. Moreover, histological evaluation revealed the formation of a thin fibrous capsule, which suggests an acceptable biocompatibility. Furthermore, as hypothesized, it was confirmed that the biodegradability can be adjusted by changing the initial polymer concentration. Collectively, the ability to fine-tune the biodegradability of RCPhC1 hydrogels demonstrates their potential for use in various clinical applications.

Collagen-gelatin scaffold impregnated with bFGF accelerates palatal wound healing of palatal mucosa in dogs.

BACKGROUND: We have developed a collagen-gelatin sponge (CGS) as a scaffold capable of the sustained release of bFGF to improve the healing process of the existing collagen scaffold. The aim of this study was to evaluate the efficacy of CGS impregnated with basic fibroblast growth factor (bFGF) in palatal wound healing in beagles. MATERIALS AND METHODS: Four standardized 6 mm diameter full-thickness wounds were made in the palate of each dog and covered with CGS impregnated with normal saline or bFGF at concentrations of 1 µg/cm2, 7 µg/cm2 and 14 µg/cm2. One and 2 wk after surgery, the wound area, neoepithelium length, thickness, area of regenerated submucosal tissue, and the number and total area of neoformed capillaries were evaluated. RESULTS: Two weeks after implantation, wounds treated with bFGF 7 µg/cm2 and 14 µg/cm2 were completely epithelized, while the length of the neoformed epithelium was significantly longer in the 7 µg/cm2 group. Groups impregnated with bFGF 7 µg/cm2 and 14 µg/cm2 showed promoted regeneration of submucosal tissue 2 wk later. The number and area of neoformed capillaries were significantly higher in the bFGF 7 µg/cm2 group than in other groups. We conclude that palatal wound healing in the bFGF 7 µg/cm2 group was promoted with good neovascularization and showed less contracture than other groups. CONCLUSIONS: Our new collagen-gelatin scaffold, CGS, impregnated with bFGF, could be a promising treatment to accelerate the regeneration of palatal mucosa.

Mathematical modeling of palatal suture pattern formation: morphological differences between sagittal and palatal sutures.

The median palatal suture serves as a growth center for the maxilla; inadequate growth at this site causes malocclusion and dental crowding. However, the pattern formation mechanism of palatal sutures is poorly understood compared with that of calvarial sutures such as the sagittal suture. In the present study, therefore, we compared the morphological characteristics of sagittal and palatal sutures in human bone specimens. We found that palatal suture width was narrower than sagittal suture width, and the interdigitation amplitude of the palatal suture was lower than that of the sagittal suture. These tendencies were also observed in the neonatal stage. However, such differences were not observed in other animals such as chimpanzees and mice. We also used a mathematical model to reproduce the differences between palatal and sagittal sutures. After an extensive parameter search, we found two conditions that could generate the difference in interdigitation amplitude and suture width: bone differentiation threshold [Formula: see text] and growth speed c. We discuss possible biological interpretations of the observed pattern difference and its cause.

A 3D analysis of growth trajectory and integration during early human prenatal facial growth.

Significant shape changes in the human facial skeleton occur in the early prenatal period, and understanding this process is critical for studying a myriad of congenital facial anomalies. However, quantifying and visualizing human fetal facial growth has been challenging. Here, we applied quantitative geometric morphometrics (GM) to high-resolution magnetic resonance images of human embryo and fetuses, to comprehensively analyze facial growth. We utilized non-linear growth estimation and GM methods to assess integrated epigenetic growth between masticatory muscles and associated bones. Our results show that the growth trajectory of the human face in the early prenatal period follows a curved line with three flexion points. Significant antero-posterior development occurs early, resulting in a shift from a mandibular prognathic to relatively orthognathic appearance, followed by expansion in the lateral direction. Furthermore, during this time, the development of the zygoma and the mandibular ramus is closely integrated with the masseter muscle.

Adipose tissue regeneration in a 3D-printed poly(lactic acid) frame-supported space in the inguinal region of rats.

BACKGROUND: Adipose tissue engineering has been studied as an alternative to current options for breast reconstruction, such as lipofilling, flap reconstruction, and silicone implants. Previously, we demonstrated that a poly(L-lactic acid) mesh containing a collagen sponge, containing neither cells nor growth factors, could be filled with the regenerated adipose tissues when implanted in rodent models. However, the main factor contributing to adipogenesis remained unclear. OBJECTIVE: We aimed to clarify whether adipogenesis can be achieved by the space provided by the mesh or by the bioactivity of collagen. METHODS: A three-dimensional (3D) poly(lactic acid) (PLA) frame, which was stiff enough to maintain its shape, was fabricated by 3D printing. The frame with (PLA+ColI) or without (PLA only) a type I collagen hydrogel was implanted in the inguinal region of rats for up to 12 months. Adipose tissue regeneration in the PLA only and PLA+ColI groups was evaluated histologically. RESULTS: The 3D PLA frame maintained its structure for 12 months in vivo and oil red O (ORO)-positive adipose tissues were regenerated in the frame. No significant difference in the ORO-positive area was detected between the PLA only and PLA+ColI groups. CONCLUSION: The space supported by the frame was a key factor in adipogenesis in vivo.

De novo adipogenesis using a bioabsorbable implant without additional cells or growth factors.

Adipose tissue regeneration in breast cancer patients without additional growth factors or adipose-tissue-derived stromal cells is desirable because of the possibility of recurrence and metastasis. We report that a poly-L-lactic acid (PLLA) mesh implant containing a collagen sponge (CS) maintained the internal space in vivo for up to 12 months and substituted for adipose tissue. We developed a PLLA capsule that maintained the internal space longer than that of PLLA mesh and compared adipose tissue formation at 12 and 24 months after implantation between the PLLA mesh with CS implant and the PLLA capsule implant with or without CS in a rabbit model. After 12 months, all implants maintained the internal space, and the adipose tissue that formed in all implant groups was larger than that in the control group. At 24 months, PLLA mesh maintained the internal space just as well as that at 12 months, while the PLLA capsule collapsed and accumulated a large number of macrophages. The formed adipose tissue in the PLLA mesh group was maintained up to 24 months; however, those in two PLLA capsule groups decreased and showed no difference from the control group. In conclusion, the internal space of the PLLA mesh implant with CS was substituted for adipose tissue at 12 months and sustained the formed adipose tissue after 24 months. The PLLA mesh implant containing CS is a desirable bioabsorbable implant that can be replaced by autologous adipose tissue after implantation in vivo without using any growth factors or cells.

[Clinical regenerative medicine: the skin].

Tissue engineering of skin is classified into acellular artificial skin and cellular artificial skin. Acellular artificial skin or artificial dermis, is composed of an inner collagen sponge and an outer silicone film. When placed on wounds, the collagen sponge is spontaneously converted into a dermis-like connective tissue. Addition of bFGF or cultured fibroblasts accelerates synthesis of the dermis-like tissue. Cultured epidermis often fails to take on a full-thickness skin defect because of lack of dermal component. Cultured skin with both epidermal and dermal components seems to be an ideal skin substitute, but its take rate is still low. Regeneration of complete skin with skin appendages, vascular networks, elastic fibers and so on is desired.

Cross-reactivity of immunoglobulin A secreted on the nasal mucosa in mice nasally inoculated with inactivated H1N1 influenza A viruses in the presence of D-octaarginine-linked polymers.

We evaluated cross-reactivity of immunoglobulin A (IgA) secreted on the nasal mucosa in mice that were nasally inoculated 4 times with a mixture of inactivated H1N1 influenza A viruses and poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) bearing d-octaarginine at 7-day intervals. Three viral strains (A/Puerto Rico/8/34, A/New Caledonia/20/99 IVR116, and A/Solomon Islands/03/2006) and D-octaarginine-linked polymers with different molecular weights were used as antigens and their carriers, respectively. Secretion of intranasal IgA was barely observed when the inactivated virus alone was administered. The polymer induced the production of intranasal IgA specific to the inoculated viruses, irrespective of the viral strain and molecular weight of the polymer. The respective antibodies cross-reacted to recombinant hemagglutinin proteins of not only the viral strain used for immunization but also other H1N1 strains, including A/Puerto Rico/8/34 strain whose hemagglutinin proteins are diverse from those of other strains. Mice with high reactivity of IgA to the inoculated viruses tended to acquire clear cross-reactivity to other viral strains. Notably, IgA induced by inactivated H1N1 A/New Caledonia/20/99 IVR116 strain with the strongest immunogenicity between 3 antigens in the presence of the polymer cross-reacted to recombinant hemagglutinin proteins of the A/Brisbane/10/2007 and A/Viet Nam/1194/2004 strains, which are categorized into H3N2 and H5N1, respectively. Our polymer is a potential candidate for an efficient antigen carrier that induces mucosal IgA having cross-reactivity to antigenically drifted variants, irrespective of the subtype of viral strains.