Nanofibrillar cellulose wound dressing in skin graft donor site treatment.

BACKGROUND: Although new therapeutic approaches for burn treatment have made progress, there is still need for better methods to enhance wound healing and recovery especially in severely burned patients. Nanofibrillar cellulose (NFC) has gained attention due to its renewable nature, good biocompatibility and excellent physical properties that are of importance for a range of applications in pharmaceutical and biomedical fields. In the present study, we investigated the potential of a wood based NFC wound dressing in a clinical trial on burn patients. Previously, we have investigated NFC as a topical functionalized wound dressing that contributes to improve wound healing in mice. METHODS: Wood based NFC wound dressing was tested in split-thickness skin graft donor site treatment for nine burn patients in clinical trials at Helsinki Burn Centre. NFC dressing was applied to split thickness skin graft donor sites. The dressing gradually dehydrated and attached to donor site during the first days. During the clinical trials, physical and mechanical properties of NFC wound dressing were optimized by changing its composition. From patient 5 forward, NFC dressing was compared to commercial lactocapromer dressing, Suprathel® (PMI Polymedics, Germany). RESULTS: Epithelialization of the NFC dressing-covered donor site was faster in comparison to Suprathel®. Healthy epithelialized skin was revealed under the detached NFC dressing. NFC dressing self-detached after 11-21days for patients 1-9, while Suprathel® self-detached after 16-28days for patients 5-9. In comparison studies with patients 5-9, NFC dressing self-detached on average 4days earlier compared with Suprathel®. Lower NFC content in the material was evaluated to influence the enhanced pliability of the dressing and attachment to the wound bed. No allergic reaction or inflammatory response to NFC was observed. NFC dressing did not cause more pain for patients than the traditional methods to treat the skin graft donor sites. CONCLUSION: Based on the preliminary clinical data, NFC dressing seems to be promising for skin graft donor site treatment since it is biocompatible, attaches easily to wound bed, and remains in place until donor site has renewed. It also detaches from the epithelialized skin by itself.

FGF signalling in craniofacial development and developmental disorders.

The Fgf signalling pathway is highly conserved in evolution and plays crucial roles in development. In the craniofacial region, it is involved in almost all structure development from early patterning to growth regulation. In craniofacial skeletogenesis, the Fgf signal pathway plays important roles in suture and synchondrosis regulation. Mutations of FGF receptors relate to syndromatic and non-syndromatic craniosynostosis. The Fgf10/Fgfr2b signal loop is critical for palatogenesis and submandibular gland formation. Perturbation of the Fgf signal is a possible mechanism of palatal cleft. Fgf10 haploinsufficiency has been identified as the cause of autosomal dominant aplasia of lacrimal and salivary glands. The Fgf signal is also a key regulator of tooth formation: in the absence of Fgfr2b tooth development is arrested at the bud stage. Fgfr4 has recently been identified as the key signal mediator in myogenesis. In this review, these aspects are discussed in detail with a focus on the most recent advances.

Slit1 is specifically expressed in the primary and secondary enamel knots during molar tooth cusp formation.

The shape and diversity of the mammalian molar teeth is suggested to be regulated by the primary and secondary enamel knots, which are putative epithelial signaling centers of the tooth. In search of novel molecules involved in tooth morphogenesis, we analyzed mRNA expression of Slit1, -2 and -3, earlier characterized as secreted signals needed for axonal pathfinding and their two receptors Robo1 and -2 (Roundabout1 and -2) in the developing mouse first molar. In situ hybridization analysis showed that Slit1 mRNAs were expressed in the primary enamel knot of the bud and cap stage tooth germ and later the expression continued in the secondary enamel knots of the late cap and bell stage tooth. In contrast, expression of Slit2 and -3 as well Robo1, and -2 was largely restricted to mesenchymal tissue components of the tooth until the bell stage. At the late bud stage, however, Robo1 transcripts were evident in the primary enamel knot, and at the cap stage a pronounced expression was noted in the middle of the tooth germ covering the primary enamel knot and dental papilla mesenchyme. During the bell stage, Robo1 and Slit2 expression became restricted to the dental epithelia, while Slit3 continued in the dental mesenchyme. Prior to birth, Robo1 and -2 were co-localized in the predontoblasts. These results indicate that Slits and Robos display distinct, developmentally regulated expression patterns during tooth morphogenesis. In addition, our results show that Slit1 is the second known gene specifically located in the primary and secondary enamel knots.

Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice.

The LKB1 tumor suppressor gene, mutated in Peutz-Jeghers syndrome, encodes a serine/threonine kinase of unknown function. Here we show that mice with a targeted disruption of Lkb1 die at midgestation, with the embryos showing neural tube defects, mesenchymal cell death, and vascular abnormalities. Extraembryonic development was also severely affected; the mutant placentas exhibited defective labyrinth layer development and the fetal vessels failed to invade the placenta. These phenotypes were associated with tissue-specific deregulation of vascular endothelial growth factor (VEGF) expression, including a marked increase in the amount of VEGF messenger RNA. Moreover, VEGF production in cultured Lkb1(-/-) fibroblasts was elevated in both normoxic and hypoxic conditions. These findings place Lkb1 in the VEGF signaling pathway and suggest that the vascular defects accompanying Lkb1 loss are mediated at least in part by VEGF.

Expression of class 3 semaphorins and neuropilin receptors in the developing mouse tooth.

The semaphorins are a large family of secreted or cell-bound signals needed for the development of the nervous system. We compared mRNA expression of class 3 semaphorins (Sema3A, 3B, 3C and 3F) and their two receptors (Neuropilin-1 and -2) in the embryonic mouse first molar tooth germ (E10-18) by radioactive in situ hybridization. All genes showed distinct developmentally regulated expression patterns during tooth organogenesis. Interestingly, Sema3A and 3C were first detected in the early dental epithelium, and later both genes were present in the epithelial primary enamel knot, a putative signaling center of the embryonic tooth regulating tooth morphogenesis. Prior to birth, Sema3A was also observed in tooth-specific cells, preodontoblasts, which later differentiate into odontoblasts secreting dentin, and in the mesenchymal dental follicle cells surrounding the tooth germ. Sema3B appeared transiently in the dental mesenchyme in the bud and cap stage tooth while Sema3F was expressed in both epithelial and mesenchymal components of the tooth. Of note, Npn-1 expression pattern was largely complementary to that of Sema3A, and transcripts were restricted to the dental mesenchymal cells. Npn-1 expression was first seen in the developing dental follicle, and later transcripts also appeared in the dental papilla mesenchyme. In contrast, Npn-2 signal was seen in both epithelial and mesenchymal tissues such as in the primary enamel knot and preodontoblasts.

Developmentally regulated expression of Smad3, Smad4, Smad6, and Smad7 involved in TGF-beta signaling.

Transforming growth factor-beta (TGF-beta) signaling is mediated from serine/threonine kinase receptors to transcriptional responses via Smad proteins. Here comparison of mRNA expression of Smad3-7 in mouse embryos (E9-E15) revealed developmentally regulated distinct expression patterns for Smad3, 4, 6, and 7. Smad3 was prominently expressed in the differentiating (from E10) central nervous system, but also in developing bones, branchial arches and epithelium of various tissues. Smad4 mostly showed ubiquitous expression, but in E15 embryos, a pronounced signal appeared in epithelial crypts of the gut. Inhibitory Smad6 and Smad7 were coexpressed at high levels in developing cardiovascular system from the earliest stages studied. In contrast, Smad6 was selectively expressed at high levels, e.g. in intramembranous bone whereas Smad7 was prominent in seminiferous tubules of the testis, demonstrating distinct expression of these genes in non-cardiovascular tissues.

CLP-36 PDZ-LIM protein associates with nonmuscle alpha-actinin-1 and alpha-actinin-4.

The PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP-36) has been suggested to act as adapters that direct LIM-binding proteins to the cytoskeleton. Most interactions of PDZ-LIM proteins with the cytoskeleton have been identified in striated muscle, where several PDZ-LIM proteins are predominantly expressed. By contrast, CLP-36 mRNA is expressed in several nonmuscle tissues, and here we demonstrate high expression of CLP-36 in epithelial cells by in situ hybridization analysis. Our subcellular localization studies indicate that in nonmuscle cells, CLP-36 protein localizes to actin stress fibers. This localization is mediated via the PDZ domain of CLP-36 that associates with the spectrin-like repeats of alpha-actinin. Interestingly, immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis indicate that both nonmuscle alpha-actinin-1 and alpha-actinin-4 form complexes with CLP-36. The high expression of alpha-actinin-4 in the colon, together with these results, suggests a specific function for the alpha-actinin-4-CLP-36 complex in the colonic epithelium. More generally, results presented here demonstrate that the association of PDZ-LIM proteins with the cytoskeleton extends to the actin stress fibers of nonmuscle cells.

Expression of LKB1 and PTEN tumor suppressor genes during mouse embryonic development.

Germ-line mutations of LKB1 and PTEN tumor suppressor genes underlie the phenotypically related Peutz-Jeghers syndrome (PJS) and Cowden disease (CD), respectively. To analyze possible developmental roles of PTEN and LKB1, we have studied their mRNA expression during mouse embryonic development (E7-17.5) by in situ hybridization. Ubiquitous expression of both genes during early stages (E7-11) became more restricted in later embryonic development (E15-19) where LKB1 and PTEN showed prominent overlapping expression in e.g. gastrointestinal tract and lung. In contrast, LKB1 was selectively expressed at high levels in testis and PTEN was prominently expressed in skin epithelium and underlying mesenchyme. These results indicate that LKB1 and PTEN display largely overlapping expression patterns during embryonic development. Moreover, a high expression of these genes was observed in the tissues and organs affected in PJS and CD patients and in PTEN+/- mice.

Retarded growth and deficits in the enteric and parasympathetic nervous system in mice lacking GFR alpha2, a functional neurturin receptor.

Glial cell line-derived neurotrophic factor (GDNF) and a related protein, neurturin (NTN), require a GPI-linked coreceptor, either GFR alpha1 or GFR alpha2, for signaling via the transmembrane Ret tyrosine kinase. We show that mice lacking functional GFR alpha2 coreceptor (Gfra2-/-) are viable and fertile but have dry eyes and grow poorly after weaning, presumably due to malnutrition. While the sympathetic innervation appeared normal, the parasympathetic cholinergic innervation was almost absent in the lacrimal and salivary glands and severely reduced in the small bowel. Neurite outgrowth and trophic effects of NTN at low concentrations were lacking in Gfra2-/- trigeminal neurons in vitro, whereas responses to GDNF were similar between the genotypes. Thus, GFR alpha2 is a physiological NTN receptor, essential for the development of specific postganglionic parasympathetic neurons.

Neurturin mRNA expression suggests roles in trigeminal innervation of the first branchial arch and in tooth formation.

Neurturin (NTN) is a recently characterized member of the glial cell line-derived neurotrophic factor (GDNF)-family which, like GDNF, can promote the survival of certain populations of neuronal cells in peripheral and central nervous systems. To elucidate the roles of NTN and a novel glycosyl-phosphatidylinositol (GPI)-linked receptor protein GFRalpha-3, a member of GDNF-family receptor alpha, in the regulation of peripheral trigeminal innervation and tooth formation, their expression patterns during mouse embryonic (E) and early postnatal (P) development (E10-P5) of the first branchial arch were analyzed by in situ hybridization. NTN mRNAs were observed in oral and cutaneous epithelia of the mandibular process at all studied stages and expression became gradually restricted to the suprabasal epithelial cells. In addition, transcripts were also detected in the epithelium of whisker follicles. In the developing first molar tooth germ, NTN showed a developmentally regulated, spatiotemporally changing expression pattern, which partially correlated with the development of innervation. During the initiation of tooth formation NTN mRNAs were expressed in dental epithelium and during later embryonic development transcripts appeared in the dental papilla mesenchyme. In addition, some transcripts were seen in the dental follicle. During postnatal development, NTN expression was restricted to the dental follicle of the incisor tooth germs. GFRalpha-3 mRNAs were not detected in teeth, but an intense expression was seen in non-neuronal cells surrounding trigeminal nerve fibers and in the trigeminal ganglia during E11-E15. Ganglion explant cultures showed that trigeminal neurons start to respond to exogenous NTN at E12, which correlates to the earlier reported appearance of the Ret-tyrosine kinase receptor in the trigeminal ganglion. Local application of NTN with beads on isolated dental mesenchyme did not stimulate cell proliferation or prevent apoptotic cell death. In addition, exogenous NTN had no effects on tooth morphogenesis in in vitro cultures. Taken together, because trigeminal neurons respond to NTN after first axons have reached their primary epithelial target fields, NTN is apparently not involved in the guidance of pioneer trigeminal nerves to their peripheral targets. However, our results show that NTN is a potent neuritogenic factor and, therefore, may act as a target-field-derived neurotrophic factor for trigeminal nerves during innervation of the cutaneous and oral epithelia as well as dental follicle surrounding the developing tooth. In addition, although NTN appears not to be directly involved in the regulation of tooth morphogenesis, it may have non-neuronal, organogenetic functions during tooth formation.

Neuronal cells and neurotrophins in odontogenesis.

There is evidence from lower animals that in addition to oral ectoderm and cranial neural crest, tooth formation depends on neuronal cells. To analyze the possible neural influence on mammalian tooth formation, peripheral nerve fibers and neuronal cells were localized in the area of the developing rat first molar tooth germ. Moreover, to study whether factors needed for neuronal development might be involved in the regulation of tooth formation and innervation, expression of NGF-related neurotrophic factors and their receptors were localized by in situ hybridization. The data suggest that although peripheral nerve fibers appear not to be required for odontogenesis, neuronal cells associated with the embryonic rat tooth may participate in the regulation of tooth formation. Localization of neurotrophins and their receptors suggests that besides their apparent roles in the regulation of tooth innervation, they may serve non-neuronal, organogenetic functions during tooth formation. Moreover, it is possible that neuronal characteristics of the dental mesenchymal cells and the presence of neuronal cells in the tooth germs may explain the specific ability of neural crest-derived, but no other mesenchymal, cells to contribute to mammalian tooth formation.

Neurotrophin mRNA expression in the developing tooth suggests multiple roles in innervation and organogenesis.

To analyze the roles of neurotrophins during early development of rat teeth, we studied the expression of neurotrophin mRNAs from the initiation of first molar formation to the completion of crown morphogenesis. With RNAase protection assay all neurotrophin mRNAs were detected in embryonic teeth. In situ hybridization analysis revealed developmentally changing, distinct expression patterns for nerve growth factor (NGF) and neurotrophin-3 (NT-3), which were shown not to be regulated by or dependent on peripheral innervation. NGF mRNAs appeared in the mesenchymal target field of the tooth at the time of the trigeminal axon ingrowth (embryonic days 14-15: E14-E15), and they were also present along the pathway taken by growing trigeminal axons. NT-4/5 mRNAs were uniformly expressed in all epithelial cells, but brain-derived neurotrophic factor (BDNF) transcripts were not detected. All neurotrophins induced neurite outgrowth from E13-E16 trigeminal ganglion explants. These results suggest that NGF is involved in the guidance of trigeminal axons to embryonic teeth. In postnatal teeth, expression of NGF mRNAs, but not other neurotrophins, correlated with trigeminal axon ingrowth, proposing that NGF is involved in local sprouting and establishment of the final innervation pattern of the dental papilla and dentin. These results suggest that NGF is required for tooth innervation and that other neurotrophins may also have regulatory roles. In addition, the expression patterns of NGF, NT-3, and NT-4/5 as well as of neurotrophin receptors suggest that the neurotrophin system may also serve non-neuronal functions during tooth development.

Localization of nerve cells in the developing rat tooth.

Earlier studies have shown that mammalian tooth formation can take place in the absence of peripheral nerve fibers. This has been taken to indicate that neurons are not needed for mammalian tooth development. However, our recent localization of peripherin, which is a neuronal cell marker, has suggested that neuronal cell bodies may be associated with developing teeth. In this study, we have analyzed in vivo and in vitro the presence of neuronal cells in developing rat tooth germs. When E14 and E16 rat first molars (thickening of presumptive dental epithelium and bud-stage tooth germ, respectively) were cultured in vitro, peripheral trigeminal axons degenerated. However, with antibodies against peripherin and L1 neural cell adhesion protein, we detected neuronal cell bodies and their axons in the explants. Next, the expression of neurofilament light-chain (NF-L) mRNAs was studied by in situ hybridization of embryonic E12 first branchial arches and tooth germs from initiation to completion of crown morphogenesis (E13, five-day post-natal teeth). NF-L transcripts were first seen at the bud stage (E15) next to the dental epithelium at the buccal side of the tooth germ. At the cap stage (E18), NF-L mRNAs were located under the oral epithelium at some distance from dental epithelium. These expression patterns correlate to the previous localization of peripherin-positive cells and suggest that NF-L expression also revealed neuronal cells. Taken together, these results demonstrate that, in addition to projections of peripheral neurons, neuronal cells are associated with the developing teeth. Hence, it is possible that neuronal cells may participate in the regulation of mammalian tooth formation.

Immunohistochemical localization of nerve fibres during development of embryonic rat molar using peripherin and protein gene product 9.5 antibodies.

Nerve fibres were localized during the initiation and early morphogenesis of the first molar tooth in rat embryos by immunoperoxidase detection of the intermediate-filament protein peripherin and protein gene product 9.5 (PGP 9.5). Nerve fibres from the trigeminal ganglion were detected in the developing first branchial arch of E12-14 embryos. Nerves were not seen in the vicinity of the developing tooth germ before the buid stage (E15), when they were seen around the condensed dental mesenchyme. During transition from the bud to the cap stage (E15), nerve fibres were detected not only in the area of the future dental follicle but also in the mesenchyme next to dental epithelium on the buccal side of the tooth germ. During later cap and bell stages nerve fibres persisted in the dental follicle, but they were not seen in the epithelial dental organ or dental papilla mesenchyme. Absence of trigeminal nerve fibres from the presumptive tooth-bearing area indicates that they are not involved in the initiation of rat tooth development. In addition, the localization of nerve fibres shows that there are some differences in the innervation of rat teeth compared with human and mouse teeth. These results provide data for further studies on the regulation of embryonic rat tooth innervation.

Expression of GDNF and its receptors in developing tooth is developmentally regulated and suggests multiple roles in innervation and organogenesis.

Glial cell line-derived neurotrophic factor (GDNF) is a recently identified survival factor for several populations of neurons in the central and peripheral nervous system that also regulates kidney development. To study the roles of GDNF in the regulation of tooth innervation and formation, we analyzed by in situ hybridization the expression patterns of GDNF and its receptors Ret, GDNF family receptor alpha-1 (GFRalpha-1), and GFRalpha-2 from the initiation of first molar formation to the completion of crown morphogenesis. At the time of trigeminal axon ingrowth, GDNF mRNAs were expressed in the mesenchyme around the tooth germ (i.e., target field of the dental innervation), suggesting that it is involved in the regulation of the embryonic tooth innervation. This hypothesis was supported by the ability of GDNF to induce neurite outgrowth from embryonic day 12 (E12) to E15 trigeminal ganglia. This timing correlated with the appearance of Ret in the subset of cells in the trigeminal ganglion at E12, whereas GFRalpha-1 and GFRalpha-2 receptors were constantly expressed in trigeminal ganglion during E11-E15. After birth, GDNF expression showed apparent correlation with the ingrowth and presence of trigeminal nerve fibers in the tooth, suggesting that GDNF is involved in the regulation of innervation of the dental papilla and dentin postnatally. Ret, GFRalpha-1, and GFRalpha-2 mRNAs were expressed in the dental epithelial and mesenchymal cells at stages when epithelial-mesenchymal signalling regulates critical steps of tooth morphogenesis. Ret and GFRalpha-2 were colocalized in the dental mesenchyme during bud and cap stages. Expression of GFRalpha-1 associated with the formation of the epithelial enamel knot, which is a putative embryonic signalling center regulating tooth shape. During postnatal development, GDNF and its receptors were expressed in dental papilla mesenchyme. In addition, GDNF and GFRalpha-1 transcripts were seen in the preodontoblasts and odontoblasts, suggesting that they may be involved in differentiation and maintenance of functional properties of the odontoblasts. Taken together, these results suggest that GDNF acts as a target-derived neurotrophic factor during tooth innervation. In addition, GDNF and its receptors may have nonneuronal organogenetic functions during tooth morphogenesis.

Expression of neurotrophin receptors during rat tooth development is developmentally regulated, independent of innervation, and suggests functions in the regulation of morphogenesis and innervation.

Low-affinity neurotrophin receptor (LANR) and trk receptor tyrosine kinases (trks) serve as low- and high-affinity receptors for neurotrophins. Besides promoting the development and maintenance of the mammalian nervous system, it has been suggested that neurotrophins may have broader functions in the development of non-neuronal tissues. To evaluate the possible roles of neurotrophic factors in tooth development, we performed a detailed examination of the expression patterns of neurotrophin receptors during development of the rat tooth from initiation to completion of crown morphogenesis. mRNA expression was studied by in situ hybridisation and LANR protein was localised by immunohistochemistry. Furthermore, dissected tooth germs were cultured in vitro to examined the role of trigeminal innervation in the expression of neurotrophin receptors. mRNAs for LANR, trkB, and trkC, but not trkA, were detected in developing teeth. LANR and the truncated form of trkB, which lacks the intracellular tyrosine kinase domain, were expressed throughout tooth morphogenesis and their expression patterns were largely non-overlapping and changed spatio-temporally. trkC was expressed after birth, and it was restricted to dental papilla mesenchyme. The expression of all receptors correlated with the development of innervation, but, in addition, the expression of LANR and trkB appeared to be associated with cell differentiation and epithelial-mesenchymal interactions. The patterns of LANR, trkB, and trkC in teeth which underwent morphogenesis in organ culture were similar to those in vivo, which indicates that the expression of these neurotrophin receptors is not regulated by and does not depend on trigeminal innervation. The data suggest that neurotrophin receptors have roles in the development of tooth innervation, but that they also have non-neuronal, organogenetic functions.

Neurotrophins in odontogenesis.

Neurotrophins (NTFs) are a family of structurally related proteins with specific effects on the developing nervous system and a wide range of non-neuronal differentiating cells. To date, four NTFs have been characterized: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). To perform their biological effects, the NTFs must bind to appropriate receptors on the surface of responsive cells. High- and low-affinity receptors for NTFs have been identified. The high-affinity receptors are members of the trk protein tyrosine kinase receptor family. The low-affinity neurotrophin receptor gp75NTFR is a common receptor for all NTFs. Here we summarize some of our previous findings on the expression patterns of NGF, gp75NTFR, TrkB, and TrkC in the developing molar tooth of the rat. Both NGF and gp75NTFR are localized in dental epithelium and mesenchyme but often their expression patterns differ. Concomitant expression of NGF and gp75NTFR in mesenchyme is correlated with odontoblast differentiation. The trkB and trkC receptors show distinct cell-specific expression patterns in developing tooth, suggesting that other NTFs, apart from NGF, may be involved in odontogenesis. These data demonstrate that NTFs participate in the cascade of molecular events that direct tooth development, and support the notion that NTFs may have multiple and distinct roles in dental tissues.