Repigmentation of cutaneous scars depends on original wound type.

Cutaneous scarring is currently an inevitable outcome following skin injury. Abnormal pigmentation within scars makes them more noticeable, causing distress for patients, particularly as there is no reliable and effective treatment available to date. The Duroc pig, known to scar badly, was used to investigate repigmentation of scars resulting from three different wound types: incisional, partial thickness excisional and full thickness excisional. Wounds were created on the backs of Duroc pigs and the resulting scars harvested at days 35, 56, 70 and 90 days post-injury. Scars were processed for histology and immunohistochemistry, quantitatively analysed using image analysis software and subjected to statistical analysis. Photographs of the macroscopic appearance of scars were scored for pigmentation using a visual analogue scale. Results demonstrated temporal and spatial differences in melanocyte repopulation and function within scars from different wound types. The microscopic pigment deposition did not correlate with macroscopic appearances in mature scars. Pigmentation of scars is dependent on the width and depth of wounds. This study has provided important information on which we can base future studies to investigate factors controlling the repigmentation of scars.

Denervation affects regenerative responses in MRL/MpJ and repair in C57BL/6 ear wounds.

The MRL/MpJ mouse displays the rare ability amongst mammals to heal injured ear tissue without scarring. Numerous studies have shown that the formation of a blastema-like structure leads to subsequent tissue regeneration in this model, indicating many parallels with amphibian limb regeneration and mammalian embryogenesis. We have recently shown that the MRL/MpJ mouse also possesses an enhanced capacity for peripheral nerve regeneration within the ear wound. Indeed, nerves are vital for the initial phase of blastema formation in the amphibian limb. In this study we investigated the capacity for wound regeneration in a denervated ear. The left ears of MRL/MpJ mice and C57BL/6 (a control strain known to have a poorer regenerative capacity) were surgically denervated at the base via an incision and nerve transection, immediately followed by a 2-mm ear punch wound. Immunohistochemical analysis showed a lack of neurofilament expression in the denervated ear wound. Histology revealed that denervation prevented blastema formation and chrondrogenesis, and also severely hindered normal healing, with disrupted re-epithelialisation, increasing wound size and progressive necrosis towards the ear tip. Denervation of the ear obliterated the regenerative capacity of the MRL/MpJ mouse, and also had a severe negative effect on the ear wound repair mechanisms of the C57BL/6 strain. These data suggest that innervation may be important not only for regeneration but also for normal wound repair processes.

TGF ß 3 immunoassay standardization: comparison of NIBSC reference preparation code 98/608 with avotermin lot 205-0505-005.

Juvista™ drug product contains human recombinant active transforming growth factor beta 3 (TGFß3; avotermin). Juvista is being developed for the prevention and reduction of human scarring. Phase II and III clinical and development batches of Juvista were assayed for content by an immunoenzymometric assay (IEMA) using a National Institute for Biological Standards and Control (NIBSC) TGFß3 reference material (98/608) and avotermin standard (Lot 205-0505-005). Paired Juvista TGFß3 data were compared directly, pooled, and processed using the statistical analysis described by Bland and Altman. A direct comparison of the two standards was also made. The Bland-Altman result was 1.958, the best estimate of the relationship between Lot 205-0505-005 and reference material 98/608. By IEMA, reference material 98/608 has approximately 50% of the immunoreactivity of Lot 205-0505-005. During clinical development, no change in Juvista TGFß3 dosage was made, but the standard used for Juvista TGFß3 assay was changed from 98/608 to 205-0505-005. The stated amount of Juvista TGFß3 in phase III trials was approximately one-half of that in phase II trials. This article highlights the importance of early adoption of an appropriate and representative standard to achieve accurate quantification of protein drug during clinical development.

A synthetic gene increases TGFß3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule.

Human transforming growth factor-ß3 (TGFß3) is a new therapeutic protein used to reduce scarring during wound healing. The active molecule is a nonglycosylated, homodimer comprised of 13-kDa polypeptide chains linked by disulphide bonds. Expression of recombinant human TGFß3 in chloroplasts and its subsequent purification would provide a sustainable source of TGFß3 free of animal pathogens. A synthetic sequence (33% GC) containing frequent chloroplast codons raised accumulation of the 13-kDa TGFß3 polypeptide by 75-fold compared to the native coding region (56% GC) when expressed in tobacco chloroplasts. The 13-kDa TGFß3 monomer band was more intense than the RuBisCO 15-kDa small subunit on Coomassie blue-stained SDS-PAGE gels. TGFß3 accumulated in insoluble aggregates and was stable in leaves of different ages but was not detected in seeds. TGFß3 represented 12% of leaf protein and appeared as monomer, dimer and trimer bands on Western blots of SDS-PAGE gels. High yield and insolubility facilitated initial purification and refolding of the 13-kDa polypeptide into the TGFß3 homodimer recognized by a conformation-dependent monoclonal antibody. The TGFß3 homodimer and trace amounts of monomer were the only bands visible on silver-stained gels following purification by hydrophobic interaction chromatography and cation exchange chromatography. N-terminal sequencing and electronspray ionization mass spectrometry showed the removal of the initiator methionine and physical equivalence of the chloroplast-produced homodimer to standard TGFß3. Functional equivalence was demonstrated by near-identical dose-response curves showing the inhibition of mink lung epithelial cell proliferation. We conclude that chloroplasts are an attractive production platform for synthesizing recombinant human TGFß3.

Peripheral nerve regeneration in the MRL/MpJ ear wound model.

The MRL/MpJ mouse displays an accelerated ability to heal ear punch wounds without scar formation (whereas wounds on the dorsal surface of the trunk heal with scar formation), offering a rare opportunity for studying tissue regeneration in adult mammals. A blastema-like structure develops and subsequently the structure of the wounded ear is restored, including cartilage, skin, hair follicles and adipose tissue. We sought to assess if the MRL/MpJ strain also possessed an enhanced capacity for peripheral nerve regeneration. Female MRL/MpJ and C57BL/6 mice were wounded with a 2-mm excisional biopsy punch to the centre of each ear and two 4-mm excisional biopsy punches to the dorsal skin. Immunohistochemical dual staining of pan-neurofilament and CD31 markers was used to investigate reinnervation and vascularisation of both the dorsal surface of the trunk and ear wounds. The MRL/MpJ mouse ear exhibited a significantly (P > 0.01) higher density of regenerated nerves than C57BL/6 between 10 and 21 days post-wounding when the blastema-like structure was forming. Unlike dorsal skin wounds, nerve regeneration in the ear wound preceded vascularisation, recapitulating early mammalian development. Immunohistochemical data suggest that factors within the blastemal mesenchyme, such as aggrecan, may direct nerve regrowth in the regenerating ear tissue.

The reinnervation and revascularization of wounds is temporarily altered after treatment with interleukin 10.

Denervated wounds fail to heal normally, and hypertrophic scars are abnormally innervated. Wounds can be manipulated with cytokines to reduce subsequent scarring. Wounds treated with the antiscarring cytokine interleukin 10 (IL10) were investigated to assess if the treatment alterered patterns of reinnervation and revascularization as the wounds matured into scars. Thirty CD1 mice underwent intradermal injection of 100 µL phosphate-buffered saline (PBS) containing 125 ng IL10 or placebo at the margins of 1 cm(2) full thickness dorsal skin excisions at the time of wounding and at 24 hours after wounding. Wounds were not dressed. Six IL10-treated and six control were harvested days 7, 14, 21, 42 and 84 postoperatively. Sections underwent histological scar assessment along with immunohistochemical staining for protein gene product 9.5 (PGP9.5), a pan-neuronal marker, and the sensory neuropeptides calcitonin gene related peptide (CGRP) and substance P (SP). The endothelial marker von Willebrand factor (VWF) was used to allow co-localization and quantification of blood vessels. Quantitative analysis was performed on the periphery and center of wounds. Wounds treated with IL10 healed with dermal collagen organized into a pattern more closely resembling normal skin than control wounds. IL10 changed the pattern of CGRP reinnervation during the healing process, but at 84 days, the density levels of all nerve fiber types were similar to controls. Wounds treated with IL10 were more vascular than untreated wounds during healing, but by 84 days, VWF density was that of unwounded skin.

Recommendations on clinical proof of efficacy for potential scar prevention and reduction therapies.

Cutaneous scarring is an enormous medical problem with approximately 100 million patients acquiring scars each year. Scar prevention/reduction represents a significant, and largely unmet, clinical need. Research into the prophylactic modulation of scar outcome differs from research into other disease processes as the scar is not present at the start of the study; measurements of changes from baseline are impossible. Final scar morphology is influenced by many variables. A fundamental principle that should be observed in the prospective evaluation of scar prevention/reduction therapies is that, if left untreated, wounds in treatment and control groups should have healed with identical scars. Observation of this principle will allow the detection of true treatment effects. The many variables that influence scar morphology mean that the evaluation of potential pharmaceutical products for this indication favors the use of self-controlled designs in clinical trials. In this article, we review variables that affect scar morphology and recommend the self-controlled design for clinical trials aiming to establish proof of efficacy of scar prevention and reduction pharmaceuticals. With no pharmaceutical products currently licensed for this indication, this represents a new therapeutic area. The principles discussed will also have direct relevance to the wider fields of wound healing and regenerative medicine.

Discovery and development of avotermin (recombinant human transforming growth factor beta 3): a new class of prophylactic therapeutic for the improvement of scarring.

Scarring in the skin following surgery or trauma may be associated with adverse aesthetic, functional, growth and psychological effects, such that both physicians and patients regard it as important to minimize the appearance of scars. The prophylactic improvement of cutaneous scar appearance represents a significant opportunity to improve the well-being of patients. Human recombinant transforming growth factor beta 3 (avotermin) is the first in a new class of therapeutic agents to address this medical need. Herein we describe scar-free healing in early embryonic development, including the identification of the cellular and molecular mechanisms underpinning the scarring process. This understanding has led to the discovery of novel therapeutics such as transforming growth factor beta 3, which can be administered to improve scar appearance in human subjects through pharmacological action. We discuss the pioneering development of transforming growth factor beta 3 in this new therapeutic area showing how it has been possible to translate preclinical concepts into clinical application, namely the improvement of scar appearance following surgery.

Prophylactic administration of avotermin for improvement of skin scarring: three double-blind, placebo-controlled, phase I/II studies.

BACKGROUND: Research into mechanisms of skin scarring identified transforming growth factor beta3 (TGFbeta3) as a potential antiscarring therapy. We assessed scar improvement with avotermin (recombinant, active, human TGFbeta3). METHODS: In three double-blind, placebo-controlled studies, intradermal avotermin (concentrations ranging from 0.25 to 500 ng/100 microL per linear cm wound margin) was administered to both margins of 1 cm, full-thickness skin incisions, before wounding and 24 h later, in healthy men and women. Treatments (avotermin and placebo or standard wound care) were randomly allocated to wound sites by a computer generated randomisation scheme, and within-participant controls compared avotermin versus placebo or standard wound care alone. Primary endpoints were visual assessment of scar formation at 6 months and 12 months after wounding in two studies, and from week 6 to month 7 after wounding in the third. Investigators, participants, and scar assessors were blinded to treatment. Efficacy analyses were intention to treat. These studies are registered with ClinicalTrials.gov, numbers NCT00847925, NCT00847795, and NCT00629811. RESULTS: In two studies, avotermin 50 ng/100 microL per linear cm significantly improved median score on a 100 mm visual analogue scale (VAS) by 5 mm (range -2 to 14; p=0.001) at month 6 and 8 mm (-29 to 18; p=0.0230) at month 12. In the third, avotermin significantly improved total scar scores at all concentrations versus placebo (mean improvement: from 14.84 mm [95 % CI 5.5-24.2] at 5 ng/100 microL per linear cm to 64.25 mm [49.4-79.1] at 500 ng/100 microL per linear cm). Nine [60%] scars treated with avotermin 50 ng/100 microL per linear cm showed 25% or less abnormal orientation of collagen fibres in the reticular dermis versus five [33%] placebo scars. After only 6 weeks from wounding, avotermin 500 ng/100 microL per linear cm improved VAS score by 16.12 mm (95% CI 10.61-21.63). Adverse events at wound sites were similar for avotermin and controls. Erythema and oedema were more frequent with avotermin than with placebo, but were transient and deemed to be consistent with normal wound healing. INTERPRETATION: Avotermin has potential to provide an accelerated and permanent improvement in scarring.

The cellular biology of flexor tendon adhesion formation: an old problem in a new paradigm.

Intrasynovial flexor tendon injuries of the hand can frequently be complicated by tendon adhesions to the surrounding sheath, limiting finger function. We have developed a new tendon injury model in the mouse to investigate the three-dimensional cellular biology of intrasynovial flexor tendon healing and adhesion formation. We investigated the cell biology using markers for inflammation, proliferation, collagen synthesis, apoptosis, and vascularization/myofibroblasts. Quantitative immunohistochemical image analysis and three-dimensional reconstruction with cell mapping was performed on labeled serial sections. Flexor tendon adhesions were also assessed 21 days after wounding using transmission electron microscopy to examine the cell phenotypes in the wound. When the tendon has been immobilized, the mouse can form tendon adhesions in the flexor tendon sheath. The cell biology of tendon healing follows the classic wound healing response of inflammation, proliferation, synthesis, and apoptosis, but the greater activity occurs in the surrounding tissue. Cells that have multiple "fibripositors" and cells with cytoplasmic protrusions that contain multiple large and small diameter fibrils can be found in the wound during collagen synthesis. In conclusion, adhesion formation occurs due to scarring between two damaged surfaces. The mouse model for flexor tendon injury represents a new platform to study adhesion formation that is genetically tractable.

The development of archosaurian first-generation teeth in a chicken mutant.

Modern birds do not have teeth. Rather, they develop a specialized keratinized structure, called the rhamphotheca, that covers the mandible, maxillae, and premaxillae. Although recombination studies have shown that the avian epidermis can respond to tooth-inductive cues from mouse or lizard oral mesenchyme and participate in tooth formation, attempts to initiate tooth development de novo in birds have failed. Here, we describe the formation of teeth in the talpid2 chicken mutant, including the developmental processes and early molecular changes associated with the formation of teeth. Additionally, we show recapitulation of the early events seen in talpid2 after in vivo activation of beta-catenin in wild-type embryos. We compare the formation of teeth in the talpid2 mutant with that in the alligator and show the formation of decidedly archosaurian (crocodilian) first-generation teeth in an avian embryo. The formation of teeth in the mutant is coupled with alterations in the specification of the oral/aboral boundary of the jaw. We propose an epigenetic model of the developmental modification of dentition in avian evolution; in this model, changes in the relative position of a lateral signaling center over competent odontogenic mesenchyme led to loss of teeth in avians while maintaining tooth developmental potential.

Partial thickness wound: Does mechanism of injury influence healing?

Wound healing is a complex multistep process which is temporally and spatially controlled. In partial thickness wounds, regeneration is possible from the stem cells in the edges of the wound and from the remnants of the epidermal appendages (such as hair follicles and sebaceous glands). This study examines whether the mechanism of injury influences healing of wounds of similar depth. Burn and excisional wounds were created on the back of Hampshire pigs and harvested at 7, 14, 28, 44, 57 and 70days after injury and processed for histology and immunohistochemistry. Quantitative analysis of re-epithelialisation, inflammatory response and thickness of the scar and semi-quantitative analyses of the architecture of the resultant scar were performed and subjected to statistical analysis. Results demonstrated a higher number of neutrophils, macrophages and lymphocytes present in the burn on day 7 compared to the excisional wounds. The inflammatory profile of burn wounds was higher than that of excisional wounds for the first month after injury albeit less marked than on day 7 after injury. Re-epithelialisation was markedly advanced in excisional wounds compared to burn wounds at day 7 after injury, corresponding to the higher number of hair follicles in the underlying dermis of excisional wounds at this time point. The thickness of the neo-epidermis increased with time and at day 70 after wounding, the neo-epidermis of the burn was significantly thicker than the neo-epidermis of the excisional scar. Interestingly, following partial thickness excision of skin, there was neo-dermal reformation albeit with an altered architecture, lacking the normal basket-weave pattern of collagen. The thickness of the dermis of partial thickness excisional scar was greater than that of the adjacent unwounded skin. The neo-dermis of the burn scar was even thicker, with the collagen arranged more compactly and disorganised compared to excisional scar and normal skin. This study provides evidence that the mechanism of injury does influence wound healing and the resultant scarring.

New therapeutics for the prevention and reduction of scarring.

Published literature shows that both physicians and patients are highly concerned about scarring and value even small improvements in scar appearance. Both severe and relatively minor scars can have a significant psychological impact on patients, irrespective of whether or not they are hidden by clothing. There is no universal standard of care for scarring and, currently, no marketed pharmaceuticals for the prophylactic reduction of scarring. Novel approaches are under development, with the furthest progressed being avotermin (Juvista; transforming growth factor beta 3). The scar-improvement efficacy of this agent, administered at the time of surgery, has been demonstrated in robust, well-controlled, randomized human studies. Avotermin and other agents in development represent a new class of prophylactic medicines promoting the regeneration of normal skin and improving scar appearance.

Avotermin: a novel antiscarring agent.

Published literature shows that both physicians and their patients are highly concerned about scarring, even relatively minor scars and those that can be concealed by clothing. Furthermore, both patients and their physicians value any opportunities to improve or minimize scarring. While a range of treatment paradigms have been evaluated, no single therapy has been adopted as a universally accepted standard of care and, currently, there are no marketed pharmaceuticals for the prophylactic reduction of scarring. Many of the available treatments are used empirically and most have not been evaluated in robust prospective, randomized, controlled clinical trials. To address this unmet medical need, translational research into the molecular mechanisms of scarring has led to the discovery and commercial development of a new class of prophylactic medicines that promote the regeneration of normal skin and improve scar appearance. Avotermin, the first agent identified in this class, is the clinical application of human recombinant transforming growth factor beta3 (TGFbeta3), a key protein involved in scar-free healing observed in embryos. Controlled, double-blind, randomized phase I/II clinical studies have shown that avotermin, administered as an intradermal injection at the time of surgery, leads to both short-term and longer-term (at >or=12 months) improvements in the appearance of scars compared with placebo and standard wound care.

Bone marrow-derived endothelial progenitor cells do not contribute significantly to new vessels during incisional wound healing.

OBJECTIVE: To assess the contribution of bone marrow (BM)-derived endothelial progenitor cells (EPCs) to the neovascularisation of cutaneous incisional wounds. METHODS: Lethally irradiated C57Bl/6 mice were transplanted with BM mononuclear cells from Tie2/lacZ mice, which constitutively overexpressed beta-galactosidase (beta-gal) in endothelial cells (ECs). Chimeras were wounded and the number of X-gal-stained (beta-gal(+)) BM-derived EPCs were calculated in histological wound sections. RESULTS: EPCs were measured in skin sections from unwounded BM transplant (BMT) mice, or at day 1 and 3 postwounding, at the level of 0.1 +/- 0.1 (mean +/- SEM) per skin/wound section. In day-5 to day-14 wounds, the number of EPCs increased gradually (1.3 +/- 0.5 at day 5 and 4.8 +/- 0.9 at day 10), peaking at day 14, when there was a significant increase in the number of EPCs per wound section (6.5 +/- 1.7) when compared to unwounded skin. Between days 14 and 18 postwounding, there was a rapid fall-off in the number of beta-gal(+) EPCs (0.8 +/- 0.5 at day 18) and numbers returned to baseline by day 21 (0.1 +/- 0.1). No evidence of vascular structures derived from BM-derived EPCs ("in situ" vasculogenesis) was observed and it was calculated that these cells contributed only 4.4% +/- 1.5% to total wound ECs at their peak. CONCLUSION: These findings indicate that the revascularization of dermal incisional wounds primarily occurs through angiogenesis because the low frequency and temporal expression of EPCs suggests that they do not make a significant contribution to the neovascularization process.

Bioengineering skin using mechanisms of regeneration and repair.

The development and use of artificial skin in treating acute and chronic wounds has, over the last 30 years, advanced from a scientific concept to a series of commercially viable products. Many important clinical milestones have been reached and the number of artificial skin substitutes licensed for clinical use is growing, but they have yet to replace the current "gold standard" of an autologous skin graft. Currently available skin substitutes often suffer from a range of problems that include poor integration (which in many cases is a direct result of inadequate vascularisation), scarring at the graft margins and a complete lack of differentiated structures. The ultimate goal for skin tissue engineers is to regenerate skin such that the complete structural and functional properties of the wounded area are restored to the levels before injury. New synthetic biomaterials are constantly being developed that may enable control over wound repair and regeneration mechanisms by manipulating cell adhesion, growth and differentiation and biomechanics for optimal tissue development. In this review, the clinical developments in skin bioengineering are discussed, from conception through to the development of clinically viable products. Central to the discussion is the development of the next generation of skin replacement therapy, the success of which is likely to be underpinned with our knowledge of wound repair and regeneration.

Interleukin-10 reduces scarring and enhances regeneration at a site of sciatic nerve repair.

Axonal regeneration at a site of peripheral nerve repair can be impeded by the formation of scar tissue, which creates a mechanical barrier and initiates the development of multiple branched axonal sprouts that form a neuroma. We have investigated the hypothesis that the application of a scar-reducing agent to the nerve repair site would permit better axonal regeneration. In anaesthetised C57 Black-6 mice, the left sciatic nerve was sectioned and immediately re-approximated using four epineurial sutures. In five groups of eight mice, we injected transforming growth factor-beta3 (50 or 500 ng), interleukin-10 (IL-10) (125 or 500 ng), or saline into and around the repair site, both before and after the nerve section. Another group of eight animals acted as sham-operated controls. After 6 weeks, the outcome was assessed by recording compound action potentials (CAPs), measuring collagen levels using picrosirius red staining, and counting the number of myelinated axons proximal and distal to the repair. CAPs evoked by electrical stimulation distal to the repair were significantly smaller in all repair groups except for the low-dose IL-10 group, where they were not significantly different from that in controls. The area of staining for collagen had significantly increased in all repair groups except for the low-dose IL-10 group, which was not significantly different from that in controls. The myelinated fibre counts were always higher distal to the repair site, but there were no significant differences between groups. We conclude that administration of a low-dose of IL-10 to a site of sciatic nerve repair reduces scar formation and permits better regeneration of the damaged axons.

Tissue engineering of replacement skin: the crossroads of biomaterials, wound healing, embryonic development, stem cells and regeneration.

Advanced therapies combating acute and chronic skin wounds are likely to be brought about using our knowledge of regenerative medicine coupled with appropriately tissue-engineered skin substitutes. At the present time, there are no models of an artificial skin that completely replicate normal uninjured skin. Natural biopolymers such as collagen and fibronectin have been investigated as potential sources of biomaterial to which cells can attach. The first generation of degradable polymers used in tissue engineering were adapted from other surgical uses and have drawbacks in terms of mechanical and degradation properties. This has led to the development of synthetic degradable gels primarily as a way to deliver cells and/or molecules in situ, the so-called smart matrix technology. Tissue or organ repair is usually accompanied by fibrotic reactions that result in the production of a scar. Certain mammalian tissues, however, have a capacity for complete regeneration without scarring; good examples include embryonic or foetal skin and the ear of the MRL/MpJ mouse. Investigations of these model systems reveal that in order to achieve such complete regeneration, the inflammatory response is altered such that the extent of fibrosis and scarring is diminished. From studies on the limited examples of mammalian regeneration, it may also be possible to exploit such models to further clarify the regenerative process. The challenge is to identify the factors and cytokines expressed during regeneration and incorporate them to create a smart matrix for use in a skin equivalent. Recent advances in the use of DNA microarray and proteomic technology are likely to aid the identification of such molecules. This, coupled with recent advances in non-viral gene delivery and stem cell technologies, may also contribute to novel approaches that would generate a skin replacement whose materials technology was based not only upon intelligent design, but also upon the molecules involved in the process of regeneration.

Temporal and spatial expression of Pax9 and Sonic hedgehog during development of normal mouse palates and cleft palates in TGF-beta3 null embryos.

Transforming growth factor-beta (TGF-beta3) gene disruption causes cleft secondary palate. Pax9 and Sonic hedgehog (Shh) genes are involved in the patterning of vertebrate embryonic tissues, including the facial skeleton. We investigated the expression of Pax9 and Shh genes during normal mouse palate development and in the developing cleft palates of TGF-beta3 null embryos. Whole mount in situ hybridization was conducted with use of Pax9 and Shh riboprobes for TGF-beta3 null, heterozygous and wild type mice at E12.5-E16.5. Histological analysis was processed by section in situ hybridization. In the wild type, Pax9 and Shh were expressed in the palate between E12.5-E15.5. Shh expression in the secondary palate was restricted to the rugae and the soft palate. Pax9 expression was predominantly in the palatal medial edge between E14.5 and E15.5. These patterns suggest that Shh and Pax9 may have different functions during palate development. In TGF-beta3 null mice, both genes expression patterns in the palate were different to those in wild type mice. In TGF-beta3 null mice, Pax9 expression was much reduced in the palatal medial edge at the critical time of palatal fusion (E14.5-E15.5). Shh expression in the palates of TGF-beta3 null mice was reduced throughout E12.5-E15.5, whilst Shh expression in heterozygous did not appear down regulated compared with the wild type. These results indicate that Pax9 and Shh expression are altered when the TGF-beta3 gene is deleted and suggest that Pax9 and Shh may be involved in the TGF-beta3 regulation of normal palatal fusion.