Interleukin-10 reduces scar formation in both animal and human cutaneous wounds: results of two preclinical and phase II randomized control studies.

Cutaneous scarring affects up to 100 million people per annum. There is no effective scar reducing/preventing therapeutic developed to date. Interleukin (IL)-10 is an anti-inflammatory and antifibrotic cytokine. In the embryo it is important for scarless wound repair. We investigated the effect on wound healing and scarring of a double deletion of the IL-10 and IL-4 genes in a knockout (KO) mouse model, and also the effect of exogenous addition of recombinant human (rh) IL-10 into rat and human cutaneous incisions. Mouse study: Two incisions were made on the dorsal skin of 20 double IL-4/IL-10 KO mice and 20 wild-type (WT) controls. Rat study: Three concentrations of rhIL-10 were investigated. Four incisions were made on the dorsal skin of 30 rats. Each rat received two concentrations. Each incision receiving a concentration of rhIL-10 was matched with a control incision, which received either placebo or standard care. Human study: Eight concentrations of rhIL-10 were investigated. Four incisions were made on each arm of 175 healthy volunteers. Four incisions received four different concentrations, which were matched with four control incisions that received either standard care or placebo. KO mice healed with poor scar histology and increased inflammation. rhIL-10-treated rat incisions healed with decreased inflammation, better scar histology, and better macroscopic scar appearance. rhIL-10-treated human incisions at low concentrations healed with better macroscopic scar appearance and less red scars. IL-10 is an important cytokine in wound healing and its suppression of inflammation and scarring is demonstrated in mice and rats with a translational effect in humans.

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.

Avotermin for scar improvement following scar revision surgery: a randomized, double-blind, within-patient, placebo-controlled, phase II clinical trial.

BACKGROUND: Skin scarring is associated with psychosocial distress and has a negative effect on quality of life. The transforming growth factor (TGF)-ß family of cytokines plays a key role in scarring. TGF-ß3 improves scar appearance in a range of mammalian species. This study was performed to assess the efficacy of intradermal avotermin (TGF-ß3) for the improvement of scar appearance following scar revision surgery. METHODS: Sixty patients (35 men and 25 women; age, 19 to 78 years; 53 Caucasians; scar length, 5 to 21 cm) received intradermal avotermin (200 ng/100 µl/linear cm wound margin) and placebo to outer wound segments immediately after, and again 24 hours after, complete (group 1) or staged (group 2) scar revision surgery. A within-patient design was chosen to control for interindividual factors that affect scarring. The primary efficacy variable was a total scar score derived from a visual analogue scale, scored by a lay panel from standardized photographs from months 1 through 7 following treatment. RESULTS: : Primary endpoint data from the combined surgical groups showed that avotermin significantly improved scar appearance compared with placebo (total scar score difference, 21.93 mm; p = 0.04). Profilometry showed a greater reduction in scar surface area from baseline with avotermin treatment compared with placebo, significant in group 2 at months 7 and 12 (difference, 41.99 mm and 25.85 mm, respectively; p = 0.03 for both comparisons). Histologic analysis from group 2 showed that, compared with placebo treatment, collagen organization in avotermin-treated scars more closely resembled normal skin in 14 of 19 cases. Avotermin was well tolerated. CONCLUSION: Avotermin administration following scar revision surgery is well tolerated and significantly improves scar appearance compared with placebo. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, I.(Figure is included in full-text article.).

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.

Scar-improving efficacy of avotermin administered into the wound margins of skin incisions as evaluated by a randomized, double-blind, placebo-controlled, phase II clinical trial.

BACKGROUND: The authors report on a prospective, randomized, placebo-controlled phase II trial to investigate avotermin (transforming growth factor beta-3) for reducing scarring resulting from acute incised skin wounds. METHODS: Seventy-one healthy male subjects (18 to 45 years) received avotermin at 50 or 200 ng/100 µl/linear centimeter of wound margin. Subjects received three standardized 1-cm incisional wounds on the inner aspect of each upper arm. Wounds were randomized to receive (into each margin): no injection (standard wound care only), one intradermal injection of avotermin or placebo (immediately before surgery), or two injections of avotermin or placebo (immediately before surgery and 24 hours later). The primary efficacy variable was a 10-cm visual analog scale score, which assessed how closely scars resembled normal skin, administered at month 12 by an independent external scar assessment panel (a panel of lay public individuals). RESULTS: Avotermin at 200 ng/100 µl/linear centimeter, administered once or twice, achieved significant improvements in scar appearance compared with controls (p<0.02 for all comparisons). The 50-ng dose, administered twice, achieved significant improvements in scar appearance versus placebo (p=0.043). Treatment was well tolerated. CONCLUSION: These results confirm that avotermin is the first of a new class of regenerative medicines that reduce scarring when administered once or twice to the approximated margins of acute skin incisions.

Therapeutic improvement of scarring: mechanisms of scarless and scar-forming healing and approaches to the discovery of new treatments.

Scarring in the skin after trauma, surgery, burn or sports injury is a major medical problem, often resulting in loss of function, restriction of tissue movement and adverse psychological effects. Whilst various studies have utilised a range of model systems that have increased our understanding of the pathways and processes underlying scar formation, they have typically not translated to the development of effective therapeutic approaches for scar management. Existing treatments are unreliable and unpredictable and there are no prescription drugs for the prevention or treatment of dermal scarring. As a consequence, scar improvement still remains an area of clear medical need. Here we describe the basic science of scar-free and scar-forming healing, the utility of pre-clinical model systems, their translation to humans, and our pioneering approach to the discovery and development of therapeutic approaches for the prophylactic improvement of scarring in man.

Therapies with emerging evidence of efficacy: avotermin for the improvement of scarring.

Many patients are dissatisfied with scars on both visible and non-visible body sites and would value any opportunity to improve or minimise scarring following surgery. Approximately 44 million procedures in the US and 42 million procedures in the EU per annum could benefit from scar reduction therapy. A wide range of non-invasive and invasive techniques have been used in an attempt to improve scarring although robust, prospective clinical trials to support the efficacy of these therapies are lacking. Differences in wound healing and scar outcome between early fetal and adult wounds led to interest in the role of the TGFbeta family of cytokines in scar formation and the identification of TGFbeta3 (avotermin) as a potential therapeutic agent for the improvement of scar appearance. Extensive pre-clinical and human Phase I and II clinical trial programmes have confirmed the scar improving efficacy of avotermin which produces macroscopic and histological improvements in scar architecture, with improved restitution of the epidermis and an organisation of dermal extracellular matrix that more closely resembles normal skin. Avotermin is safe and well tolerated and is currently in Phase III of clinical development, with the first study, in patients undergoing scar revision surgery, fully recruited.

Effects of avotermin (transforming growth factor ß3) in a clinically relevant pig model of long, full-thickness incisional wounds.

BACKGROUND: The pig is an accepted species for evaluating the safety of molecules in dermal wound healing indications; however, the sizes of wounds assessed have not always been comparable to large incisions encountered clinically. OBJECTIVE: To develop a clinically relevant model of incisional wounding in the Göttingen minipig for assessing the safety and tolerance of compounds in development to improve scarring. METHODS: Intradermal avotermin (recombinant transforming growth factor ß3 [TGFß3]) up to 6,000 ng/100µL was administered twice to 20 cm full-thickness incisions. RESULTS: Incisions were well tolerated in the minipig. Avotermin treatment was not associated with adverse changes in a range of clinical parameters, including wound healing and strength. Plasma TGFß3 levels were transient with ≈0.1% bioavailability. CONCLUSION: A clinically relevant model of long, full-thickness, sutured surgical incisions in the minipig is achievable. Avotermin is well tolerated in this model and does not adversely affect normal wound healing at levels that significantly exceed those doses to be used clinically in humans.

Remote wound monitoring of chronic ulcers.

Chronic wounds or ulcers are wounds that do not heal in the usual manner. This type of wound is most common in the elderly and in paraplegic patients with an estimated 1% of the population suffering from leg ulcers and the costs adding up to 4% of the annual National Health Service budget in the U.K. There is an identified need to develop a device capable of remote wound monitoring that enables patients to take charge of their wound management under clinical guidance. A new ¿wound mapping¿ device has been developed, which is based on electrical impedance spectroscopy and involves the multifrequency characterization of the electrical properties of wound tissue under an electrode array. A key feature of the prototype device is the anticipated incorporation of the measuring array into standard commercial occlusive dressings, thereby protecting the wound from interference and contamination, and thus, promoting wound healing, while monitoring the protected wound. Further development is planned including wireless transmission, thus enabling telewound monitoring as described earlier.

Avotermin for the improvement of scar appearance: a new pharmaceutical in a new therapeutic area.

Disfiguring scarring in the skin is an area of high medical need. Current treatments for scarring have variable or limited effectiveness and have typically not been evaluated in randomized, controlled, double-blind clinical trials. The prophylactic improvement in scar appearance, through administration of agents around the time of injury, represents a new therapeutic approach for which there are currently no registered pharmaceuticals. Extensive research into the mechanisms of scar-free and scar-forming healing has provided a robust scientific rationale for the development of avotermin (human recombinant TGF-beta3) as a potential therapeutic for the improvement of scar appearance in humans. The pioneering approach used for the clinical development of avotermin in this new indication has explained the efficacy and safety profile of avotermin in several, prospectively randomized, double-blind clinical studies in human volunteers and patients. These studies, which show a clear translation from preclinical efficacy models to the clinical environment, have shown that prophylactic scar improvement is pharmaceutically achievable. It is anticipated that therapeutics such as avotermin, with a sound mechanistic basis and proof of effectiveness in suitably robust clinical trials, will be available to meet the needs of patients in the foreseeable future.

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.

A new approach for the prophylactic improvement of surgical scarring: avotermin (TGF beta 3).

Patients and physicians are concerned about scarring resulting from surgery, and patients, in particular, value even small improvements in scarring. Translational research into the processes involved in scarring at the molecular, cellular, and tissue levels has facilitated the discovery and development of new biological approaches for improving scarring. This article highlights research concerning avotermin (human recombinant TGF beta 3), the first in a new class of prophylactic medicines that may promote the regeneration of normal skin and improve scar appearance.

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.

Prevention and reduction of scarring in the skin by Transforming Growth Factor beta 3 (TGFbeta3): from laboratory discovery to clinical pharmaceutical.

Scarring in the skin after trauma, surgery, burn or sports injury is a major medical problem, often resulting in adverse aesthetics, loss of function, restriction of tissue movement and/or growth and adverse psychological effects. Current treatments are empirical and unpredictable, and there are no prescription drugs for the prevention or treatment of dermal scarring. We have investigated the cellular and molecular differences between scar-free healing in embryonic wounds and scar-forming healing in adult wounds. We have identified Transforming Growth Factor beta 3 (TGFbeta3) as a key regulator of the scar-free phenotype in embryonic healing. Exogenous addition of TGFbeta3 to cutaneous wounds in pre-clinical (adult) in vivo models reduces early extracellular matrix deposition and these molecules are deposited with a markedly improved architecture in the neodermis, resembling that of normal skin. This improvement of structural organisation in the healing wound is self-propagating and leads to a reduction of subsequent scarring. TGFbeta3 has completed safety studies and entered human clinical trials. Data from these studies have demonstrated that TGFbeta3 (Juvista) in humans is safe and well tolerated. Acute, local administration of TGFbeta3 (Juvista) significantly reduces dermal scarring in a dose responsive manner resulting in the regeneration of a skin structure that is permanently improved.

Maturation of the human scar: an observational study.

BACKGROUND: The natural history of scar maturation in humans has never been formally described from either a clinical or a histologic standpoint. METHODS: The maturation of incisional scars was observed in 58 healthy male volunteers who each had 2 x 1-cm incisional wounds created on the inner aspect of both upper arms. The resulting scars were photographed digitally at monthly intervals for 12 months and excised for histologic analysis at specific time points. All histologic specimens were stained using Masson's trichrome and reviewed together with the corresponding digital clinical scar images to produce macroscopic and microscopic descriptions of the maturation process. RESULTS: Three distinct groups, each displaying a different rate of longitudinal progression of scar maturation, were identified from within the study group. The majority of volunteers belonged to a "representative" subset but distinct "poor" and "excellent" subsets were also identified. The poor subset invariably contained volunteers younger than 30 years of age, whereas the majority of the excellent subset comprised subjects older than 55 years of age. CONCLUSIONS: Scar maturation occurs as a series of defined macroscopic and microscopic stages over the course of 1 year. The rate of scar maturation varied within the study group, with older subjects (>55 years) displaying accelerated maturation, whereas a prolonged high turnover state and a retarded rate of maturation were observed in younger subjects (<30 years).

Scar redness in humans: how long does it persist after incisional and excisional wounding?

BACKGROUND: The natural history of scar redness in humans has never been formally described, and the point at which normal scar redness fades is unknown. METHODS: As part of a randomized, placebo-controlled, clinical trial investigating the effects of various doses of transforming growth factor-beta3 on scar quality, the authors observed the process of scar redness and maturation in non-drug-treated incisional and excisional wounds made on the upper inner arms of 103 volunteers. Scar photographic images were assessed by a review panel to ascertain the month during which redness faded for a particular scar. Scar histology documented the level of inflammation and angiogenesis. RESULTS: Scar redness faded at an average of 7 months. Scar redness for incisions faded significantly faster than excisions (p = 0.0001, Kruskal-Wallis test), and significant differences were also seen between anteriorly and posteriorly placed scars for incisions (p = 0.0008) and excisions (p = 0.0035), respectively. Month 12 histologic examination revealed the absence of any ongoing inflammatory processes in all scars. CONCLUSIONS: Scar redness fades on average at 7 months. This is influenced by the wound type and position. The authors advocate the use of the term "rubor perseverans" to describe the physiologic redness of a normal scar as it matures beyond the first month, a process that does not involve inflammation.

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.

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.

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.