Ethanol treatment of nanoPGA/PCL composite scaffolds enhances human chondrocyte development in the cellular microenvironment of tissue-engineered auricle constructs.

A major obstacle for tissue engineering ear-shaped cartilage is poorly developed tissue comprising cell-scaffold constructs. To address this issue, bioresorbable scaffolds of poly-ε-caprolactone (PCL) and polyglycolic acid nanofibers (nanoPGA) were evaluated using an ethanol treatment step before auricular chondrocyte scaffold seeding, an approach considered to enhance scaffold hydrophilicity and cartilage regeneration. Auricular chondrocytes were isolated from canine ears and human surgical samples discarded during otoplasty, including microtia reconstruction. Canine chondrocytes were seeded onto PCL and nanoPGA sheets either with or without ethanol treatment to examine cellular adhesion in vitro. Human chondrocytes were seeded onto three-dimensional bioresorbable composite scaffolds (PCL with surface coverage of nanoPGA) either with or without ethanol treatment and then implanted into athymic mice for 10 and 20 weeks. On construct retrieval, scanning electron microscopy showed canine auricular chondrocytes seeded onto ethanol-treated scaffolds in vitro developed extended cell processes contacting scaffold surfaces, a result suggesting cell-scaffold adhesion and a favorable microenvironment compared to the same cells with limited processes over untreated scaffolds. Adhesion of canine chondrocytes was statistically significantly greater (p ≤ 0.05) for ethanol-treated compared to untreated scaffold sheets. After implantation for 10 weeks, constructs of human auricular chondrocytes seeded onto ethanol-treated scaffolds were covered with glossy cartilage while constructs consisting of the same cells seeded onto untreated scaffolds revealed sparse connective tissue and cartilage regeneration. Following 10 weeks of implantation, RT-qPCR analyses of chondrocytes grown on ethanol-treated scaffolds showed greater expression levels for several cartilage-related genes compared to cells developed on untreated scaffolds with statistically significantly increased SRY-box transcription factor 5 (SOX5) and decreased interleukin-1α (inflammation-related) expression levels (p ≤ 0.05). Ethanol treatment of scaffolds led to increased cartilage production for 20- compared to 10-week constructs. While hydrophilicity of scaffolds was not assessed directly in the present findings, a possible factor supporting the summary data is that hydrophilicity may be enhanced for ethanol-treated nanoPGA/PCL scaffolds, an effect leading to improvement of chondrocyte adhesion, the cellular microenvironment and cartilage regeneration in tissue-engineered auricle constructs.

Efficacy of sliced nerves of different thickness in a biodegradable nerve conduit to promote Schwann cell migration and axonal growth: An experimental study in the rat model.

BACKGROUND: Using the rat sciatic nerve model, sliced nerves of different thickness was combined to a biodegradable nerve conduit and the amount of nerve fragment necessary to promote nerve regeneration was investigated. MATERIALS AND METHODS: Harvested sciatic nerve (n = 6) was processed in sliced nerve of the different width; 2, 1, 0.5 mm, respectively. Western blot analysis was carried out to determine protein expression of Erk1/2. Subsequently, a total of 246 rats were used to create a 10 mm gap in the sciatic nerve. A polyglycolic acid-based nerve conduit was used to bridge the gap, with one sliced (width; 2, 1, 0.5 mm) or two (width; 1 mm × 2) incorporated within the conduit (n = 6 at each point in each group). At 2, 4, 8, and 20 weeks after surgery, samples were resected and subjected to immune-histological, transmission electron microscopic, and motor functional evaluation for nerve regeneration. RESULTS: Western blot analysis demonstrated Erk1/2 expressions were significantly increased in the groups of 2-mm and 1-mm width and attenuated in the 0.5-mm width group (p < .05). The immune-histological study showed the migration of Schwann cells and axon elongation were significantly extended in the groups of 2-mm, 1-mm, and 1 mm × 2 width at 4 weeks (p < .01), in which nerve conduction velocity was marked at 20 weeks (p < .01) after implantation. CONCLUSION: When nerve tissue was inserted in the biodegradable nerve conduit as a sliced nerve, the method of inserting two sheets with a slice width of 1 mm most strongly accelerated motor function.

Function-preserving Surgery for Femoral Sarcoma by "In Situ Preparation" Technique with a Free Flap.

Resection of soft-tissue sarcomas near important tissues (major blood vessels, nerves, bones) is challenging. "In situ preparation" (ISP) technique enables the function of the affected limb to be maintained by preserving the tissue as much as possible. The technique is based on evaluation of the margin of resection of important tissues near the tumor during surgery. Postoperative fractures are known to frequently occur, however, in cases where bones were preserved and periosteum has been resected by the ISP. We present the case of a 51-year-old woman who required treatment for soft-tissue sarcoma close to the femur. During surgery, femoral periosteum was included in the tumor side and the femur was preserved by the ISP. We covered the femur using a vascularized latissimus dorsi free flap instead of periosteum. The flap survived completely, and 5 years after surgery, there has been no recurrence or postoperative complications and the lower limb is functional. This is the first reported case of successful combined use of the bone ISP and the vascularized latissimus dorsi free flap to preserve the function of the limb affected by femoral sarcoma suspected of bone infiltration.

Third Toe Pulp Reconstruction Using the Contralateral Second Toe Hemi-pulp Free Flap.

A hemi-pulp flap is widely known as a flap useful for aesthetic and functional reconstruction of the fingers, and rarely used for toe reconstruction. We performed third toe pulp reconstruction using a free hemi-pulp flap harvested from the contralateral second toe to repair the tissue defect following toe replantation. An 18-year-old woman was injured with complete left third toe amputation and open fracture of the proximal phalanx of the left second toe in a traffic accident. On the same day, third toe replantation was urgently performed. After surgery, the third toe was partially taken, and had a toe pulp tissue defect due to necrosis. It was reconstructed with a free hemi-pulp flap prepared from the contralateral second toe. The flap was completely taken. Three years after surgery, the reconstructed left third toe was aesthetically favorable. Perception of the flap region was restored up to S2 without pain and there was no complication such as numbness, callus, and ulceration. In the flap donor site (right second toe), the skin graft was unnoticeable without pigmentation. Toe pulp reconstruction requires a sensory flap as low-invasive as possible with excellent sensory restoration, texture, feel, and shear property. This method is considered as one of the low-invasive, aesthetic, and functional reconstruction methods.

A Comparative Study of Nail Fold Approach and Microscopic Nail Bed Approach in the Surgical Treatment of Subungual Exostosis.

ABSTRACT: Subungual exostosis is a benign tumor that occurs under the nail plate of the distal phalanx and causes nail deformity and pain. There are many reports of recurrence and postoperative nail deformity, and the surgical approach needs to be reassessed. Two typical surgical approaches are from the nail fold and the nail bed. Here, we compare 2 surgical approaches for subungual exostosis that were performed in our department. Twenty-three cases of subungual exostosis were treated between 2010 and 2019; 12 cases were treated by the nail fold approach and 11 by the microscopic nail bed approach. The nail fold approach resulted in not only scarring but also nail deformities, such as onychodystrophy. There were 2 (18.2%) cases of recurrence. In the microscopic nail bed approach, there was no case of recurrence. It was possible to preserve the thinned nail bed by dissecting under a microscope, and the thinned nail bed did not become necrotic in any cases. To solve postoperative nail deformity and a high recurrence rate, sufficient resection and meticulous surgical procedure on the nail bed and nail matrix are essential. The microscopic nail bed approach satisfied both of these criteria and was considered to be a useful method for subungual exostosis.

Use of sliced or minced peripheral nerve segments for nerve regeneration through a biodegradable nerve conduit: A preliminary study in the rat.

BACKGROUND: Using the rat sciatic nerve model, the difference in outcome using a nerve segment either sliced open or minced with a blade incorporated into a nerve conduit were compared and the relative effects upon the rate and completeness of the nerve regeneration was determined. MATERIALS AND METHODS: A 10-mm gap was created in the rat sciatic nerve and bridged with a biodegradable nerve conduit. Segments of the resected nerve (2-mm lengths) were prepared by either slicing the nerve with one longitudinal cut or by scalpel mincing of the nerve tissue, with insertion of the prepared nerve segment into the center of the conduit. Flow cytometry and Western blotting of these preparations were performed to measure viable cells and to examine the expression of Erk1/2 for neural regeneration potential with both treatments. in vivo nerve regeneration was evaluated at 2, 4, 8, and 20 weeks, using immunohistochemistry, transmission electron microscopy, muscle wet weight, and nerve conduction velocity determination. RESULTS: The sliced nerve group showed significantly greater Schwann cell migration with the subsequent axonal elongation at 4 weeks after implantation, in comparison to the minced nerve group and controls (unaltered conduit grafts). By 20 weeks anterior tibial muscle weight and nerve conduction velocity were also greater in the sliced nerve group in comparison to the other groups (p < .05). CONCLUSION: These findings suggest that insertion of a sliced section of nerve into a biodegradable nerve conduit can shorten the time for and improve the quality of nerve regeneration.

Eye Comfort and Physiological Reconstruction of an Entire Upper Eyelid Defect.

OBJECTIVE: Reconstruction of an extensive full-thickness upper eyelid defect is challenging. The purpose of this report is to introduce this procedure with emphasis on reconstruction of the eyelid margin to obtain eye comfort. METHODS: We designed a technique using a radial forearm flap for the outer layer to reconstruct the entire eyelid after resection of Merkel cell carcinoma. In additional, the inner layer and the eyelid margin were reconstructed with a buccal mucosal graft and a reverse Hughes flap. RESULTS: There has been no recurrence of the tumor, opening and closing functions of the eyelid are maintained, and the patient has not complained of eye discomfort. CONCLUSION: Maintenance of mobility, flexibility, and a good ocular surface in contact with the sensitive cornea are the main foci of upper eyelid reconstruction, with an optimal fissure height and an appropriate contour of the eyelid. In addition, to obtain eye comfort, it is important to protect the cornea without significantly restricting eyelid mobility.

Successful Intralesional Laser Therapy for Sclerotherapy-resistant Huge Venous Malformation.

Venous malformations (VMs) are compressible, blue-purple tumors that are present at birth, and are treated with either surgery or sclerotherapy, or a combination of both. Patients often experience recurrent hemorrhage, swelling, pain, or difficulty in daily life. Treatment of massive VMs can, therefore, be challenging. We applied intralesional laser photocoagulation (ILP) to a 19-year-old female patient with a huge VM who had shown resistance to sclerotherapy. It spanned from the dorsal area to the lateral chest. ILP is delivered directly into thick, deep lesions through a bare fiber delivery system. The effect on deep components is optimized without directly contacting the cutaneous or mucosal surfaces to minimize epithelial damage. We applied ILP to the lesion at 30 W in continuous mode (10 seconds) under ultrasound monitoring using an Nd:YAG laser of 1064-nm wavelength. In total, there have been 4 sessions of the same treatment; the lesion was treated with a combined 120 kJ of energy. Satisfactory regression of the huge VM was achieved by this ILP treatment. No significant complications (major bleeding, severe pain, post-perforation skin ulcer, and scar contraction) occurred during the treatment. ILP has been indicated for only sclerotherapy-resistant cases until now, but we suggest that it is a potentially safe and minimally-invasive resolution of VM without scarring or loss of normal shape, sensory feel, or function. ILP appears to be a safe and minimally-invasive resolution of VM. We propose that this technique could become a first-choice treatment modality for VM.

Influence of Periosteum Location on the Bone and Cartilage in Tissue-Engineered Phalanx.

PURPOSE: This study investigated the influence of periosteal tissue of different origins on the calcification at the diaphysis and chondrocyte maturation at the epiphysis in an engineered phalanx. We hypothesized that the periosteum from long bones would better provide donor cells for bone formation and signals for maturation of the joint cartilage. METHODS: Periosteum was harvested from 4 locations (cranium, mandible, radius, and ilium) of calf bones. A human phalangeal bone-shaped, biodegradable, 3-dimensional scaffold hydroxyapatite-poly L-lactic-ɛ-caprolactone (HA-P[LA/CL]) was prepared using a human phalangeal bone-shaped template. A bioengineered human phalanx was fabricated by combining periosteal grafts with biodegradable copolymers. The joint cartilage region (chondrocyte/polyglycolic acid [PGA] composite) was subsequently sutured to the phalangeal bone region (periosteum/HA-P[LA/CL] composite) with absorbable sutures to make a human phalangeal bone model. These were then implanted in nude mice for maturation of the constructs. Macroscopic, radiographic, histological, and immune-histochemical evaluations were carried out to determine the relative influence of the periosteal graft source on bone and cartilage formation at 10 and 20 weeks after implantation. RESULTS: Calcification localized under the periosteum was noted in the cranium, radius, and ilium groups after 10 weeks, which markedly expanded at the modelled diaphysis after 20 weeks. The width in the minor axis direction tended to increase with time after grafting in the cranium group, whereas the longitudinal length increased in the radius and ilium groups. The joint cartilage thickness changed with time depending on the type of periosteum, and periosteum collected from the radius and ilium was associated with the greatest cartilage thickness in the joint cartilage maturation process. CONCLUSIONS: These results suggest that periosteum collected from radius of calves demonstrated superior bone formation and chondrocyte maturation in the engineered phalanx compared with other sources of periosteum. CLINICAL RELEVANCE: The osteogenic capacity depends on the periosteal source regardless of intramembranous or endochondral ossification. The appropriate periosteal choice is essential in the phalangeal bone and cartilage tissue engineering. The results are important for broadening tissue engineering possibilities for clinical application.

A clinical multi-center registry study on digital nerve repair using a biodegradable nerve conduit of PGA with external and internal collagen scaffolding.

BACKGROUND: In this study, we evaluated the clinical efficacy of a biodegradable nerve conduit constructed of polyglycolic acid (PGA) tube with external and internal collagen scaffolding for digital nerve repair. PATIENTS AND METHODS: A multi-center registry study was conducted in 11 locations between July 2013 and May 2016. Multiple mechanisms of injury included clean-cut (12 patients), crush (5 patients), and avulsion (3 patients) types of injuries. These patients underwent nerve repair with a biodegradable nerve conduit, with 9 patients having a primary repair and 11 patients having delayed repair. Average nerve gap was 16.7 mm (range: 1-50 mm). An average of 13 months follow-up (range: 12-15 months) was available including sensory assessments. RESULTS: Improved s2PD was found with less severe injury as in clean-cut (7.5 ± 1.5 mm), which was statistically significant in comparison to those in crush (9.8 ± 1.9 mm, P = .0384) and in avulsion (10.7 ± 4.7 mm, P = .0013) type injuries. A meaningful recovery (S3+ or S4) was observed in 90% of the 20 digital nerve repairs with a biodegradable nerve conduit of PGA with external and internal collagen scaffolding. Avulsion injuries had significantly lower levels of meaningful recovery (67%) in comparison to those of clean-cut (P = .0291) and crush (P = .0486) types of injury. No adverse effects were reported postoperatively. CONCLUSION: These results indicate that a biodegradable nerve conduit of PGA with external and internal collagen scaffolding is suitable for digital nerve repair of short nerve gaps with high levels of sensory recovery as measured by two-point discrimination.

A basic fibroblast growth factor slow-release system combined to a biodegradable nerve conduit improves endothelial cell and Schwann cell proliferation: A preliminary study in a rat model.

BACKGROUND: A basic fibroblast growth factor (bFGF) slow-release system was combined to a biodegradable nerve conduit with the hypothesis this slow-release system would increase the capacity to promote nerve vascularization and Schwann cell proliferation in a rat model. MATERIALS AND METHODS: Slow-release of bFGF was determined using Enzyme-Linked ImmunoSorbent Assay (ELISA). A total of 60 rats were used to create a 10 mm gap in the sciatic nerve. A polyglycolic acid-based nerve conduit was used to bridge the gap, either without or with a bFGF slow-release incorporated around the conduit (n = 30 in each group). At 2 (n = 6), 4 (n = 6), 8 (n = 6), and 20 (n = 12) weeks after surgery, samples were resected and subjected to histological, immunohistochemical, and transmission electron microscopic evaluation for nerve regeneration. RESULTS: Continuous release of bFGF was found during the observation period of 2 weeks. After in vivo implantation of the nerve conduit, greater endothelial cell migration and vascularization resulted at 2 weeks (proximal: 20.0 ± 2.0 vs. 12.7 ± 2.1, P = .01, middle: 17.3 ± 3.5 vs. 8.7 ± 3.2, P = .03). Schwann cells showed a trend toward greater proliferation and axonal growth had significant elongation (4.9 ± 1.1 mm vs. 2.8 ± 1.5 mm, P = .04) at 4 weeks after implantation. The number of myelinated nerve fibers, indicating nerve maturation, were increased 20 weeks after implantation (proximal: 83.3 ± 7.5 vs. 53.3 ± 5.5, P = .06, distal: 71.0 ± 12.5 vs. 44.0 ± 11.1, P = .04). CONCLUSIONS: These findings suggest that the bFGF slow-release system improves nerve vascularization and Schwann cell proliferation through the biodegradable nerve conduit.

Preliminary Study of PGA Fabric for Seromas at Latissimus Dorsi Flap Donor Sites.

BACKGROUND: Seroma formation is a major complication following latissimus dorsi (LD) flap transfer for breast reconstruction. We implanted a nonwoven polyglycolic acid (PGA) fabric-a biodegradable polymer-in the LD flap donor site and examined its effect on postoperative seroma formation and resolution in a comparative study on 38 patients undergoing primary 1-stage breast reconstruction by LD flap. METHOD: A PGA treatment group had a PGA fabric placed in the donor wound (PGA group, n = 20), whereas a second group was treated with standard donor site closure (control group, n = 18). The incidence of seromas was comparable between the groups. RESULT: There was a significant reduction in aspiration volume by needle aspiration after drain removal (P < 0.05) and in the time until seroma resolution (P < 0.01) in the PGA group compared with the control group. CONCLUSION: These results indicate that application of PGA at the LD donor site is useful in controlling postoperative seromas.

A Novel Method to Induce Cartilage Regeneration with Cubic Microcartilage.

Cartilage tissue is characterized by its poor regenerative properties, and the clinical performance of cartilage grafts to replace cartilage defects has been unsatisfactory. Recently, cartilage regeneration with mature chondrocytes and stem cells has been developed and applied in clinical settings. However, there are challenges with the use of mature chondrocytes and stem cells for tissue regeneration, including the high costs associated with the standard stem cell isolation methods and the decreased cell viability due to cell manipulation. Previous studies demonstrated that cartilage can be regenerated from chondrocyte clusters that contain stem cells. Based upon some of the existing techniques, the goal of this study was to develop a novel and practical method to induce cartilage regeneration. A microslicer device was developed to process cartilage tissues into micron-size cartilage (microcartilage) in a minimally invasive manner. We evaluated microcartilage sizes and demonstrated 100-400 µm as optimal for generating a high cell yield with collagenase digestion. In addition, autologous intrafascial implantation of the composites of microcartilage and an absorbable scaffold with a slow-release system of basic fibroblast growth factor (bFGF) was carried out to induce cartilage regeneration. Our results demonstrated that the extent of bFGF diffusion depends on the size of microcartilage, and that cartilage regeneration was induced most effectively with 100 µm of microcartilage via SOX5 upregulation. These findings suggest that cartilage regeneration is possible with microcartilage as a source of cells without ex vivo cell expansion.

Reconstruction of Severely Crushed Fingertip Amputations with Basic Fibroblast Growth Factor Slow Release System.

Supplemental Digital Content is available in the text.

Long-term result of a biodegradable osteo-inductive copolymer for the treatment of orbital blowout fracture.

PURPOSE: We have recently reconstructed the orbital wall using a bioabsorbable osteo-inductive copolymer as a support material for the bone defects in patients with orbital blowout fracture. The purpose of this study was to investigate a 2-year follow-up after implantation. MATERIAL AND METHODS: In order to investigate the efficacy and safety of the bioabsorbable osteo-inductive copolymer HA-P(LA/CL), hydroxyapatite-poly(l-lactide-ε-caprolactone), we measured CT density of the implanted copolymers in 7 patients at such time points as 1 week, 6 months and 2 years after surgery along with the long-term outcomes with regard to postoperative complications. RESULTS: Five patients in whom the intraoperative maximum width of the bone defect was less than 15 mm demonstrated the fracture types of either linear or trap-door. All patients treated with HA-P(LA/CL) copolymer indicated good bone formation with high CT density (>500) at 2 years postoperatively. In contrast, 2 patients in whom the intraoperative maximum width of the bone defect was more than 16 mm demonstrated a punched-out type fracture. Postoperative CT of the HA-P(LA/CL) copolymer showed low density (28.0 and 173.4) at 2 years postoperatively, indicative of limited bone formation. None of the cases demonstrated significant postoperative complications such as diplopia, enophthalmos, infection and extrusion of the implant material. CONCLUSION: The results of this study demonstrate that surgical treatment of orbital blowout fractures using bioabsorbable osteo-inductive copolymer was useful in linear and trap-door fractures.

Randomized controlled trial of the application of topical b-FGF-impregnated gelatin microspheres to improve tissue survival in subzone II fingertip amputations.

We undertook a randomized controlled trial of subzone II fingertip amputations, comparing standard treatment with topical application of gelatin microspheres prepared with basic fibroblast growth factor (b-FGF) to provide a slow, sustained release of b-FGF with microsphere degradation. Forty-eight digits from 42 patients were randomized into the two study arms. The microspheres were applied as a paste on exposed tissue surfaces, whereas standard treatment was without any topical treatment. Patients were treated either with microsurgical revascularization or by simple composite graft, based on the surgeon's clinical decision. Tissue survival of the replanted fingertips was measured by a blinded evaluator 3 weeks postoperatively. A modest improvement in survival was seen with b-FGF-microsphere application for both revascularized and composite grafted fingertips, though this did not achieve statistical significance. Whether the slow release of b-FGF through a bioresorbable carrier gives any improvement in outcome in patients with subzone II fingertip amputations is unproven.

Morphologic study of normal, ingrown, and pincer nails.

BACKGROUND: Pincer nail has been confused with ingrown nail for decades. OBJECTIVE: The objectives were to analyze the circumferential length of pincer nail and the relationship between nail deformity and the underlying distal phalangeal shape. METHODS: The circumferential length of 53 pincer nails was determined at successive 5-mm intervals of the nail plate. Sixty great toes, including normal (n=20), ingrown (n=20), and pincer nails (n=20), were assessed with respect to body height, body weight, body mass index, nail height, nail width index, and nail height index. Nail angle and height of osteophyte were measured. RESULTS: Despite the presence or absence of overcurvature, the circumferential length displayed nearly identical values across the proximal to distal range of the nail. In the pincer nail group, only the correlation between the width and height indices was statistically significant; 50%, 80%, and 100% of cases were confirmed based on osteophyte presence in normal, ingrown, and pincer nails. CONCLUSION: The findings suggest that the mechanical cause may be associated with the over curved nail, which is affected by nail bed contraction. Results may support the hypothesis that an osteophyte of the distal phalanx may not be a cause of, but rather a result of, an overcurving deformity.

Tissue engineering a model for the human ear: assessment of size, shape, morphology, and gene expression following seeding of different chondrocytes.

This study examines the tissue engineering of a human ear model through use of bovine chondrocytes isolated from four different cartilaginous sites (nasoseptal, articular, costal, and auricular) and seeded onto biodegradable poly(l-lactic acid) and poly(L-lactide-epsilon-caprolactone) (50 : 50) polymer ear-shaped scaffolds. After implantation in athymic mice for up to 40 weeks, cell/scaffold constructs were harvested and analyzed in terms of size, shape, histology, and gene expression. Gross morphology revealed that all the tissue-engineered cartilages retained the initial human auricular shape through 40 weeks of implantation. Scaffolds alone lost significant size and shape over the same period. Quantitative reverse transcription-polymerase chain reaction demonstrated that the engineered chondrocyte/scaffolds yielded unique expression patterns for type II collagen, aggrecan, and bone sialoprotein mRNA. Histological analysis showed type II collagen and proteoglycan to be the predominant extracellular matrix components of the various constructs sampled at different implantation times. Elastin was also present but it was found only in constructs seeded with auricular chondrocytes. By 40 weeks of implantation, tissue-engineered cartilage of costal origin became calcified, marked by a notably high relative gene expression level of bone sialoprotein and the presence of rigid, nodular protrusions formed by mineralizing rudimentary cartilaginous growth plates. The collective data suggest that nasoseptal, articular, and auricular cartilages represent harvest sites suitable for development of tissue-engineered human ear models with retention over time of three-dimensional construct architecture, gene expression, and extracellular matrix composition comparable to normal, nonmineralizing cartilages. Calcification of constructs of costal chondrocyte origin clearly shows that chondrocytes from different tissue sources are not identical and retain distinct characteristics and that these specific cells are inappropriate for use in engineering a flexible ear model.

Molecular mechanisms of cleft lip formation in CL/Fr mice.

CL/Fr mice have a high incidence of cleft lip and the cleft lip is the result of incomplete fusion between the medial and lateral nasal prominences and the maxillary prominence at about day 11.5 of gestation. However, little is known about the molecular mechanisms that are responsible for the incomplete fusion. We made a molecular pathological investigation using 11.5-day CL/Fr embryos. Five embryos were each examined for real-time polymerase chain reaction (PCR) analysis. During the first palatal formation in normal development, an epithelial seam is formed when the medial and lateral nasal prominences first make contact. Some epithelial cells of the epithelial seam then undergo apoptosis, with remaining cells transforming into a mesenchymal phenotype (epithelial-mesenchymal transition, EMT). Mesenchymal cells of the medial and lateral nasal prominences then merge across the previous boundary of separation. In CL/Fr mice with cleft lip, neither apoptosis nor EMT occurs in the epithelial cells. Increased expression of claudin 6 mRNA is seen in epithelial cells of epithelial seam in cleft lip compared with that in normal embryos. Slug mRNA expression was also significantly reduced whereas noggin was increased in CL/Fr embryos with cleft lip. We suggest that EMT is prevented in CL/Fr mice with cleft lip by increased expression of claudin 6 and coexistent downregulation of slug in cells of the epithelial seam, and these altered concentrations of transcription factors/repressors prevent fusion of the medial and lateral nasal prominences, leading to clefts of the lip.