Hirohisa Kusuhara

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‐ɛ‐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.

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. The authors have indicated no significant interest with commercial supporters.

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.

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.

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.