Shinichi Asamura

Tissue engineering of an auricular cartilage model utilizing cultured chondrocyte-poly(L-lactide-epsilon-caprolactone) scaffolds.

To determine the potential development in vivo of tissue-engineered auricular cartilage, chondrocytes from articular cartilage of bovine forelimb joints were seeded on poly(L-lactic acid-epsilon-caprolactone) copolymer scaffolds molded into the shape of a human ear. Copolymer scaffolds alone in the same shape were studied for comparison. Chondrocyte-seeded copolymer constructs and scaffolds alone were each implanted in dorsal skin flaps of athymic mice for up to 40 weeks. Retrieved specimens were examined by histological and molecular techniques. After 10 weeks of implantation, cell-seeded constructs developed cartilage as assessed by toluidine blue and safranin-O red staining; a vascular, perichondrium-like capsule enveloped these constructs; and tissue formation resembled the auricular shape molded originally. Cartilage matrix formation increased, the capsule persisted, and initial auricular configuration was maintained through implantation for 40 weeks. The presence of cartilage production was correlated with RT-PCR analysis, which showed expression of bovine-specific type II collagen and aggrecan mRNA in cell-seeded specimens at 20 and 40 weeks. Copolymer scaffolds monitored only for 40 weeks failed to develop cartilage or a defined capsule and expressed no mRNA. Extensive vascularization led to scaffold erosion, decrease in original size, and loss of contour and shape. These results demonstrate that poly(L-lactic acid-epsilon-caprolactone) copolymer seeded with articular chondrocytes supports development and maintenance of cartilage in a human ear shape over periods to 40 weeks in this implantation model.

Bone regeneration using a bone morphogenetic protein-2 saturated slow-release gelatin hydrogel sheet: evaluation in a canine orbital floor fracture model.

Bone regeneration methods using bone inductive cytokines show promise, however, due to early diffusion and absorption of single applications of these cytokines, the bone inductive effects are limited. In this study, such a system was applied, using gelatin hydrogel as a carrier to slowly release (bone morphogenetic proteins) BMP-2 over a relatively long period in vivo. By coupling this slow-release system with a biodegradable copolymer, this composite was evaluated by grafting into bone defect sites of a canine orbital floor fracture model. Radio-iodinated BMP-2 incorporated into the gelatin hydrogel carrier and subcutaneously implanted into nude mice showed a similar slow release (approximately, 60% at 3 days and 80% at 14 days) as the radiolabeled hydrogel carrier alone. In contrast, greater than 90% of fluid-injected BMP-2 was lost in the injection site within the first 8 hours. Using a dog model of orbital floor fracture, a complex of BMP-2-saturated gelatin hydrogel and a polylactide-based biodegradable copolymer was implanted into the orbital bone defect. Bone structural analysis, using radiography, histologic examination, and microfocus CT, showed greatly enhanced new bone formation and defect healing at 5 weeks in comparison to implanted biodegradable copolymer directly saturated with the same amount of BMP-2 (no slow-release hydrogel carrier). A trabecular structure resembling that normal bone tissue was restored in the new bone tissue generated by the slow-release constructs. Thus study demonstrates the potential of slow-release BMP-2 for bone healing of difficult defects.

Treatment of orbital floor fracture using a periosteum—polymer complex

Various materials for the reconstruction of bone defects in orbital floor fractures have been developed and applied clinically. Recently, reconstruction using polymers, in place of autologous bone and artificial materials, has been actively introduced, but there are problems, such as the size of reconstructable bone defects and the decomposition rate of polymers. A basic study was performed on bone regeneration using a periosteum-polymer complex produced by attaching periosteum to a biodegradable polymer sheet. In this study, patients with orbital floor fractures were evaluated clinically who had undergone reconstruction of orbital floor defects of the using a periosteum-polymer complex produced by applying periosteum to an Hydroxyapatite-[poly (l-lactide-epsilon-caprolactone)](HA-P (CL/LA)) sheet and the ilium in the previous 3 years. A bone defect of less than 2.5cm(2) area was defined as small, that of 2.5-4cm(2) as intermediate, and that of more than 4cm(2) as a large bone defect. For small bone defects, hypoaesthesia in the infraorbital nerve was observed in one patient each of the periosteum-polymer complex and ilium groups. Regarding intermediate bone defects, diplopia and hypoaesthesia in the infraorbital nerve were observed in one patient in each of the two groups. For large bone defects, diplopia was observed in one patient each for the periosteum-polymer complex and ilium groups, and hypoaesthesia of the infraorbital nerve was only detected in one patient of the former group. Not more than 2mm of enophthalmos was detected in any patient. The anatomical eyeball position and eyeball movement were normal after surgical treatment using the periosteum-polymer complex, just as in reconstruction using autologous bone.

Cytokine-Rich Autologous Serum System for Cartilaginous Tissue Engineering

Animal serum used for tissue engineering approaches has unacceptable risk for contamination with infectious agents. In this study, a cytokine-rich autologous serum (CRAS) system was developed. Canine auricular chondrocytes were cultured in medium supplemented with either fetal bovine serum (FBS) or autologous canine serum, alone or supplemented with basic fibroblast growth factor (b-FGF). Cell proliferative capacity was higher in the CRAS cultures than in those cultured in FBS, with greater expression of aggrecan and type II collagen in the b-FGF-supplemented CRAS group. The chondrocytes were seeded onto an ear-shaped biodegradable polymer (poly-l-lactide:ϵ-caprolactone, 50:50) and cultured in a Bioflow reactor for 1 week, using the 3 different culture media indicated above, and subsequently implanted into nude mice. The best outcome (cartilage gene expression and morphologic properties) was seen with tissue-engineered constructs precultured in the b-FGF-supplemented CRAS media. These findings indicate a clinically realizable approach for tissue engineering of cartilaginous structures.

Evaluation of nanofiber-based polyglycolic acid scaffolds for improved chondrocyte retention and in vivo bioengineered cartilage regeneration.

Background: In conventional studies on the regeneration of auricle-shaped cartilage in autogenous models using large animals, there were problems with the cartilage regeneration induction capacity and long-term retention of geometric shape. In this study, the authors sought to improve on outcome in these regards: a nonwoven fabric of polyglycolic acid was developed through nanotechnology, and the effect of nanofiber diameter on in vitro cell-seeding efficiency and the in vivo response after implantation in an autogenous large-animal model were evaluated. Methods: Canine chondrocytes were isolated and seeded onto polyglycolic acid fabric ranging from 0.5 to 20 &mgr;m in average diameter. Cell seeding efficiency was highest for mid-range polyglycolic acid fibers (average diameter, 0.8, 3.0, and 7.0 &mgr;m). Flat and auricle-shaped scaffolds were constructed using polypropylene structural support, sandwiching a nonwoven polyglycolic acid fabric that contained autogenous chondrocytes together with basic fibroblast growth factor–laden particles and an exterior fibrin sealant. Scaffolds were then implanted autogenously and evaluated at 5 and 20 weeks. Results: Biomechanical strength was optimal for polyglycolic acid fiber diameters of 0.8 to 3.0 &mgr;m. Optimal cell maintenance and neocartilage response were seen with polyglycolic acid fiber diameters in the same mid-range for nanofiber constructs. Conclusion: These findings demonstrate the potential for nanoscale modulation of auricle-shaped cartilage regeneration in a large-animal model.

What is the best strategy for Asians with involutional entropion

BackgroundEven though many different procedures have been proposed to involutional entropion, there is no established criterion standard in terms of the choice of the operative method. Considering the anatomical difference of the lower eyelid structure between Asians and whites, we evaluated the effectiveness of our surgical approach to involutional entropion exclusively for Japanese patients. MethodsTwenty-one patients complained of discomfort around the eye, and they also suffered from foreign body sensation, ocular pain, epiphora, and photophobia. All the patients with the involutional entropion were surgically treated with combined procedures (the lower eyelid retractors advancement, the modified Hotz method, and the modified Wheeler method). ResultsTwenty-three lower eyelids of 21 patients with involutional entropion underwent surgery. There were 10 men and 11 women with a mean age of 78 years (range, 69–94 years). All patients we operated on were completely satisfied with any residual ocular symptoms. There was no case of recurrence following the primary procedure during 16 months of the mean follow-up period. ConclusionBased on the anatomical difference in various ethnic groups, we propose the best strategy for Asian patients with the involutional entropion.

Is it truly necessary to add epicanthoplasty for correction of the epiblepharon

BackgroundThere are many modified variations of the original Hotz procedure for the repair of the epiblepharon. No matter which procedure is used, there must be some factors that may cause recurrence. One of possible causes of these unsatisfactory results can be due to the presence of epicanthal folds (EFs) among the oriental population. It is important to determine whether patients should be repaired with the simple epiblepharon or if it should be combined with epicanthoplasty especially for actively growing children. MethodsAll the patients were between 4 and 7 years old and had both epiblepharon and EF. The EFs were classified in 3 types, and all patients were operated on with the modified Hotz procedure. A “good” outcome was defined to be no contact between the eyelash and eyeball, and a “fair” outcome was defined to be several eyelashes contact with the eyeball, without any annoying symptoms. A “poor” outcome was defined to be most of the eyelids still in contact with the eyeball, and these patients persistently complain of irritation or keratitis. ResultsThe study included 46 lower eyelids of 23 patients (14 females, 9 males; mean age, 5.7 years) who underwent operation. Thirty-five eyelids (76.1%) were assessed to have a “good” outcome, 9 eyelids (19.6%) were assessed to have a “fair” outcome, and 2 eyelids (4.3%) were assessed to have a “poor” outcome. ConclusionsWe firmly believe that epicanthoplasty is not necessarily performed routinely for all epiblepharon unless there is any specific reason to justify the combined procedure.

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.

Frontalis sling procedure for ocular myasthenia gravis

A 39-year-old woman was diagnosed with myasthenia gravis when she was 8 years old. Although many treatments – such as cholinesterase inhibitors and steroids – had been given to the patient, her condition did not improve sufficiently. As she demonstrated bilateral 3 mm levator function without any eye movement disturbances, bilateral frontalis sling procedures were performed with an autologous fascia lata. One year after the operation, the operated upper eyelids showed symmetrically appropriate heights with good functional outcome. A sling procedure with an autologous fascia lata was suitable for correcting poor levator function of an ocular myasthenia gravis case.

What Is the Best Way to Handle the Involutional Blepharoptosis Repair

AbstractThere are many different operations to correct involutional blepharoptosis (IB); however, the outcome of the corrective surgery is rather unpredictable, regardless of the procedure employed. A reasonably predictable outcome can be achieved with careful intraoperative evaluation of the condition, with measuring of the margin reflex distance-1 (MRD-1) in supine position of the patients. With these prepositions, we collected data that indicated that our approach can achieve a predictable outcome.This was a prospective study of 21 consecutive patients (8 men and 13 women) involving 42 eyelids with IB. IB was defined as an MRD-1 of <2 mm. All 21 patients were informed of the purposes of the study, and underwent levator aponeurosis advancement. The MRD-1 was measured intraoperatively with the patients in a supine position and in the 3-month postoperative inspection with the patients in a sitting position. Statistical analyses using paired t-tests were performed.From intraoperative measurement, mean MRD-1 values were 4.31 mm on the right side (range 3.0–4.5) and 4.29 mm on the left side (range 3.5–5.0). Three months after the operations, mean MRD-1 values were 3.07 mm on the right side (range 1.5–4.0) and 3.07 mm on the left side (range 2.0–4.0). Compared with the intraoperative MRD-1 measurements, those of the postoperatives were significantly 1.2 mm reduced (right: P < 0.01, left: P < 0.01).The intraoperative measurement of MRD-1 without changing position of patients could result in successful outcome of the operation.