Noritaka Isogai

Formation of phalanges and small joints by tissue-engineering.

BACKGROUND This report describes the formation of small phalanges and whole joints from three types of bovine-cell sources transplanted onto biodegradable polymer matrices. The resulting structures had the shape and composition of human phalanges with joints. METHODS Fresh bovine periosteum was wrapped around a copolymer of polyglycolic and poly-L-lactic acid. Separate sheets of polyglycolic acid polymer were then seeded with chondrocytes and tenocytes isolated from the shoulders of freshly killed calves. The gross form of a composite tissue structure was constituted in vitro by assembling the parts and suturing them to create models of a distal phalanx, a middle phalanx, and a distal interphalangeal joint. RESULTS Subcutaneous implantation of the sutured composite tissues into athymic mice resulted in the formation, after twenty weeks, of new tissue with the shape and dimensions of human phalanges with joints. Histological examination revealed mature articular cartilage and subchondral bone with a tenocapsule that had a structure similar to that of human phalanges and joints. There was continuous cell differentiation at the ectopic site even after extended periods. CONCLUSIONS These findings suggest that the formation of phalanges and small joints is possible with the selective placement of periosteum, chondrocytes, and tenocytes into a biodegradable synthetic polymer scaffold.

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.

Experimental use of fibrin glue to induce site-directed osteogenesis from cultured periosteal cells.

The purpose of this study was to determine whether a combination of fibrin glue and cultured periosteal cells will result in new bone formation at heterotopic sites in nude mice. Growing cells and developing matrices surrounding periosteal explants from the diaphyses of radii of newborn calves were minced and mixed with fibrin glue in a syringe. The cell/matrix-fibrin glue admixture was then injected into the subcutaneous space on the dorsum of athymic nude mice. After 12 weeks of implantation, gross morphology and histologic investigations showed newly formed bone structures in all cell/matrix-fibrin glue admixtures, but none in fibrin glue injected alone and used as control samples. Osteopontin, a protein important in bone development, was identified by a Western blot assay of the cell/matrix-fibrin glue composite. This study supports the feasibility of initiating site-directed formation of bone structures at heterotopic tissue sites by means of injection of cultured periosteal cells and matrix in a fibrin glue carrier. (Plast. Reconstr. Surg. 105: 953, 2000.)

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.

Quantitative analysis of the reconstructed breast using a 3-dimensional laser light scanner.

Postoperative cosmesis of the reconstructed breast depends on the interrelation of shape, size (volume), and symmetry. In this study, reconstructed breasts were analyzed with 3-dimensional projections generated by laser light scanning. Fifty-one cases of breast reconstruction following mastectomy (16 cases of rectus abdominis flap, 15 cases of latissimus dorsi flap, and 20 cases of tissue expansion) were evaluated 6 months postoperatively. Shape, volume, and symmetry were quantitatively evaluated. Captured images of the normal breast were mirror-reversed and superimposed on images of the reconstructed breast. Differences in the generated Moire patterns were used to quantitatively compare breasts. The method was rapid, reproducible, and accurate in comparison to thermoplaster casts. It was found that rectus abdominis flaps applied following total mastectomy and latissimus dorsi flaps applied following partial mastectomy gave the best results for their relatively low degree of asymmetry. Application of tissue expansion led to greater asymmetry and poorer overall cosmesis. In conclusion, a 3-dimensional laser light scanning system makes it possible to quantitate the cosmetic outcome following breast reconstruction.

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.

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.

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.

Application of medical thermography to the diagnosis of Frey's syndrome.

In Freys syndrome, the secretory parasympathetic fibers of the parotid gland are thought to communicate with the sympathetic nerve fibers of sweat glands and blood vessels of the skin following parotidectomy. Miscommunication results in subjective gustatory sweating and facial flushing, which appear early with postoperative mastication. In this study, we compared the efficacy of medical thermography to the Minors starch‐iodine test to determine the presence of gustatory sweating in Freys syndrome.

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.