Novel ultrasonic evaluation of tissue-engineered cartilage for large osteochondral defects--non-invasive judgment of tissue-engineered cartilage.

Although numerous methods for regenerating articular cartilage have been investigated, the regenerated tissue showed various histological findings from hyaline-like cartilage to fibrous tissue. Without biopsy, we are unable to know whether the cartilage regeneration method was histologically successful or not. We developed a new ultrasonic evaluation system for articular cartilage using the maximum magnitude (MM) from ultrasonic analysis. The purpose of this study was to investigate the usefulness of ultrasonic judgment of the cartilage regeneration procedure. Using our system we quantitatively evaluated tissue-engineered cartilage in rabbit cartilage defects. The specimens were retrospectively divided into two groups on the basis of histological findings and investigated whether significant differences in ultrasonic analysis could be found between the two (group H: hyaline-like cartilage group, successful; group F: fibrous tissue group, failure). In the ultrasonic findings, the MM was 1.11+/-0.32 in group H and 0.65+/-0.18 in group F and these differences were significant (P=0.00061). Our results suggest that the ultrasonic evaluation system used in the present study is capable of judging the success or failure of cartilage regeneration procedures, and therefore, it could be a valuable tool arthroscopic diagnosis of cartilage regeneration.     

Biomechanical properties of tissue-engineered cartilage from human and rabbit chondrocytes.

OBJECTIVE: To describe tissue-engineered cartilage from rabbit and human chondrocytes. STUDY DESIGN AND SETTING: Chondrocytes from rabbit and human ears were seeded onto a template and implanted for 8 or 16 weeks of in vivo incubation. RESULTS: For the 8-week and 16-week groups, the UTS for cartilage was 3.8 MPa and 3.7 MPa, stiffness was 62.4 MPa and 51.8 MPa, and resilience was 181.8 J/m(3) and 109.1 J/m(3), respectively. Experimental cartilage was significantly different from controls. From 5 human specimens, the UTS was 5.4 MPa, stiffness was 6.6 MPa, and resilience was 2.0 J/m(3). The control had UTS of 8.8 MPa, stiffness of 12.2 MPa, and resilience of 2.9 J/m(3). Histology showed mature cartilage but with a fibrovascular infiltrate and increased cellularity. CONCLUSIONS: Mechanical properties of tissue-engineered cartilage can be quantified and are less than that of controls.     

Analysis and isolation of renal tubular cells by flow cytometry.

The cells of the renal cortex have rich heterogeneity of structure and function. Flow cytometry, the technique of rapid laser-based single cell analysis, can give information about cellular mixtures not obtainable by any other means. We examined a variety of fluorescent markers to identify populations of renal cells by flow cytometry. Cellular digests of rat cortex were fluorescently stained with either enzymatic activity probes, or polyclonal antibodies. Fluorescent staining for the proximal marker gamma-glutamyl-transpeptidase (tau-GT) was an order of magnitude brighter than autofluorescence, and stained 71 +/- 11% of the cells. Second, we colocalized enzymatic and antibody markers. There was tight colocalization of tau-GT enzyme activity, detected with fluorogenic substrates, with specific surface binding of tau-GT antibodies. Third, populations of fluorescently labelled cells can be rapidly isolated by flow cytometry sorting. Flow cytometry sorting isolated 10(7) cells positive for the proximal tubular marker tau-GT in a little under one hour. The sorted cells were viable with 99 +/- 2% trypan blue exclusion (N = 8). Sodium-dependent phloridzin-inhibitable glucose uptake was present in sorted cells, with greater uptake/mg protein than in unsorted controls. The sorted cells grew in culture as a monolayer of tightly adherent cuboidal cells. Hence, flow cytometry allows us to quantitate the heterogeneity in mixed renal cellular digests. Flow cytometry allows us to rapidly isolate millions of cells according to fluorescently tagged markers. The isolated cells are viable, retain sodium-dependent transport properties, and grow in culture.     

鼻中隔软骨及耳软骨在鼻尖延长中的应用

目的 探讨自体鼻中隔软骨联合耳软骨治疗短鼻的临床疗效.方法 采集自体鼻中隔软骨、耳软骨分别制成鼻中隔尾侧延伸移植物、鼻小柱支撑移植物及鼻尖移植物,重塑鼻翼软骨支架.结果 对263例患者随访1～2年,鼻尖形态自然,支撑稳固,延长明显,外形显著改善,均未出现鼻尖相关并发症.结论 采用自体鼻中隔软骨及耳软骨延长鼻尖,其重塑的鼻翼软骨支架符合正常鼻尖解剖结构,能更好地控制鼻尖形态,并避免使用异体材料而出现相关并发症.是一种效果确切、安全有效的方法.     

Identification of histologically undetectable parathyroid hyperplasia by flow cytometry.

Recurrent hyperparathyroid disease is a continuing surgical problem resulting in a controversy about how much parathyroid tissue to excise initially. We studied the DNA content of parathyroid cells and analyzed the differences found in normal, hyperplastic, and adenomatous glands with the new technique of flow cytometry.By staining DNA with an intercalating fluorescent dye, propidium iodide, and passing the nuclei through an exciting laser beam, the amount of DNA per nucleus can be measured. The resultant DNA histogram can be analyzed to provide information on the per cent of cells in the cell cycle phases (G0 + G1, S, and G2 + M).Histograms from normal parathyroids (parathyroids from nonhyperparathyroid patients) were statistically compared with histograms from adenomatous parathyroids. A significant difference (p < 0.04) in the tetraploid region (G2 + M) was identified. The computer exploited this difference to calculate the per cent chance of any sample being an adenoma. In all parathyroid samples tested from 54 patients, there were no false positives in the normal parathyroid group and twelve false negatives (15 per cent) in the adenomatous group. Biopsies from histologically and grossly “normal” glands from hyperparathyroid patients in which an adenoma had been excised showed varied DNA patterns typical of both groups. Seventy-one per cent of these parathyroid glands appeared normal and had virtually no activity in the tetraploid region. Some glands were in an indeterminate range and a few definitely had DNA content typical of an adenomatous parathyroid.This study describes a clinically adaptable, quantitative method for analyzing biopsied parathyroid glands that has the potential of identifying previously undetectable hyperplasia at the initial surgical procedure. The ability to selectively excise these hyperplastic glands may reduce the late recurrence sometimes seen with excision of only an enlarged adenoma and prevent hypoparathyroidism after excessive ablation of normal parathyroid tissue.     

Osteochondritis dissecans of the talus treated by the transplantation of tissue-engineered cartilage

Management of osteochondral lesions of the joint has been difficult, because articular cartilage has a poor healing capacity as a result of its lack of vessels, nerve supply, and its isolation of systemic regulation. Although a lot of basic research and surgical treatments for cartilage repair have focused on osteochondral lesions in the knee joint, orthopedic surgeons have recently diverted their attention to osteochondral lesions in the ankle joint, partly because of the widespread introduction of arthroscopy in ankle surgery. There have been many attempts to treat articular cartilage defects in the ankle joint as well as in the knee joint. However, no treatment has achieved efficient healing with hyaline cartilage. Recently, tissue engineering technique for cartilage repair has been gaining much attention in the orthopedic field. In this study, we reported on a patient with osteochondritis dissecans of the talar dome, successfully treated by transplantation of tissue-engineered cartilage made ex vivo using atelocollagen gel and low tibial osteotomy.     

Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage.

Cartilaginous constructs have been grown in vitro with use of isolated cells, biodegradable polymer scaffolds, and bioreactors. In the present work, the relationships between the composition and mechanical properties of engineered cartilage constructs were studied by culturing bovine calf articular chondrocytes on fibrous polyglycolic acid scaffolds (5 mm in diameter, 2-mm thick, and 97% porous) in three different environments: static flasks, mixed flasks, and rotating vessels. After 6 weeks of cultivation, the composition, morphology, and mechanical function of the constructs in radially confined static and dynamic compression all depended on the conditions of in vitro cultivation. Static culture yielded small and fragile constructs, while turbulent flow in mixed flasks yielded constructs with fibrous outer capsules; both environments resulted in constructs with poor mechanical properties. The constructs that were cultured freely suspended in a dynamic laminar flow field in rotating vessels were the largest, contained continuous cartilage-like extracellular matrices with the highest fractions of glycosaminoglycan and collagen, and had the best mechanical properties. The equilibrium modulus, hydraulic permeability, dynamic stiffness, and streaming potential correlated with the wet-weight fractions of glycosaminoglycan, collagen, and water. These findings suggest that the hydrodynamic conditions in tissue-culture bioreactors can modulate the composition, morphology, mechanical properties, and electromechanical function of engineered cartilage.     

Analysis and sorting of prostate cancer cell types by flow cytometry.

BACKGROUND: Prostate tumor heterogeneity as manifested by differential expression of markers can be attributed to multiple types of cancer cells populating a tumor. Does the composition differ between primary tumor and metastasis? How can one isolate the different cancer cell types to study? What is the relationship among cancer cell types? METHODS: Flow cytometry keying on the prostate epithelial cell surface markers CD57 and CD44 was applied to analyze and sort single cells prepared from tumor tissue samples by collagenase digestion. In normal tissue, CD57 is found on luminal cells and CD44 on basal cells. RESULTS: CD57(+) and CD44(+) cells were sorted from various prostate tumor tissue specimens. The CD57(+) cancer cell type was found to predominate in primary tumors, while the CD44(+) cancer cell type was found to predominate in two visceral metastases. All tumors could be characterized by a ratio of CD57(+) and CD44(+) cancer cells. CONCLUSIONS: Two types of prostate cancer cells, CD57(+) and CD44(+), were identified. The finding that most primary tumors contain a predominantly CD57(+) cancer cell population agrees with the argument that cancer cells arise from the transformation of CD57(+) luminal cells. However, CD44(+) cancer cells are also present in some primary tumors; and in some metastases, they, and not CD57(+) cells, constitute a predominant population.     

Synchronous lung cancers defined by deoxyribonucleic acid flow cytometry.

When two pulmonary tumors are seen simultaneously in patients with lung cancer, it always raises a question as to whether the lesions are synchronous lung cancers or lung cancer with intrapulmonary metastasis. To settle this issue, we used deoxyribonucleic acid flow cytometry. Deoxyribonucleic acid ploidy patterns of tumors were able to be analyzed in 14 patients with two simultaneous pulmonary tumors resected by operation. These two tumors, with completely different patterns of deoxyribonucleic acid ploidy, were defined as synchronous lung cancers. Tumors were defined as lung cancer with intrapulmonary metastasis when both tumors showed diploidy or when at least one deoxyribonucleic acid index of abnormal clones between two aneuploid tumors was the same or almost identical. Tumors of the five patients with stage I disease were classified as synchronous lung cancers according to the criteria involving deoxyribonucleic acid flow cytometry. Both tumors were adenocarcinomas in four patients and large-cell and squamous cell carcinomas in one. Both tumors in four patients were located in the same lobe but different segments. All but one patient with different histologic types are alive without recurrence from 24 to 100 months after operation. Tumors of the nine patients with stage III disease in whom intrapulmonary metastasis was clinically suspected were classified as lung cancer with intrapulmonary metastasis according to the criteria. These data suggest that deoxyribonucleic acid flow cytometry of tumors may have diagnostic value in determining synchronous lung cancers.     

鼻中隔软骨及耳软骨在鼻尖整形中的联合应用

目的：探讨鼻中隔软骨及耳软骨在鼻尖整形手术中的联合应用的方法和优点。方法：对56例就医者施行了此项手术,应用自体鼻中隔软骨做鼻中隔延伸移植物,耳软骨做鼻尖移植物,结合各种鼻翼软骨缝合技术进行鼻尖整形。结果：所有鼻外形获得显著改善,鼻尖形态自然、圆润而俊俏,同时无皮肤或粘膜破损、假体外露、感染等并发症的发生,随访3个月～1年,鼻尖形态稳定,效果满意。结论：联合应用鼻中隔软骨及耳软骨进行鼻尖整形的方式,更接近正常鼻解剖结构,更好控制鼻尖形态,更容易获得预期的效果。     

Anatomical features of auricular and retroauricular skin

Summary A comparative study is presented on the anatomy of auricular and retroauricular skin. Auricular skin is very thin and well furnished with vascular plexuses arranged parallel to the surface. The layer of subcutaneous fat is scanty, with no columnae adiposae extending into the dermis; the reticular layer of the dermis is evenly interspersed with adipose tissue. The organisation of adipose tissue is perivascular to the skin vessels. At the insertion of the auricle, the structure of retroauricular skin corresponds to that of auricular skin. Towards the hair line, the retroauricular skin gradually assumes the characteristics of the scalp. Consideration is given to the way in which the anatomy of the skin from the region of the ear influences the healing of such skin in free grafting.     

Hyaluronic acid-based polymers as cell carriers for tissue-engineered repair of bone and cartilage.

Culture-expanded bone marrow-derived mesenchymal progenitor cells differentiate into chondrocytes or osteoblasts when implanted subcutaneously in vivo in combination with an appropriate delivery vehicle. This in vivo implantation technique is used to test new materials as putative delivery vehicles in skeletal tissue-engineering models. HYAFF 11 and ACP sponges, two biomaterials based on hyaluronic acid modified by esterification of the carboxyl groups of the glucuronic acid, were tested as osteogenic or chondrogenic delivery vehicles for rabbit mesenchymal progenitor cells and compared with a well characterized porous calcium phosphate ceramic delivery vehicle. The implant materials were examined by scanning electron microscopy for differences in pore structure or cellular interactions, were quantified for their ability to bind and retain mesenchymal progenitor cells, and were examined histologically for their ability to support osteogenesis and chondrogenesis after subcutaneous implantation into nude mice. The ACP sponge bound the same number of cells as fibronectin-coated ceramic, whereas the HYAFF 11 sponge bound 90% more. When coated with fibronectin, ACP and HYAFF 11 bound, respectively, 100 and 130% more cells than the coated ceramics. HYAFF 11 sponge composites retained their integrity after the 3 or 6-week incubation period in the animals and were processed for histomorphometric analysis. As a result of rapid degradation or resorption in vivo, ACP sponges could not be recovered after implantation and could not be analyzed. HYAFF 11 sponges presented more area available for cell attachment and more available volume for newly formed tissue. Following loading with mesenchymal progenitor cells and implantation, the pores of the sponges contained more bone and cartilage than the pores of ceramic cubes at either time point. Thus, relative to ceramic, HYAFF 11 sponges allow incorporation of twice as many cells and produce a 30% increase in the relative amount of bone and cartilage per unit area. Hence, the hyaluronic acid-based delivery vehicles are superior to porous calcium phosphate ceramic with respect to the number of cells loaded per unit volume of implant, and HYAFF 11 sponges are superior to the ceramics with regard to the amount of bone and cartilage formed. Additionally, hyaluronic acid-based vehicles have the advantage of degradation/resorption characteristics that allow complete replacement of the implant with newly formed tissue.     

Posttransplant antidonor antibodies and graft rejection. Evaluation by two-color flow cytometry

The posttransplant production of antibodies against cryopreserved donor cells was studied in 50 consecutive cadaveric kidney graft recipients and in 23 additional patients selected for acute rejection. Serum was obtained twice weekly during the first 3 weeks posttransplant and then monthly for 6 months. IgM and IgG anti-T cell Abs were measured by 2-color flow cytometry. Results were analyzed in conjunction with the patients' demographics, previous sensitization, HLA-matching, posttransplant blood transfusions, incidence of delayed function, rejection episodes, and biopsy results. Antidonor antibodies, predominantly IgG, were detected in 19/48 (40%) of the patients proximate to the time of rejection. In contrast, antibodies were seen in only 2/22 (9%) of nonrejecting patients, and these antibodies were exclusively IgM. Younger patients were more likely to have antibody-mediated rejections. Cytotoxic antibody reactivity against panel cells developed or increased posttransplant in some patients, but it did not correlate with rejection. Previous sensitization and posttransplant transfusions favored the development of posttransplant panel reactivity but not of antidonor antibodies. Most rejections, including those associated with antidonor antibodies, were reversed by antirejection therapy. We conclude that antidonor antibodies are involved in a significant proportion of rejection episodes and that the damage induced does not necessarily culminate with loss of the graft.     

Detection and quantitation of sperm-bound antibodies by flow cytometry of human semen.

Detection of sperm surface antibodies is important for the diagnosis and treatment of infertility. The authors have investigated the methodologic aspects of flow cytometry (FCM) to detect sperm-bound antibodies and to quantitate the sperm antibody load (antibody molecules/spermatozoa). To obtain reliable results, dead spermatozoa must be excluded from analysis because they can bind antibody nonspecifically, and comprise 10% to 58% (n = 28) of the ejaculate in subfertile men. Flow cytometry estimation of dead cells correlates (r = 0.83) significantly with the manual Eosin Y method. After staining washed sperm samples (n = 26) with fluorescein-isothiocyanate-conjugated F(ab')2 fragments of anti-IgG and IgA antibodies, the sperm load was 11,500 +/- 8,600 IgG molecules and 13,200 +/- 9,500 IgA molecules per spermatozoa. The sperm antibody load measured on different occasions could be compared between patients or in the same patient by the use of calibration standards. Since the inter- and intra-assay variation of the FCM assays was less than 10%, FCM has the potential reliably and objectively to monitor the sperm antibody load during corticosteroid treatment.