Expression of myosin heavy chain isoforms in Duchenne muscular dystrophy patients and carriers

The expression of MHC isoforms in the skeletal muscles of nine patients with Duchenne muscular dystrophy (DMD) (from 2.5 to 15 yr of age) and three DMD carriers was studied using different specific anti-MHC MAbs. We also analyzed muscle fiber size and fiber reactivity with acridine orange and/or with a surface antigen marker. One-quarter of all fibers of DMD patients, or less with age, were of normal size and contained only adult slow MHC. Half of the muscle fibers contained adult and developmental MHCs. Only half of these fibers were representative of an active regenerative process. MHC co-expression also altered the proportion of normal fast or slow fibers. Adult fast MHCs were expressed as unique MHC only in small and very small fibers in the oldest DMD patients. In DMD carrier muscles, the greatest alterations in MHC expression were observed in patients with the most reduced dystrophin expression. However, MHC changes in dystrophin-positive fibers were similar to those observed in dystrophin-free fibers. In conclusion, disruptions or delays in the switching of all genes coding for adult fast and slow MHC and developmental MHC coincided with dystrophin deletion and with perturbations in its expression.     

Myeloid regeneration after bone-marrow transplantation monitored by serum measurements of myeloperoxidase, lysozyme and lactoferrin

Bone-marrow regeneration after chemo- and radiotherapy-induced aplasia can be monitored by serum levels of myeloperoxidase (MPO), lysozyme (LYS) and lactoferrin (LF). In 10 patients with leukemia, serum measurements were performed before and after bone-marrow transplantation. Bone-marrow regeneration was suggested by increments in serum MPO and LYS 5 and 4 days prior to the increase in mononuclear cells (Mono) and 10 and 9 d before the increase in polymorphonuclear leukocytes (PMN) in the peripheral blood. LF started to rise 4.5 d before detectable circulating PMNs. 2 patients with early relapses of leukemia post transplantation are shown to display atypical patterns of serum MPO and LYS. We conclude that serum measurements of MPO, LYS and LF may be used as early and sensitive means to monitor bone-marrow activity during hematological regeneration. However, the findings also strongly support the earlier proposal that MPO alone may be used to reflect myeloid activity in the bone-marrow in general.     

自体骨膜游离移植的实验研究--不同龄动物大块关节软骨缺损修复的比较

目的研究比较自体骨膜移植软骨再生修复不同龄动物大块关节软骨缺损。方法用52只不同龄家兔自体骨膜游离移植修复大块关节软骨缺损,比较移植骨膜生发层朝向关节腔与松质骨时再生软骨的差别。结果经不同时期肉眼和组织学检查证实,幼年兔和成年兔的骨膜移植都能生成软骨,修复大块关节软骨缺损。在成年兔骨膜再生的软骨与成年兔本身周围正常软骨的厚度、组织结构一样。移植骨膜生发层朝向关节腔与松质骨二者间再生软骨结果无明显差别。结论骨膜具有再生软骨的能力,可用来移植修复关节软骨的缺损。骨膜移植生发层不同朝向对软骨再生无明显影响。成年后骨膜移植修复关节软骨缺损能够生成与自身相适应的软骨。     

Chemical traumatization of adult mouse olfactory epithelium in situ stimulates growth and differentiation of olfactory neurons in vitro

This study demonstrates that ZnSO₄ induced chemical trauma results in an in situ regeneration of the olfactory epithelium which, when maintained in vitro, provides an enriched population of olfactory neurons. Therefore, the ability of the olfactory epithelium to respond to chemical trauma with increased mitotic activity can be used to increase growth of neurons in culture. Tissue obtained from normal or vehicle-treated adult mice produced few olfactory neurons, when maintained in culture, compared to cultures established from tissue following an in situ ZnSO₄ trauma. Maximal neuronal yields were obtained in cultures established from tissue that was removed 4–6 days following chemical trauma. The morphological appearance and the presence of cell specific intermediate filament proteins were used to classify the cell types in these olfactory epithelial cultures. Single cells and aggregates of cells which were immunopositive for keratin, but immunonegative for neurofilament protein and GFAP, were identified as epithelioid. Flattened polygonal cells immunopositive for GFAP were identified as glia. A small population of flattened cells was immunonegative for all of the antibodies used in this study. Cells that had processes were immunonegative for GFAP and keratin. Some were immunopositive for 200 kDa and 160 kDa neurofilament proteins but immunonegative for the 68 kDa neurofilament protein. A few of these cells showed positive immunoreactivity with the olfactory marker protein (OMP) antibody and most likely represented the most mature olfactory neurons in the cultures. This trauma-induced culture model using olfactory tissue from adult mice can serve as a source of CNS neurons for comparison with cultured embryonic neurons.     

Research review: DNA polymerases as molecular markers of the regenerating capacity of hepatocytes

Hepatocyte regeneration has been widely investigated, with the mitotic index and the incorporation of [³H]thymidine being used as regeneration markers. We focused on the induction of DNA replication enzymes, particularly DNA polymerases (pol) α, δ, and ε. Using rat models, we have shown that the activity of pol α in crude liver extract well represents the regenerating capacity of hepatocytes. Using pol α as an indicator, we analyzed liver regeneration in rat models under various conditions: obstructive jaundice, external or internal biliary drainage, and the obstruction of portal vein branches. It has been revealed that the ligation of the common bile duct alone induces a certain amount of hepatocyte proliferation. It was striking that external biliary drainage suppressed regeneration capacity in cholestatic rat liver after partial hepatectomy. The strong regeneration in nonligated lobes induced by portal branch ligation was similar to the liver regeneration seen after partial hepatectomy with respect to the induction of DNA polymerases. Taken together, the aspects of DNA replication, particularly the induction of DNA polymerases, may contribute to shedding new light on the regeneration of human liver. This work was supported in part by a Grant-in-Aid for General Scientific Research and for Cancer Research from the Ministry of Education, Science and Culture, Japan, and by grants from the Uehara Memorial Foundation     

Histological comparison of patellar cartilage degeneration between chondromalacia in youth and osteoarthritis in aging

The histological findings of the patellar cartilage were compared between cases of chondromalacia, which occurs predominantly in young persons (22 patients, average age 19.8 years) and cases of osteoarthritis, which is common among the elderly (21 patients, average age 65.4 years). The histological findings of cartilage in the chondromalacia were characterized by increased density and vigorous fibrous metaplasia of chondrocytes. These findings may be considered to represent a reactive change in the chondrocyte. Cartilage degeneration in osteoarthritis, by contrast, is regressive and presents a clearly different histological picture from that of chondromalacia patellae. We conclude that chondromalacia does not easily lead to osteoarthritis. On the other hand, the cartilage was characteristically softened, as observed by gross inspection, and showed rarefaction of the cartilage matrix. It should be noted that the change was not observed in aging, but showed a pattern of cartilage degeneration peculiar to young patients with chondromalacia patellae.     

Chondrocyte-seeded hydroxyapatite for repair of large articular cartilage defects. A pilot study in the goat

The aim of this study was to evaluate the potential for restoration of a large cartilage defect in the goat knee with hydroxyapatite (HA) loaded with chondrocytes. Isolated chondrocytes were suspended in fibrin glue, seeded on top of the HA, and then the composite graft was implanted in the defect. After transplantation, cell behaviour, newly synthesised matrix and the HA–glue interface were assessed histologically after 2, 4, 12, 26 and 52 weeks. Special attention was paid to the incorporation process of HA in the subchondral bone and interactions between this biomaterial and the fibrin-glue–chondrocyte suspension.

Chondrocytes in the glue proved to survive the transplantation procedure and produced new metachromatically stained matrix two weeks after implantation. The glue–cell suspension had penetrated the superficial porous structure of the HA. Four weeks after surgery, islands of hyaline-like cartilage were observed at the HA–glue interface. A layer of fibrous tissue was formed surrounding the HA graft, resulting in a relatively instable fixation of the HA in the defect. This instability of the graft in the defect, possibly together with early weight bearing, resulted in a gradual loss of the newly formed hyaline cartilage-like repair tissue. Progressive resorption of the HA occurred without any sign of active bone remodelling from the host site. One year after surgery part of the defect which extended down to the cancellous bone had been predominantly restored with newly formed lamellar bone. Only small HA remnants were still present at the bottom of the original defect. Resurfacing of the joint had occurred with fibrocartilaginous repair tissue.

The absence of adequate fixation capacity of the HA near the joint space resulted in a relative instability of the graft with progressive resorption. Therefore, HA is not a suitable biomaterial to facilitate the repair of large articular cartilage defects.     

Study of nerve regeneration in centrocentral anastomosis

Summary Nerve regeneration was studied in a model of centrocentral anastomosis (CCA) performed on the sciatic nerve of the rat. Experimental CCA was made by suturing the proximal end of the peroneal branch on the proximal end of the sural branch, placing between them a peroneal nerve graft (Group I, 20 rats) or a silicone chamber (Group II, 12 rats). Nerve grafts had a length of 5mm and silicone chambers 7 mm. In six silicone chambers an 1 mm nerve graft was placed in the centre of the tube. In group I animals anterograde degeneration was studied by cutting the graft 60 days after surgery. In group II, nerve regeneration was studied 2, 4 and 8 weeks after surgery. Results indicate that in CCA: 1) regenerated axons coming from one nerve end grow into the graft but do not cross the contralateral suture line; 2) regeneration is poorer in silicone chambers than in nerve grafts; and, 3) in silicone chambers regeneration is related to time. The reduction in the regenerative capability in CCA seems to be related to the alteration of nerve sprouts aiming for the peripheral targets.     

Comparative effects of plateletderived growth factor-BB and insulin-like growth factor-I, individually and in combination, on periodontal regeneration in Macaca fascicularis

Platelet-derived growth factor (PDGF) and insulin-like growth factor I (IGF-I) in combination have previously been shown to enhance periodontal regeneration. The objective of this study was to further characterize the biological effects of this combination of growth factors in non-human primates and compare the effects to those of each growth factor individually. Ligature-induced periodontitis was initiated in 10 cynomolgus monkeys. After periodontal lesions were established, surgery was performed, and either a methylcellulose gel vehicle or vehicle containing 10 μg each of either PDGF-BB, IGF-I or both PDGF-BB and IGF-I was applied to exposed root surfaces. Biopsies were taken 4 and 12 wk after treatment and the extent of periodontal regeneration was assessed by histomorphometry. At both 4 and 12 wk vehicle-treated lesions generally revealed minimal osseous defect fill (ODF) (8.5±2.1% and 14.5±5.7%, respectively) and new attachment (NA) (34.1±5.2% and 26.6±10.5%, respectively). IGF-I treatment did not significantly alter healing compared to vehicle in any parameter at both 4 and 12 wk. PDGFBB-treated sites exhibited significant (p<0.05) regeneration of NA (69.6±12.0%) at 12 wk; trends for PDGF-BB treatment effect were also observed in other parameters at 4 and 12 wk. although these increases were not statistically significant. Treatment with PDGF-BB/IGF-I resulted in 21.6±5.1 % and 42.5±8.3% ODF at 4 and 12 wk, respectively, and 64.1±7.7% and 74.6±7.4% NA at 4 and 12 wk, respectively (all significantly greater than vehicle, p<0.05). The results from this study demonstrated that: 1) IGF-1 alone at the dose tested did not significantly alter periodontal wound healing; 2) PDGF-BB alone significantly stimulated NA, with trends of effect on other parameters; and 3) the PDGF-BB/IGF-I combination resulted in significant increases in NA and ODF above vehicle at both 4 and 12 wk.     

游离骨膜移植修复关节软骨缺损的实验研究

在家兔的股骨内髁上作一关节软骨全层缺损,且从同侧胫骨近端取下一片骨膜骨块,将骨膜生发层向外包绕骨块,嵌入缺损处,观察其作用。实验表明,骨膜在一定条件下可以化生成软骨;滑液环境与关节运动在软骨化生过程中起重要作用;透明软骨化生和再生率与运动量及开始时间相关;自体骨可作为一种将游离骨膜固定在软骨缺损内的填充材料。