Seong-Keun Cho

Preventing collapse in early osteonecrosis of the femoral head. A randomised clinical trial of core decompression

We performed a randomised trial on 37 hips (33 patients) with early-stage osteonecrosis (ON). After the initial clinical evaluation, including plain radiography and MRI, 18 hips were randomly assigned to a core-decompression group and 19 to a conservatively-treated group. All the patients were regularly followed up by clinical evaluation, plain radiography and MRI at intervals of three months. Hip pain was relieved in nine out of ten initially symptomatic hips in the core-decompression group but persisted in three out of four initially painful hips in the conservatively-treated group at the second assessment (p < 0.05). At a minimum follow-up of 24 months, 14 of the 18 core-decompressed hips (78%) and 15 of the 19 non-operated hips (79%) developed collapse of the femoral head. By survival analysis, there was no significant difference in the time to collapse between the two groups (log-rank test p = 0.79). Core decompression may be effective tin symptomatic relief, but is of no greater value than conservative management in preventing collapse in early osteonecrosis of the femoral head.

Tibial bone defects treated by internal bone transport using the Ilizarov method.

Abstract.We reviewed 27 cases of tibial bone defects treated by internal bone transport using the Ilizarov method. The causes of the bone defects were open fractures in 14 segments and infected non-unions in 13. The average length of the defects was 8.3 cm (range, 3–20 cm). There were 21 one-level tibial transports, 3 two-level tibial transports, 1 one-level tibial transport with fibular transport, and 2 fibular transports. At the docking site, 25 segments underwent bone grafting. Eleven of the 25 were Papineau-type open cancellous bone grafts. Acute shortening or docking was performed in 10 segments. Bone union was obtained in every instance. The average time of external fixation was 8 months and the average time to union was 7.1 months. Bone grafting at the docking site is recommended in order to shorten the duration of treatment and to prevent refracture and non-union.Résumé.Nous avons passé en revue 27 cas de perte de substance du tibia traités par «transport osseux interne» conformément à la méthode Ilizarov. Ces anormalies provenaient de fractures ouvertes dans 13. La longeur moyenne des defect osseux était de 8,3 cm (entre 3 et 20 cm). On a effectué 21 «transports tibiaux» de niveau 1, 3 «transports tibiaux» de niveau 2, 1 «transport tibial» avec «transport du péroné», ainsi que 2 «transport du péroné». Dans 25 cas une greffe osseuse a été pratiquée sur le site de réduction. 11 cas des 25 greffes pratiquées étaitent du type Papinau. D’importances réduction ont dûétre effectuées dans 10 cas. Des fusions ont été obtenues dans tous les cas. Le temps moyen de fixation externe était de 8 mois et celui de fusion de 7,1 mois. La greffe osseuse est recommandée sur le site de réduction afin de raccourcir la durée du traitement et de prévenir toute récidive de fracture ou de non-consolidation.

Serial Cloning of Pigs by Somatic Cell Nuclear Transfer: Restoration of Phenotypic Normality During Serial Cloning

Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first‐generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second‐generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third‐generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age‐ and sex‐matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clones cells during its gestational development. Developmental Dynamics 236:3369–3382, 2007.

Nuclear remodelling and the developmental potential of nuclear transferred porcine oocytes under delayed-activated conditions

It is still unclear whether nuclear envelope breakdown and premature chromosome condensation are essential for the reprogramming of the donor nucleus following somatic nuclear transfer. To address this, we determined the ability of delayed-activated or simultaneously activated porcine oocytes to undergo nuclear remodelling and development following somatic cell nuclear transfer. A small microtubule aster was observed in association with decondensed chromatin following nuclear transfer, suggesting the introduction of a somatic cell centrosome. In the delayed-activated condition, most fibroblast nuclei divided into two chromosome masses and two pronuclear-like structures following transfer into oocytes. In contrast, fibroblast nuclei in the simultaneously activated condition formed a large, swollen, pronuclear-like structure. Microtubule asters were organised in the vicinity of the nucleus regardless of the number of nuclei. More reconstructed oocytes developed to the blastocyst stage in the delayed-activated condition than in the simultaneously activated condition (p < 0.05). Nine piglets were born from two recipient sows following transfer of delayed-activated reconstructed oocytes, while none developed to full term in the simultaneously activated condition. Fingerprint analysis showed that the PCR-RFLP patterns of the nine offspring were identical to that of the donor pig. These results suggest that the activation of recipient oocytes during nuclear transfer probably relates to the nuclear remodelling process, which can affect the ability of embryos created by somatic cell nuclear transfer to develop.

Proteomic Analysis of the Extraembryonic Tissue from Cloned Porcine Embryos

Cloned animals developed from somatic cell nuclear transfer (SCNT) embryos are useful resources for agricultural and medical applications. However, the birth rate in the cloned animals is very low, and the cloned animals that have survived show various developmental defects. In this report, we present the morphology and differentially regulated proteins in the extraembryonic tissue from SCNT embryos to understand the molecular nature of the tissue. We examined 26-day-old SCNT porcine embryos at which the sonogram can first detect pregnancy. The extraembryonic tissue from SCNT embryos was abnormally small compared with the control. In the proteomic analysis with the SCNT extraembryonic tissue, 39 proteins were identified as differentially regulated proteins. Among up-regulated proteins, Annexins and Hsp27 were found. They are closely related to the processes of apoptosis. Among down-regulated proteins, Peroxiredoxins and anaerobic glycolytic enzymes were identified. In the Western blot analysis, antioxidant enzymes and the antiapoptotic Bcl-2 protein were down-regulated, and caspases were up-regulated. In the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay with the placenta from SCNT embryos, apoptotic trophoblasts were observed. These results demonstrate that a major reason for the low birth rate of cloned animals is due to abnormal apoptosis in the extraembryonic tissue during early pregnancy.

Production of germline transgenic chickens expressing enhanced green fluorescent protein using a MoMLV-based retrovirus vector

The Moloney murine leukemia virus (MoMLV) ‐based retrovirus vector system has been used most often in gene transfer work, but has been known to cause silencing of the imported gene in transgenic animals. In the present study, using a MoMLV‐based retrovirus vector, we successfully generated a new transgenic chicken line expressing high levels of enhanced green fluorescent protein (eGFP). The level of eGFP expression was conserved after germline transmission and as much as 100 g of eGFP could be detected per 1 mg of tissue protein. DNA sequencing showed that the transgene had been integrated at chromosome 26 of the G1 and G2 generation transgenic chickens. Owing to the stable integration of the transgene, it is now feasible to produce G3 generation of homozygous eGFP transgenic chickens that will provide 100% transgenic eggs. These results will help establish a useful transgenic chicken model system for studies of embryonic development and for efficient production of transgenic chickens as bioreactors.—Koo, B. C., Kwon, M. S., Choi, B. R., Kim, J‐H., Cho, S‐K., Sohn, S. H., Cho, E. J., Lee, H. T., Chang, W., Jeon, I., Park, J‐K., Park, J. B., Kim, T. Production of germline transgenic chickens expressing enhanced green fluorescent protein using a MoMLV‐based retrovirus vector. FASEB J. 20, 2251–2260 (2006)

Total knee arthroplasty in bony ankylosis in gross flexion

Between June 1993 and December 1994, we performed total knee arthroplasty (TKA) on 27 knees in 24 patients with spontaneous bony ankylosis in severe flexion. The mean age at operation was 43.5 years (30 to 60). No patient had preoperative pain. Three were unable to walk and 21 could manage less than five blocks. The mean duration of the ankylosis was 18.7 years (13 to 25) and its mean position was 105 degree flexion (75 to 135). The preoperative Hospital for Special Surgery Knee Score of 60 points was improved to 87 at the final follow-up three to five years later. All knees were free from pain. The mean range of active flexion in 24 knees was 97 degrees (78 to 115) and the mean arc of movement 91 degrees (78 to 98). The mean fixed flexion deformity was 6 degrees (0 to 25) and the extension lag 8 degrees (0 to 25). Angular deformity was corrected to between 0 degrees and 10 degrees of valgus. Four patients were able to walk one block and 20 five to seven blocks. Thirteen knees (48%) showed some necrosis at the skin edge; one knee required arthrodesis and another resection arthroplasty. One had a recurrence of tuberculous infection requiring arthrodesis. One patient had a rupture of the quadriceps tendon. To date no prosthesis has required revision for loosening. Radiolucency of 1 mm or less about the tibial prosthesis was observed at follow-up in four of the 24 knees. Our results have shown that one-stage TKA and skeletal traction after operation can achieve correction of severe flexion deformity of the knee with marked improvement in the function and quality of life.

Role of Thrombotic and Fibrinolytic Disorders in Osteonecrosis of the Femoral Head

Some studies have suggested that thrombotic and fibrinolytic disorders may be etiologic causes of osteonecrosis of the femoral head. A case-control study was done to determine whether these disorders are associated with osteonecrosis of the femoral head in East Asian patients with nontraumatic osteonecrosis of the femoral head. Twenty-four consecutive patients who had been diagnosed as having nontraumatic osteonecrosis of the femoral head were matched with 24 control subjects for gender, age (1-year range), and the time of presentation (1-year range). Thrombotic factors including protein C activity, protein S activity, antithrombin III, anticardiolipin antibody immunoglobulins G and M, and lupus antibody were investigated. Fibrinolytic factors including tissue-plasminogen activator, plasminogen activator inhibitor-1, tissue-plasminogen activator and plasminogen activator inhibitor-1 ratio, lipoprotein (a), and plasminogen also were investigated. There were no significant differences in the levels of thrombotic and fibrinolytic factors. In eight patients with idiopathic osteonecrosis, anticardiolipin antibody immunoglobulin G, an antiphospholipid antibody which is associated with thrombotic phenomena, was lower than that in respective control subjects. These data do not confirm an etiologic role for thrombotic and fibrinolytic disorders in East Asian patients with nontraumatic osteonecrosis of the femoral head.

Enhanced cryosurvival of bovine blastocysts produced in vitro in serum-free medium.

AbstractPurpose: Culture systems affect the development of IVP embryos and consequently their cryosurvival potential. The viability of postthawed bovine IVP embryos developed from IVM/IVC medium in the presence or absence of serum was compared. Methods: Cumulus-oocyte complexes were matured in IVM medium supplemented with or without serum. Some oocytes were evaluated for nuclear maturation status and others were inseminated with semen. Presumptive zygotes were cultured in IVC medium supplemented with or without serum for 9 days. Blastocysts were cryopreserved with 1.5 M ethylene glycol in PBS. Results: No difference was observed in the nuclear maturation status and cleavage rates in both groups, but significantly (P < 0.05) higher in blastocyst rates in the serum-supplemented group. After freezing, survival of blastocysts was higher in the serum-free group. At 36 h culture after thawing, blastocysts developed without serum had significantly (P < 0.05) higher cell number than those cultured with serum. Conclusions: We conclude that serum-free culture system enhances the viability of frozen-thawed bovine embryos.

Improvement in post-thaw viability of in vitro-produced bovine blastocysts vitrified by glass micropipette (GMP)

The purpose of this study was to investigate the use of a glass micropipette (GMP) as a vessel for vitrification of in vitro-produced (IVP) bovine blastocysts and to compare the results with post-thaw survival rate of bovine blastocysts frozen in GMP with those frozen in open pulled straw (OPS) that have been previously investigated. The GMP vessel permitted higher freezing and warming rates than the OPS due to the higher heat conductivity of the glass and the lower mass of the solution that contained the embryos. Groups of three bovine IVP blastocysts were sequentially placed into vitrification solution before being loaded into either the OPS or GMP vessels and they immersed into LN(2) within 20-25 s. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in a holding medium (HM: D-PBS supplemented with 5% FCS) and then in TCM-199 for 5 min in both cases. They was then cultured in TCM 199 supplemented with 10% FCS for 24 or 48 h. The rate of blastocyst re-expansion was significantly different between OPS (79.6%) and GMP (90.4%) methods. Neither was the hatching rate significantly different among OPS (51.8%), GMP (57.1%) methods and non-vitrified group (67.3%). Only the rate of post-thaw re-expanding of blastocysts loaded in narrow column was significantly higher than that of the wide column (83.3% versus 56.7%) (P < 0.05), although the GMP straw was loaded with three blastocysts per vessel. These results indicated that the GMP vessels provided high survival rates of bovine IVP blastocysts. The location of the embryos loaded into a narrow or wide portion was considered to be a limiting factor to the viability of bovine IVP embryos.