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Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity

  • Kim, Young June;Ahn, Kwang Sung;Kim, Minjeong;Kim, Min Ju;Ahn, Jin Seop;Ryu, Junghyun;Heo, Soon Young;Park, Sang-Min;Kang, Jee Hyun;Choi, You Jung;Shim, Hosup
    • Asian-Australasian Journal of Animal Sciences
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    • v.30 no.3
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    • pp.439-445
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    • 2017
  • Objective: Production of alpha-1,3-galactosyltransferase (${\alpha}GT$)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. Heterozygous ${\alpha}GT$ knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce ${\alpha}GT$-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Methods: Miniature pig fibroblasts were transfected with ${\alpha}GT$ gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous ${\alpha}GT$ gene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-${\alpha}$-1,3-galactose, an epitope produced by ${\alpha}GT$. Using magnetic activated cell sorting, cells with monoallelic disruption of ${\alpha}GT$ were removed. Remaining cells with LOH carrying biallelic disruption of ${\alpha}GT$ were used for the second round NT to produce homozygous ${\alpha}GT$ gene-targeted piglets. Results: Monoallelic mutation of ${\alpha}GT$ gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous ${\alpha}GT$ gene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygous ${\alpha}GT$ knockout piglets. Conclusion: The present study demonstrates that the time required for the production of ${\alpha}GT$-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a substantial length of time for breeding.

Effects of a traditional Chinese medicine formula and its extraction on muscle fiber characteristics in finishing pigs, porcine cell proliferation and isoforms of myosin heavy chain gene expression in myocytes

  • Yu, Qin Ping;Feng, Ding Yuan;He, Xiao Jun;Wu, Fan;Xia, Min Hao;Dong, Tao;Liu, Yi Hua;Tan, Hui Ze;Zou, Shi Geng;Zheng, Tao;Ou, Xian Hua;Zuo, Jian Jun
    • Asian-Australasian Journal of Animal Sciences
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    • v.30 no.11
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    • pp.1620-1632
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    • 2017
  • Objective: This study evaluated the effects of a traditional Chinese medicine formula (TCMF) on muscle fiber characteristics in finishing pigs and the effects of the formula's extract (distilled water, ethyl acetate and petroleum ether extraction) on porcine cell proliferation and isoforms of myosin heavy chain (MyHC) gene expression in myocytes. Methods: In a completely randomized design, ninety pigs were assigned to three diets with five replications per treatment and six pigs per pen. The diets included the basal diet (control group), TCMF1 (basal diet+2.5 g/kg TCMF) and TCMF2 (basal diet+5 g/kg TCMF). The psoas major muscle was obtained from pigs at the end of the experiment. Muscle fiber characteristics in the psoas major muscle were analyzed using myosin ATPase staining. Cell proliferation was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye and cytometry. Isoforms of MyHC gene expression were detected by real-time quantitative polymerase chain reaction. Results: The final body weight and carcass weight of finishing pigs were increased by TCMF1 (p<0.05), while the psoas major muscle cross-sectional area was increased by TCMF (p<0.05). The cross-sectional area and diameter of psoas major muscle fiber Ι, IIA, and IIB were increased by TCMF2 (p<0.05). The cross-sectional area and fiber diameter of psoas major muscle fiber IIA and IIB were increased by diet supplementation with TCMF1 (p<0.05). Psoas major muscle fiber IIA and IIB fiber density from the pigs fed the TCMF1 diet and the type IIB fiber density from the pigs fed the TCMF2 diet were lower compared to pigs fed the control diet (p<0.05). Pigs fed TCMF2 had a higher composition of type Ι fiber and a lower percentage of type IIB fiber in the psoas major muscle (p<0.05). The expression levels of MyHC Ι, MyHC IIa, and MyHC IIx mRNA increased and the amount of MyHC IIb mRNA decreased in the psoas major muscle from TCMF2, whereas MyHC Ι and MyHC IIx mRNA increased in the psoas major muscle from TCMF1 (p<0.05). Peroxisome proliferator-activated receptor ${\gamma}$ $coactivator-1{\alpha}$ and CaN mRNA expression in the psoas major muscle were up-regulated by TCMF (p<0.05). Porcine skeletal muscle satellite cell proliferation was promoted by $4{\mu}g/mL$ and $20{\mu}g/mL$ TCMF water extraction (p<0.05). Both $1{\mu}g/mL$ and $5{\mu}g/mL$ of TCMF water extraction increased MyHC IIa, MyHC IIb, and MyHC IIx mRNA expression in porcine myocytes (p<0.05), while MyHC Ι mRNA expression in porcine myocytes was decreased by $5{\mu}g/mL$ TCMF water extraction (p<0.05). Porcine myocyte MyHC Ι and MyHC IIx mRNA expression were increased, and MyHC IIa and MyHC IIb mRNA expression were down-regulated by $5{\mu}g/mL$ TCMF ethyl acetate extraction (p<0.05). MyHC Ι and MyHC IIa mRNA expression in porcine myocytes were increased, and the MyHC IIb mRNA expression was decreased by $1{\mu}g/mL$ TCMF ethyl acetate extraction (p<0.05). Four isoforms of MyHC mRNA expression in porcine myocytes were reduced by $5{\mu}g/mL$ TCMF petroleum ether extraction (p<0.05). MyHC IIa mRNA expression in porcine myocytes increased and MyHC IIb mRNA expression decreased by $1{\mu}g/mL$ in a TCMF petroleum ether extraction (p<0.05). Conclusion: These results indicated that TCMF amplified the psoas major muscle cross-sectional area through changing muscle fiber characteristics in finishing pigs. This effect was confirmed as TCMF extraction promoted porcine cell proliferation and affected isoforms of MyHC gene expression in myocytes.

Stress Patterns in the Reconstructed Double Bundles of the Anterior Cruciate Ligament in Response to an Anterior Tibial Load and Rotatory Load: an Analysis using a 3-Dimensional Finite Element Model (삼차원 유한 요소 모델을 이용한 전방십자인대 이중다발 재건술 후 전방 전위 및 회전 부하에 따른 이식건 응력 양상 분석)

  • Seo, Young-Jin;Song, Si Young;Ahn, Jung Tae;Kim, Yoon-Sang;Ko, Jun Ho;Jang, Seong-Wook;Yoo, Yon-Sik
    • Journal of the Korean Arthroscopy Society
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    • v.16 no.2
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    • pp.160-166
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    • 2012
  • Purpose: The aim of this study was to determine the patterns of the stress distribution within the reconstructed anterior cruciate ligament (ACL) double bundles in response to an anterior tibial load and rotatory load at $45^{\circ}$ flexed knee model by use of a 3-dimensional finite element analysis (FEM). Materials and Methods: The $0^{\circ}$ and $45^{\circ}$ flexed 3-D knee model were reconstructed based on the high resolution computed tomography (CT) images from the right knee of a healthy male subject. To simulate double bundle ACL reconstruction, in $0^{\circ}$ analytic model, four 7 mm diameter tunnels were created at the center of each anteromedial (AM) and posterolateral (PL) footprints on the femur and tibia. The grafts were inserted into the corresponding bone tunnels and then reconstructed knee model was flexed to $45^{\circ}$. As a next step, the 5 mm anterior tibial load and internal rotational load of $10^{\circ}$ were applied on the final Computer aided design (CAD) model. And then stress patterns of each bundle were assessed using a finite element analysis. Results: In response to the 5 mm of anterior tibial load, the AM bundle showed increased stresses around the tibial and femoral attachment sites; especially in the anterior aspect of the bundle. In the PL bundle, the highest stress concentration was also noticed on the anterior aspect of the bundle. Under $10^{\circ}$ internal rotational load, the stress concentration was predominant around the anterior aspect of the tibial attachment site within the AM bundle. The PL bundle also showed highest stress concentration on the anterior aspect of the bundle. Conclusion: Although the stress patterns were not identical among the AM and PL bundle, there were common trends in the stress distribution. The stress concentration was predominant on the anterior aspect of both bundles in response to the anterior tibial load and rotatory load.

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Arthroscopic Osteochondroplasty of Osteochondral Lesion of the Talus (거골 골연골 병변에 대한 관절경적 골연골성형술)

  • Lee, Myung Jin;Kim, Sung Soo;Wang, Lih;Lee, Chul Won;You, Sung Gon;Hwang, Jin Soo
    • Journal of the Korean Arthroscopy Society
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    • v.16 no.2
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    • pp.134-139
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    • 2012
  • Purpose: To report the treatment results of arthroscopic osteochondroplasty in osteochondral lesion of the talus. Materials and Methods: This study included 32 patients who had osteochondral lesion of the talus treated with arthroscopic osteochondroplasty and were able to be followed up over 3 years from 1999 to 2007. The mean follow up period was 48 (38-108) months, and 22 male patients and 10 female patients with a mean age of 34 (20-56) years were included. Locations of lesion were evaluated with simple X-ray and magnetic resonance imaging (MRI), and severities of lesions were classified by Berndt and Harty classification. All cases were treated by arthroscopic debridement or microfracture. The treatment results were assessed by American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot scale and subjective and functional score of Kailkkonen. The group treated by arthroscopic microfracture was compared with the group treated by arthroscopic debridement. Results: The mean AOFAS ankle-hindfoot scale was significantly improved 53 (${\pm}7$) points preoperatively to 84 (${\pm}8$) points postoperatively (P<0.001). The mean subjective and functional score was also improved 55 (${\pm}8$) points preoperatively to 88 (${\pm}8$) points postoperatively with statistical significant (P<0.001). There were no relations between the severity of lesions and the postoperative functional score of ankle (P>0.05). There were no significant differences in the clinical results between the group treated with arthroscopic microfracture and the group treated with arthroscopic debridement (P>0.05). Conclusion: Arthroscopic osteochondroplasty of osteochondral lesion of the talus shows excellent result in terms of patients' satisfaction. There were no significant differences in the clinical results between arthroscopic microfracture group and debridement group.

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Arthroscopic Anterior Debridement and Mini-Open Posterior Resection for Primary Osteoarthritis of the Elbow (주관절 원발성 골성 관절염의 관절경적 전방 변연 절제술 및 최소 절개 후방 절제술)

  • Kim, Young-Kyu;Moon, Sung-Hoon;Cho, Seung-Hyun;Oh, Won-Seok
    • Journal of the Korean Arthroscopy Society
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    • v.16 no.1
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    • pp.40-46
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    • 2012
  • Purpose: This study evaluated the clinical outcome of arthroscopic debridement of anterior compartment and mini-open resection of posterior osteophyte for the treatment of primary osteoarthritis of the elbow. Materials and Methods: Between March 2003 and Feburary 2010, 19 cases who were performed arthroscopic debridement of anterior compartment and resection of posterior osteophyte through mini-open procedure for refractory osteoarthritis of the elbow were enrolled. Average follow-up period was 19 months. Mean age was 49 years old. Clinical results were evaluated by the scoring system of Andrew-Carson Rating Scale (ACRS) and Mayo Elbow Performance Score (MEPS). Results: In the range of motion, flexion contracture was improved from $28.7^{\circ}$ preoperatively to $17.9^{\circ}$ postoperatively, further flexion was improved from $105.1^{\circ}$ to $121.8^{\circ}$. In the scoring system of MEPS, score was improved from 51.1 points preoperatively to 87.9 points in last follow up, 3 cases had in excellent result, 13 good and 3 fair. According to the scoring system of ACRS, score was improved from 92.9 points to 168.2 points, 3 excellent, 14 good and 2 fair. Except one case, all cases returned to preoperative ordinary daily living activity and their own job. Conclusion: For the treatment of refractory osteoarthritis of the elbow, arthroscopic debridement of the anterior compartment and mini-open resection of posterior osteophyte would be helpful on pain relief and functional recovery of the elbow. But this procedure was required long term follow-up in aspect of recurrence of osteophytes and progress of arthritis of the elbow.

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Effect of Release of the Superficial Medial Collateral Ligament in Repair of the Posterior Medial Meniscus Root Tear (내측 반월상 연골 후각 부착부 봉합술 시 표층 내측측부인대 유리술의 효과)

  • Yang, Byung Se;Lee, Dhong Won;Nam, Sang Wook;Ha, Jeong Ku;Kim, Jin Goo
    • Journal of the Korean Arthroscopy Society
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    • v.16 no.2
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    • pp.114-120
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    • 2012
  • Purpose: The purpose of the study was to evaluate the usefulness and the stability of the superficial medial collateral ligament (MCL) release in posterior medial meniscus root repair. Materials and Methods: We compared 20 patients who underwent posterior medial meniscus root repair with superficial MCL preserved (PM) and 32 patients who underwent posterior medial meniscus root repair combined with superficial MCL release (RM) from April 2006 to September 2010. We excluded the patients combined with other surgery. To evaluate the postoperative valgus instability in RM group, we examined direct tenderness on MCL insertion, the subjective feeling of instability and valgus stress test at 3 months and 1 year follow-up. We compared the tourniquet time between PM group and RM group, and the clinical results were assessed by Lysholm score and International Knee Documentation Committee (IKDC) for the usefulness. Results: All patients had no clinically significant complication related to the superficial MCL release. Three months and 1 year follow-up, there were no positive tenderness test, no subjective symptoms and no significant increase of valgus instability although 5 patients examined grade I valgus instability. The mean tourniquet time was $41.3{\pm}12.7$ minutes in RM group and $53.5{\pm}13.6$ minutes in PM group. There was a significant difference in the tourniquet time between the two groups (P<0.05). Average Lysholm score was $56.8{\pm}5.5$ (range, 44-70) preoperatively and $85.1{\pm}5.8$ (range, 77-94) postoperatively in PM group, and was $56.2{\pm}5.4$ (range, 45-67) preoperatively and $87.4{\pm}3.9$ (range, 82-95) postoperatively in RM group (P<0.001). No significant difference of Lysholm score was found in both groups (P<0.05). Average IKDC scores was $42.6{\pm}3.9$ (range, 30-53) preoperatively and $77.2{\pm}6.3$ (range, 68-92) postoperatively in PM group, and was $42.7{\pm}5.7$ (range, 30-53) preoperatively and $89.6{\pm}2.9$ (range, 84-95) postoperatively in RM group (P<0.05). There was also no significant difference of IKDC score in both groups (P<0.05). Conclusion: The superficial MCL release in posterior medial meniscus root repair is useful to gain a wide surgical field and reduces the tourniquet time and does not lead to postoperative valgus instability. It can be considered clinically useful and safe procedure in medial meniscus posterior root repair.

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Comparison of Single-Incision, Single-Bundle Posterior Cruciate Ligament Reconstruction Using Allograft Tendon (동종건을 이용한 단일 절개, 단일 다발 후방십자인대 재건술의 비교)

  • Kyung, Hee-Soo;Cheon, Sang-Ho;Park, Kyung-Hyun;Jung, Jae-Wook
    • Journal of the Korean Arthroscopy Society
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    • v.16 no.2
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    • pp.107-113
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    • 2012
  • Purpose: The purpose of this study was to evaluate the posterior cruciate ligament (PCL) reconstruction with single bundle, single-incision technique using Achilles tendon and tibialis anterior allograft with ligament remnant preservation. Materials and Methods: Twenty six patients underwent PCL reconstruction was included. There were 21 males and 5 females. Mean age was 32 years. Used graft was a fresh frozen Achilles tendon allograft (group I, 14 cases) and tibialis anterior allograft (group II, 12 cases). Arthroscopic PCL reconstruction was performed using transtibial, single-incision and single bundle technique with remnant preserving as possible. For clinical evaluation, range of motion, posterior drawer test, Lysholm score, Tegner activity scale, International Knee Documentation Committee (IKDC) grade and posterior stress radiograph were used. The mean follow-up period was 21.6 months (12-40 months). Associated injuries were 5 medial collateral ligament injuries, which were treated by conservative method. Results: Range of motion (ROM) was returned to normal range in 24 cases, but ROM deficit under $10^{\circ}$ flexion was 2 cases at final follow-up period. Preoperative posterior drawer test was 17 cases in grade II and 9 cases in grade III. At final follow-up 13 cases returned within normal grade, 7 cases grade I and 6 cases grade II posterior instability. Lysholm mean score was improved from preoperatively 62 to 90 at final follow-up period. Tegner activity mean scale improved from preoperatively 3.5 to 5.6 at final follow-up period. IDKC grade was grade A was 3 cases, grade B 17 cases, grade C 6 cases. In posterior stress radiograph, posterior displacement was improved from mean 12 mm preoperative to 4.5 mm at final follow-up. There were no statistical differences between two groups in clinical evaluations. There were two cases of re-rupture of graft at the bone-tendon junction in group I. Conclusion: We had successful results of PCL reconstruction with single-incision, single bundle technique using Achilles and tibialis anterior allograft without difference between two groups in patients with PCL injury. There were more re-rupture of graft in Achilles tendon group.

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Arthroscopic Pull-out Suture Repair of Posterior Root Tear of the Medial Meniscus - Minimum 5 Years Follow-up Results - (내측 반월상 연골판 후방 부착부 파열의 관절경적 견인 봉합술 - 최소 5년 추시결과 -)

  • SaKong, Hyub;Shin, Hong Kwan;Lee, Young-Kook;Bae, Ki Cheor;Cho, Chul Hyun;Lee, Kyung Jae;Son, Eun-Seok;Kim, Doo Han
    • Journal of the Korean Arthroscopy Society
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    • v.16 no.2
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    • pp.153-159
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    • 2012
  • Purpose: This study aims at evaluating our results with a 5-year follow up of arthroscopic pull-out suture repair of posterior root tear of the medial meniscus. Materials and Methods: This study enrolled 30 cases who underwent a arthroscopic pull-out suture repair to treat the posterior root tear of the medial meniscus from January 2001 to May 2005 and followed up at least 5 years. The average follow-up period was 76 months (range, 60-90 months). Clinical results by use of the Lysholm knee score and radiographic grade by use of the Kellgren-Lawrence classification were evaluated and second-look arthroscopy was done in all cases. The clinical results were compared with trauma history, obesity, varus deformity and time to operation. Results: At the last follow up, the Lysholm knee score improved from 55 points to 86 points. For the radiological results, 23 cases displayed maintenance or improvement of the medial joint space on the follow up X-ray, but 7 cases displayed decrease of the medial joint space, and 1 case was lately performed total knee replacement due to progressive osteoarthritic change. Conclusion: Arthroscopic pull-out suture repair has a good result at 27 cases (90%) in minimum 5 years follow-up. The clinical improvement was significantly reduced in more severe varus angle.

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Arthroscopic Management for Pyogenic Arthritis with Positive Culture in the Knee Joint (배양 검사로 증명된 화농성 슬관절염의 관절경적 치료)

  • Baek, Seung-Hoon;Kim, Se Sik
    • Journal of the Korean Arthroscopy Society
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    • v.16 no.2
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    • pp.167-174
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    • 2012
  • Purpose: The purpose of this study is investigation of clinical and functional outcomes in homogeneous group with positive culture after arthroscopic management for pyogenic knee arthritis and analysis of factors affecting those outcomes. Materials and Methods: Thirty-two patients with positive culture after arthroscopic management were included. Mean follow-up period was 41.6 months. Clinical evaluation included death related to infection, recurrence, time to normalize erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), duration of administration of intravenous antibiotics and readmission. Radiographic evaluation was performed according to Kellgren and Lawrence. The prevalence of total knee arthroplasty was investigated and functional evaluation included modified Lysholm, Tegner activity and Korean version of the Western Ontario and McMaster Universities (K-WOMAC) score. Results: Staphylococcus aureus was identified in 21 patients. Time to normalize ESR and CRP was 78.0 and 67.6 days, respectively. Two patients died while there were six recurrences and five readmissions. Rate of recurrence was significantly high in patients with chronic renal failure (P=0.034) and incidence of readmission was associated with higher radiographic grade of osteoarthritis and rate of reoperation (P=0.032 and P=0.006, respectively). At the final follow-up, radiographic grade worsened in 21 patients and was associated with those at first visit. Five arthroplasties were performed. Average modified Lysholm score, Tegner activity score and K-WOMAC score were 53.5, 2.7, 44.2 points, respectively. Conclusion: The severity of osteoarthritis on final radiographs was associated with those at first visit. Patients with higher grade of osteoarthritis at first visit showed higher incidence of readmission and those with chronic renal failure demonstrated higher chances of recurrence.

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Measurement of the Nursing Workload by Patient Classification System in a Secondary Hospital;As a Preliminary Step for Computerization of Nursing Staffing and Scheduling (환자분류에 의한 일개 2차 의료기관의 간호업무량 조사;전산화를 위한 기초작업으로서)

  • Park, Jung-Ho;Joe, Hyon;Park, Hyeoun-Ae;Han, Hye-Rah
    • Journal of Korean Academy of Nursing Administration
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    • v.1 no.1
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    • pp.132-146
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    • 1995
  • Even though Korean medical law stipulates that number of patients attended by a nurse is 2.5 for hospitalization and 30 for ambulatory care, the number of patients cared by a nurse per day is much greater than the standard prescribed by the medical law. Current productivity of nurses is not desirable unless the quality of care is considered. And nursing manpower staffing based on neither current nurses' productivity nor standard of medical law cannot respond properly to dynamic situation of the medical services. Under this background, the necessity of more efficient management of nursing manpower occupying 1/3 of total hospital workers has been recognized by many nursing administrators. Many nursing researchers have studied to foretell the nursing manpower objectively on the basis of measured nursing workload according to patient classification as well. Most of These researches, however, have been conducted in the tertiary hospitals, so it is imperative to conduct other researches to predict necessary nursing manpower in the secondary and the primary hospitals. The study was performed to measure nursing workload and predict pertinent nursing manpower to a secondary hospital with 400beds. Nursing workload was surveyed using measuring tool for direct and indirect care hours in a surgical unit and a medical unit. Survey was conducted from Sep.10 to Sep.16 and from Oct.5 to Oct.11, 1994 respectively by two skilled nurses, Subjects were patients, patients' family members and nursing personnels. Results are follows : 1. Patient classification distributed as 22% of class I (mildly ill patient), 57% of class II (moderately ill patient), and 21% of class III (acutely ill patient) in the medical nursing unit, while 23% of class I, 29% of class II, 12% of class III, and 36% of classIV (critically ill patient) in the surgical nursing unit. There was no difference of inpatient number between weekday and weekend. Bed circulation rate was 89% in both units and average patients number per day was 37.4 (total 42beds) in the medical nursing unit, 32.9 (total 37beds) in the medical nursing unit. 2. Direct care hours per day measured as 2.8hrs for class I, 3.3hrs for class II, and 3.5hrs for class III in the medical nursing unit, while 3.1hrs for class I, 3hrs for class II, 2.7hrs for class III, and 2.2hrs for classIV in the surgical nursing unit. Meanwhile, hours for nursing assistant activities per patient by patients' family members were 11mins and 200mins respectively. Direct care hour rate by shift was day 36%, evening 25%, and night 39% in the medical nursing unit, while 40%, 29%, and and 31% respectively in the surgical nursing unit. 3. Measurement and observation activity held 44.2% of direct care activities of nurses and medication 36.7%, communication 11.7%, exercise 1.8%, treatment 1.3%, hygiene 1.3%, elimination and irrigation 1.1%, suction 1%, nutrition 0.5%, thermotherapy 0.3%, oxygen therapy 0.1% in order. 4. Indirect care hours per day were 294.2mins in the medical nursing unit, and 273.9mins in the surgical nursing unit. By shift, evening was the highest in both units. Indirect care hours for each patient were 44.5mins in the medical nursing unit and 46mins in the surgical nursing unit. 5. checking activities including doctor's order, medication, and delivering patients to the next shift occupied 39.7% of indirect care activities, and preparation 26%, recording 23.8%, communication and conference 6.7%, managing equipments 2.1%, messenger activity 1.7% in order. 6. On the ground of these results, nursing manpower needed in a secondary hospital was estimated ; 27 nursing personnels for the medical nursing unit of 37beds, and 20 nursing personnels for the surgical nursing unit of 33beds.

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