• Journal of Veterinary Science
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• v.21 no.1
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• pp.9.1-9.15
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• 2020

Regenerative therapy holds great promise in the development of cures of some untreatable diseases such as cardiovascular diseases, and pluripotent stem cells (PSCs) including induced PSCs (iPSCs) are the most important regenerative seed cells. Recently, differentiation of human PSCs into functional tissues and cells in vitro has been widely reported. However, although porcine reports are rare they are quite essential, as the pig is an important animal model for the in vitro generation of human organs. In this study, we reprogramed porcine embryonic fibroblasts into porcine iPSCs (piPSCs), and differentiated them into cluster of differentiation 31 (CD31)-positive endothelial cells (ECs) (piPSC-derived ECs, piPS-ECs) using an optimized single-layer culture method. During differentiation, we observed that a combination of GSK3β inhibitor (CHIR99021) and bone morphogenetic protein 4 (BMP4) promoted mesodermal differentiation, resulting in higher proportions of CD31-positive cells than those from separate CHIR99021 or BMP4 treatment. Importantly, the piPS-ECs showed comparable morphological and functional properties to immortalized porcine aortic ECs, which are capable of taking up low-density lipoprotein and forming network structures on Matrigel. Our study, which is the first trial on a species other than human and mouse, has provided an optimized single-layer culture method for obtaining ECs from porcine PSCs. Our approach can be beneficial when evaluating autologous EC transplantation in pig models.

• Korean Journal of Radiology
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• v.22 no.5
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• pp.782-791
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• 2021

Objective: To evaluate the signal intensity of the periosteum using ultrashort echo time pulse sequence with three-dimensional cone trajectory (3D UTE) with or without fat suppression (FS) to distinguish from artifacts in porcine tibias. Materials and Methods: The periosteum and overlying soft tissue of three porcine lower legs were partially peeled away from the tibial cortex. Another porcine tibia was prepared as three segments: with an intact periosteum outer and inner layer, with an intact periosteum inner layer, and without periosteum. Axial T1 weighted sequence (T1 WI) and 3D UTE (FS) were performed. Another porcine tibia without periosteum was prepared and subjected to 3D UTE (FS) and T1 WI twice, with positional changes. Two radiologists analyzed images to reach a consensus. Results: The three periosteal tissues that were partially peeled away from the cortex showed a high signal in 3D UTE (FS) and low signal on T1 WI. 3D UTE (FS) showed a high signal around the cortical surface with an intact outer and inner periosteum, and subtle high signals, mainly around the upper cortical surfaces with the inner layer of the periosteum and without periosteum. T1 WI showed no signal around the cortical surfaces, regardless of the periosteum state. The porcine tibia without periosteum showed changes in the high signal area around the cortical surface as the position changed in 3D UTE (FS). No signal was detected around the cortical surface in T1 WI, regardless of the position change. Conclusion: The periosteum showed a high signal in 3D UTE and 3D UTE FS that overlapped with artifacts around the cortical bone.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.39 no.3
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• pp.120-126
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• 2013

Objectives: This study sought to elucidate the effect of autogenous tooth bone material by experimenting on minipig's maxillary sinus and performing histological and histomorphometric analyses. Materials and Methods: Five 18-24 month-old male minipigs were selected, and right maxillary sinuses were grafted with bone graft material made of their respective autogenous teeth extracted eight weeks earlier. The left sides were grafted with synthetic hydroxyapatite as control groups. All minipigs were sacrificed at 12 weeks after bone graft, which was known to be 1 sigma (${\sigma}$) period for pigs. Specimens were evaluated histologically under a light microscope after haematoxylin-eosin staining followed by semi-quantitative study via histomorphometric analysis. The ratio of new bone to total area was evaluated using digital software for calculation of area. Results: All specimens were available, except one on the right side (experimental group), which was missing during specimen preparation. This study demonstrated new bone at the periphery of the existing bone in both groups, showing evidence of bone remodeling, however, encroachment of new bone on the central part of the graft at the 1 ${\sigma}$ period was observed only in the autogenous tooth bone group (experimental group). Histomorphometric analysis showed more new bone formation in the experimental group compared to the control group. Although the difference was not statistically significant (P>0.05), the mean percentage area for new bone for the experimental and control groups were $57.19%{\pm}11.16%$ and $34.07%{\pm}13.09%$, respectively. Conclusion: The novel bone graft material using autogenous tooth is a good alternative to autogenous bone, comparable to autogenous bone, and outperforming synthetic hydroxyapatite bone graft materials in terms of bone regeneration capacity. Augmentation with autogenous tooth bone materials will reduce donor site morbidity without hampering the safety of the autogenous bone graft.

• Advances in biomechanics and applications
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• v.1 no.1
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• pp.15-22
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• 2014

Computed tomography (CT) is being utilized in orthopaedics and related research to estimate bone strength. These applications benefit from calibration of Hounsfield units to mineral density typical of long bone, up to $1750mg/cm^3$. This study describes a method for establishing repeatable calibration of Hounsfield units to density, and determines the effects of imaging medium on calibration accuracy. Four hydroxyapatite standards were imaged in air on 7 occasions over 19 weeks using a helical multi-slice CT scanner. Each standard was scanned 5 times in different media: porcine soft tissue, water, and air. Calibrated densities were highly repeatable (CV<3.5%). No difference in density was observed between water and soft tissue conditions (p>0.08). This work provides a model for determining repeatable scanner-specific density calibration, demonstrates that the linear relationship between Hounsfield units and density extends to values typical of cortical bone, and supports the practice of imaging calibration standards in an environment similar to that of the target bone.

• Journal of Embryo Transfer
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• v.27 no.3
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• pp.183-187
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• 2012

Adult stem cell transplantation has been increased every year, because of the lack of organ donors for regenerative medicine. Therefore, development of reliable and safety cryopreservation and bio-baking method for stem cell therapy is urgently needed. The present study investigated safety of dimethyl sulfoxide (DMSO) such as common cryoprotectant on porcine bone marrow derived mesenchymal stem cells (pBM-MSCs) by evaluating the activation of Caspase-3 and -7, apoptosis related important signal pathway. pBM-MSCs used for the present study were isolated density gradient method by Ficoll-Paque Plus and cultured in A-DMEM supplemented 10% FBS at $38.5^{\circ}C$ in 5% $CO_2$ incubator. pBM-MSCs were cryopreserved in A-DMEM supplemented either with 5%, 10% or 20% DMSO by cooling rate at $-1^{\circ}C$/min in a Kryo 360 (planner 300, Middlesex, UK) and kept into $LN_2$. Survival rate of cells after thawing did not differ between 5% and 10% DMSO but was lowest in 20% DMSO by 0.4% trypan blue exclusion. Activation of Caspase-3 and -7 by Vybrant FAM Caspase-3 and -7 Assay Assay Kit (Molecular probes, Inc.OR, USA) was analyzed with a flow cytometer. Both of cryopreserved and control groups (fresh pBM-MSCs) were observed after the activation of Caspase-3 and -7. The activation did not differ between 5% and 10% DMSO, but was observed highest in 20% DMSO. Therefore 5% DMSO can be possibly used for cell cryopreservation instead of 10% DMSO.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.43 no.6
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• pp.373-387
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• 2017

Objectives: The purpose of this study was to introduce our three experiments on bone morphogenetic protein (BMP) and its carriers performed using the critical sized segmental defect (CSD) model in rat fibula and to investigate development of animal models and carriers for more effective bone regeneration. Materials and Methods: For the experiments, 14, 16, and 24 rats with CSDs on both fibulae were used in Experiments 1, 2, and 3, respectively. BMP-2 with absorbable collagen sponge (ACS) (Experiments 1 and 2), autoclaved autogenous bone (AAB) and fibrin glue (FG) (Experiment 3), and xenogenic bone (Experiment 2) were used in the experimental groups. Radiographic and histomorphological evaluations were performed during the follow-up period of each experiment. Results: Significant new bone formation was commonly observed in all experimental groups using BMP-2 compared to control and xenograft (porcine bone) groups. Although there was some difference based on BMP carrier, regenerated bone volume was typically reduced by remodeling after initially forming excessive bone. Conclusion: BMP-2 demonstrates excellent ability for bone regeneration because of its osteoinductivity, but efficacy can be significantly different depending on its delivery system. ACS and FG showed relatively good bone regeneration capacity, satisfying the essential conditions of localization and release-control when used as BMP carriers. AAB could not provide release-control as a BMP carrier, but its space-maintenance role was remarkable. Carriers and scaffolds that can provide sufficient support to the BMP/carrier complex are necessary for large bone defects, and AAB is thought to be able to act as an effective scaffold. The CSD model of rat fibula is simple and useful for initial estimate of bone regeneration by agents including BMPs.

• The Journal of the Korean bone and joint tumor society
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• v.1 no.1
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• pp.7-16
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• 1995

In countries where confucianism is popular, it is extremely hard to get fresh cadaver bone for allograft. Therefore in Korea, the reimplantation of resected autoclaved autogenous bone segments has been increasingly performed for limb reconstruction of extremities with malignancies. To preserve the bone morphogenetic protein and mechanical strength of heated bone, many studies have reported that pasteurization of bone is far better than autoclaving over $100^{\circ}C$. Based on this assumption, replacement with a pasteurized autogenous bone graft after resection of a malignant bone tumor was deemed feasible for reconstruction. However, little is known about how high a temperature and how much time for pasteurization is needed to make tumors completely necrotic and to maintain the mechanical strength of bone. Consequantly, experimental studies were carried out to test heat conductivity of human bone and torsional strength of porcine tibia after pasteurization. First, two pairs of human proximal tibia and distal femur were used. We used T-type thermocoples to check core temperature of the bone and a computerized data acquisition system to record results. Without reaming of the medullary cavity, in a $60^{\circ}C$-thermostatic saline tub, it took 32 minutes and 50 seconds to raise the core temperature of human proximal tibia from $20^{\circ}C$ to $58^{\circ}C$, and 30 minutes for distal femur. In a $80^{\circ}C$ saline tub, it took 12 minutes and 50 seconds for proximal tibia, and 11 minutes and 10 seconds for distal femur. In contrast, using porcine tibia whose cortical thickness is similar to that of human tibia, after reaming of the medullary canal, it took less than 3 minutes and 30 seconds in a $60^{\circ}C$ saline tub, less than 1 minute and 45 seconds in a $70^{\circ}C$ tub, and less than 1 minute in a $80^{\circ}C$ tub to elevate core temperature from $20^{\circ}C$ to $58^{\circ}C$. Second, based on data of the heat conductivity test, we compared the torsional strength before and after pasteurization. Twenty matched pairs of porcine tibia were used, The left one was used as a non-heated control group and the right one as a pasteurized experimental group. Wighout reaming of the medullary cavity, there was no statistical difference in torsional strength between the pasteurization of the $60^{\circ}C$-35minute and of $80^{\circ}C$-15minute. With reaming, we also found no statistical difference among pasteurization of $60^{\circ}C$-15 minute, of $70^{\circ}C$-15 minute, and of $80^{\circ}C$-15 minute groups. In conclusion, reaming of the medullary canal is very helpful in saving pasteurization time. And, in a $60^{\circ}C$ saline tub, no significant weakness in torsional strength occurs with pasteurization of the bone for up to 35 minutes. Also no significant weakness in torsional strength occurs with an exposure of 15 minutes to the $80^{\circ}C$ saline tub.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.31 no.3
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• pp.198-206
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• 2009

Purpose: The aims of this study were to assess the in vitro co-culturing pattern of isolated skin-derived precursor cells (SKPs) with a mixed demineralized bone (DMB) and fibrin glue scaffold and to evaluate in vivo osteogenesis after transplantation of autogenous SKPs with a these mixed scaffold in the animal's mandibular defects. Materials and Methods: We isolated SKPs from the ears of adult 4 miniature pigs. The isolated SKPs were co-cultured with a mixed DMB and fibrin glue scaffold in a non-osteogenic medium for 1, 2, and 4 weeks. Histological characteristics of in vitro co-cultured cells and scaffold were evaluated. $1{\times}10^7\;cells/100\;{\mu}l$ of autogenous porcine SKPs were grafted into the mandibular defects with a DMB and fibrin glue scaffold. In the control sites, only a scaffold was grafted, without SKPs. After two animals each were euthanized at 2 and 4 weeks after grafting, the in vivo osteogenesis was evaluated with histolomorphometric and osteocalcin immunohistochemical studies. Results: Homogeneously shaped skin-derived cells were isolated from porcine ear skin after 3 or 4 weeks of primary culture. In vitro osteogenic differentiation of SKPs was observed after co-culturing with a DMB and fibrin glue scaffold in a non-osteogenic medium. Von Kossa-positive bone minerals were also noted in the co-cultured medium at 4 weeks. As the culture time progressed, the number of observable cells increased. Trabecular new bone formation and osteocalcin expression were more pronounced in the SKP-grafted group compared to the control group. Conclusion: These findings suggest that autogenous SKP grafting with a DMB and fibrin glue scaffold can serve as a useful alternative to bone grafting technique.

• Journal of Periodontal and Implant Science
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• v.40 no.5
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• pp.232-238
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• 2010

Purpose: To prolong the degradation time of collagen membranes, various cross-linking techniques have been developed. For cross-linking, chemicals such as formaldehyde and glutaraldehyde are added to collagen membranes, but these chemicals could adversely affect surrounding tissues. The aim of this study is to evaluate the ability of porous non-chemical cross-linking porcine-derived collagen nanofibrous membrane to enhance bone and associated tissue regeneration in one-wall intrabony defects in beagle dogs. Methods: The second and third mandibular premolars and the first molars of 2 adult beagles were extracted bilaterally and the extraction sites were allowed to heal for 10 weeks. One-wall intrabony defects were prepared bilaterally on the mesial and distal side of the fourth mandibular premolars. Among eight defects, four defects were not covered with membrane as controls and the other four defects were covered with membrane as the experimental group. The animals were sacrificed 10 weeks after surgery. Results: Wound healing was generally uneventful. For all parameters evaluating bone regeneration, the experimental group showed significantly superior results compared to the control. In new bone height (NBh), the experimental group exhibited a greater mean value than the control ($3.04{\pm}0.23\;mm/1.57{\pm}0.59$, P=0.003). Also, in new bone area (NBa) and new bone volume (NBv), the experimental group showed superior results compared to the control (NBa, $34.48{\pm}10.21%$ vs. $5.09{\pm}5.76%$, P=0.014; and NBv, $28.04{\pm}12.96$ vs. $1.55{\pm}0.57$, P=0.041). On the other hand, for parameters evaluating periodontal tissue regeneration, including junctional epithelium migration and new cementum height, there were no statistically significant differences between two groups. Conclusions: Within the limitations of this study, this collagen membrane enhanced bone regeneration at one-wall intrabony defects. On the other hand, no influence of this membrane on periodontal tissue regeneration could be ascertained in this study.

• Journal of Periodontal and Implant Science
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• v.25 no.1
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• pp.67-75
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• 1995

The progress of periodontal disease and the wound healing process after treatment result in alveolar bone bone change. So, detection of it is very important in the diagnosis and the radiograph of periodontal disease. Various effects have been made to assess the subtle alveolar bone change and digital subtraction radiography (DSR) has been reported to be the best method in evaluating it qualitatively and quantitatively. The present study was performed to estimate the detectable alveolar bone change qualitatively with digital subtraction radiography. For the in vitro study, 10 intraoral standard radiographs were taken from porcine dry mandible which a rectangular cortical bone chip of 0.1mm to 1.0mm thickness with 0.1mm increment was attached on the buccal surface. The radiographs without and with bone plates were reviewed at the same time by 10 observers and requested to detect the presence of cortical bone plates. Digital Subtraction radiograph was reviewed subsequently by using the DSR system(digital converter-256 grey-levels,DT 2851,Data Translation Co., U.S.A;IBM 386 ; CCD camera, FOTOVIX, Tamrom Co., Japan). The detectable thickness of cortical bone plate was O.4mm on the intraoral radiograph and 0.2mm on the subtaction images. For the human study, radiographs were taken from patients by using intraoral film holding device and aluminum reference wedge before and 3 month after bone graft and 1 week after osteoplasty. The grey level change was estimated in the subtraction images and calculated to aluminum equivalent thickness. The grey level of the grafted site was higher that that of healthy controls. Average grey levels of change on healthy controls were O.48mm aluminum equivalent. However, the amount of changes in grafted sites were 1.87mm aluminum thickness equivalent and in the site of osteoplasty were -1.49mm aluminum thickness equivalent. In conclusion, digital subtraction radiography was more effective in detecting as subtle change of alveolar bone than intraoral standard radiography. With the aid of quantitative analysis of digital subtraction radiography, alveolar bone resorption of apposition can be estimated during diagnosis and treatment of periodontally diseased patients.