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Stem Cells Seeded on Multilayered Scaffolds Implanted into an Injured Bladder Rat Model Improves Bladder Function

Kshitiz Raj SHRESTHA ; Seung Hwan JEON ; Ae Ryang JUNG ; In Gul KIM ; Ga Eun KIM ; Yong Hyun PARK ; Soo Hyun KIM ; Ji Youl LEE

Tissue Engineering and Regenerative Medicine.2019;16(2):201-212. doi:10.1007/s13770-019-00187-x

BACKGROUND: To investigate whether human adipose-derived stem cells (hADSCs) seeded on multilayered poly (L-lactide-co-ε-caprolactone) (PLCL) sheets improve bladder function in a rat model of detrusor smooth muscle-removed bladder. METHODS: Male rats were randomly divided into 4 groups: Normal, injury (detrusor smooth muscle-removed bladder), PLCL (detrusor smooth muscle-removed bladder implanted with PLCL sheets), and PLCL + ADSC (detrusor smooth muscle-removed bladder implanted with PLCL sheets seeded with hADSCs). Four weeks after the treatment, physiological, histological, immunohistochemical, and immunoblot analyses were performed. RESULTS: hADSCs were compatible with PLCL sheets. Further, the physiological study of PLCL + ADSC group showed significant improvement in compliance and contractility suggesting the functional improvement of the bladder. Histological, immunohistochemical and immunoblot analyses revealed the uniform distribution of hADSCs in between PLCL sheets as well as differentiation of hADSCs into smooth muscle cells (SMC) which is illustrated by the expression of SMC markers. CONCLUSION: hADSCs seeded on the multilayered PLCL sheets has the potential to differentiate into SMC, thus facilitating the recovery of compliance and contractility of the injured bladder.
Animals ; Clothing ; Compliance ; Humans ; Male ; Models, Animal ; Muscle, Smooth ; Myocytes, Smooth Muscle ; Rats ; Stem Cells ; Tissue Engineering ; Urinary Bladder

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Treatment of Periodontal Bone Defects with Stem Cells from Inflammatory Dental Pulp Tissues in Miniature Swine

Ye LI ; Xi NAN ; Tian Yu ZHONG ; Tong LI ; Ang LI

Tissue Engineering and Regenerative Medicine.2019;16(2):191-200. doi:10.1007/s13770-018-00175-7

BACKGROUND: Containing a certain proportion of mesenchymal stem cells, inflammatory dental tissue showed great tissue regeneration potential in recent years. However, whether it is applicable to promote tissue regeneration in vivo remains to be elucidated. Therefore, we evaluated the feasibility of stem cells from inflammatory dental pulp tissues (DPSCs-IPs) to reconstruct periodontal defects in miniature pigs. METHODS: The autologous pig DPSCs-IPs were first cultured, appraised and loaded onto β-tricalcium phosphate (β-TCP). The compounds were then engrafted into an artificially-created periodontal defect. Three months later, the extent of periodontal regeneration was evaluated. Clinical examination, radiological examination and immunohistochemical staining were used to assess periodontal regeneration. RESULTS: The data collectively showed that DPSCs-IPs from miniature pigs expressed moderate to high levels of STRO-1 and CD146 as well as low levels of CD34 and CD45. DPSCs-IPs have osteogentic, adipogenic and chondrogenic differentiation abilities. DPSCs-IPs were engrafted onto β-TCP and regenerated bone to repair periodontal defects by 3 months' post-surgical reconstruction. CONCLUSION: Autologous DPSCs-IPs may be a feasible means of periodontal regeneration in miniature pigs.
Dental Pulp ; Mesenchymal Stromal Cells ; Periodontitis ; Regeneration ; Stem Cells ; Swine ; Swine, Miniature

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D-RADA16-RGD-Reinforced Nano-Hydroxyapatite/Polyamide 66 Ternary Biomaterial for Bone Formation

WeiKang ZHAO ; Bin HE ; Ao ZHOU ; Yuling LI ; Xiaojun CHEN ; Qiming YANG ; Beike CHEN ; Bo QIAO ; Dianming JIANG

Tissue Engineering and Regenerative Medicine.2019;16(2):177-189. doi:10.1007/s13770-018-0171-5

BACKGROUND: Nano-hydroxyapatite/polyamide 66 (nHA/PA66) is a composite used widely in the repair of bone defects. However, this material is insufficient bioactivity. In contrast, D-RADA16-RGD self-assembling peptide (D-RADA16-RGD sequence containing all D-amino acids is Ac-RADARADARADARADARGDS-CONH2) shows admirable bioactivity for both cell culture and bone regeneration. Here, we describe the fabrication of a favorable biomaterial material (nHA/PA66/D-RADA16-RGD). METHODS: Proteinase K and circular dichroism spectroscopy were employed to test the stability and secondary structural properties of peptide D-RADA16-RGD respectively. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the surface of these materials. Confocal laser scanning (CLS), cell counting kit-8 tests (CCK-8), alizarin red S staining, cell immunofluorescence analysis and Western blotting were involved in vitro. Also biosafety and bioactivity of them have been evaluated in vivo. RESULTS: Proteinase K and circular dichroism spectroscopy demonstrated that D-RADA16-RGD in nHA/PA66 was able to form stable-sheet secondary structure. SEM and TEM showed that the D-RADA16-RGD material was 7–33 nm in width and 130–600 nm in length, and the interwoven pore size ranged from 40 to 200 nm. CLS suggests that cells in nHA/PA66/D-RADA16-RGD group were linked to adjacent cells with more actin filaments. CCK-8 analysis showed that nHA/PA66/D-RADA16-RGD revealed good biocompatibility. The results of Alizarin-red S staining and Western blotting as well as vivo osteogenesis suggest nHA/PA66/D-RADA16-RGD exhibits better bioactivity. CONCLUSION: This study demonstrates that our nHA/PA66/D-RADA16-RGD composite exhibits reasonable mechanical properties, biocompatibility and bioactivity with promotion of bone formation.
Actin Cytoskeleton ; Blotting, Western ; Bone Regeneration ; Cell Count ; Cell Culture Techniques ; Circular Dichroism ; Endopeptidase K ; Fluorescent Antibody Technique ; In Vitro Techniques ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Osteogenesis ; Sincalide ; Spectrum Analysis

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Differentiation Capacity of Monocyte-Derived Multipotential Cells on Nanocomposite Poly(e-caprolactone)-Based Thin Films

Iro KOLIAKOU ; Eleni GOUNARI ; Maria NERANTZAKI ; Eleni PAVLIDOU ; Dimitrios BIKIARIS ; Martha KALOYIANNI ; George KOLIAKOS

Tissue Engineering and Regenerative Medicine.2019;16(2):161-175. doi:10.1007/s13770-019-00185-z

BACKGROUND: Lonocyte-derived multipotential cells (MOMCs) include progenitors capable of differentiation into multiple cell lineages and thus represent an ideal autologous transplantable cell source for regenerative medicine. In this study, we cultured MOMCs, generated from mononuclear cells of peripheral blood, on the surface of nanocomposite thin films. METHODS: For this purpose, nanocomposite Poly(e-caprolactone) (PCL)-based thin films containing either 2.5 wt% silica nanotubes (SiO2ntbs) or strontium hydroxyapatite nanorods (SrHAnrds), were prepared using the spin-coating method. The induced differentiation capacity of MOMCs, towards bone and endothelium, was estimated using flow cytometry, real-time polymerase chain reaction, scanning electron microscopy and fluorescence microscopy after cells' genetic modification using the Sleeping Beauty Transposon System aiming their observation onto the scaffolds. Moreover, Wharton's Jelly Mesenchymal Stromal Cells were cultivated as a control cell line, while Human Umbilical Vein Endothelial Cells were used to strengthen and accelerate the differentiation procedure in semi-permeable culture systems. Finally, the cytotoxicity of the studied materials was checked with MTT assay. RESULTS: The highest differentiation capacity of MOMCs was observed on PCL/SiO2ntbs 2.5 wt% nanocomposite film, as they progressively lost their native markers and gained endothelial lineage, in both protein and transcriptional level. In addition, the presence of SrHAnrds in the PCL matrix triggered processes related to osteoblast bone formation. CONCLUSION: To conclude, the differentiation of MOMCs was selectively guided by incorporating SiO2ntbs or SrHAnrds into a polymeric matrix, for the first time.
Autografts ; Beauty ; Cell Line ; Cell Lineage ; Durapatite ; Endothelium ; Flow Cytometry ; Human Umbilical Vein Endothelial Cells ; Mesenchymal Stromal Cells ; Methods ; Microscopy, Electron, Scanning ; Microscopy, Fluorescence ; Nanocomposites ; Nanotubes ; Osteoblasts ; Osteogenesis ; Polymers ; Real-Time Polymerase Chain Reaction ; Regenerative Medicine ; Silicon Dioxide ; Strontium ; Wharton Jelly

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Characterization of Tendon-Specific Markers in Various Human Tissues, Tenocytes and Mesenchymal Stem Cells

Chris Hyunchul JO ; Hyun Ju LIM ; Kang Sup YOON

Tissue Engineering and Regenerative Medicine.2019;16(2):151-159. doi:10.1007/s13770-019-00182-2

BACKGROUND: Unlike bone, cartilage, or muscle, tendon-specific markers are not well established. The purpose of the study was to investigate expression pattern and level of 6 well-known tendon-specific markers, in various human musculoskeletal tissues, tenocytes, and mesenchymal stem cells (MSCs). METHODS: Musculoskeletal tissue samples of tendon, bone, cartilage, nerve, muscle, and fat were obtained from patients undergoing orthopedic surgery. Tenocytes, MSCs from bone marrow, adipose tissue, and umbilical cord were isolated from each tissue and cultured. Six tendon-specific markers, scleraxis (Scx), tenomodulin (TNMD), thrombospondin-4 (TSP-4), tenascin-C (TNC), type I collagen (Col I), and type III collagen (Col III) were investigated in tendon tissue, tenocytes, and MSCs. RESULTS: mRNA levels of 6 tendon-specific markers were significantly higher in tendon tissue that in other connective tissues levels of Scx, TNMD, TSP-4, and Col III immediately decreased after plating tenocytes in culture dishes whereas those of TNC and Col I did not. In comparison with tendon tissue, mRNA levels pattern of Scx, TNMD, and TSP-4 in tenocytes were significantly higher than that in MSCs, but lower than in tendon tissue whereas expression pattern of TNC, Col I and III showed different pattern with each other. CONCLUSION: This study demonstrated that 6 commonly used tendon-specific markers were mainly expressed in tendon tissue, but that expression level and pattern of the tendon-specific markers with respect to kinds of tissues, culture duration of tenocytes and sources of MSCs.
Adipose Tissue ; Biomarkers ; Bone Marrow ; Cartilage ; Collagen Type I ; Collagen Type III ; Connective Tissue ; Humans ; Mesenchymal Stromal Cells ; Orthopedics ; RNA, Messenger ; Tenascin ; Tendons ; Umbilical Cord

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Conditioned Medium Enhances Osteogenic Differentiation of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells

Siying ZHONG ; Xufeng HE ; Yuexia LI ; Xiangxin LOU

Tissue Engineering and Regenerative Medicine.2019;16(2):141-150. doi:10.1007/s13770-018-0173-3

BACKGROUND: Recent studies have shown that induced pluripotent stem cells (iPSCs) could be differentiated into mesenchymal stem cells (MSCs) with notable advantages over iPSCs per se. In order to promote the application of iPSC-MSCs for osteoregenerative medicine, the present study aimed to assess the ability of murine iPSC-MSCs to differentiate into osteoblast phenotype. METHODS: Osteogenic differentiation medium, blending mouse osteoblast-conditioned medium (CM) with basic medium (BM) at ratio 3:7, 5:5 and 7:3, were administered to iPSC-MSCs, respectively. After 14 days, differentiation was evaluated by lineage-specific morphology, histological stain, quantitative reverse transcription-polymerase chain reaction and immunostaining. RESULTS: The osteogenesis-related genes, alp, runx2, col1 and ocn expressions suggest that culture medium consisting of CM:BM at the ratio of 3:7 enhanced the osteogenic differentiation more than other concentrations that were tested. In addition, the alkaline phosphatase activity and osteogenic marker Runx2 expression demonstrate that the combination of CM and BM significantly enhanced the osteogenic differentiation of iPSC-MSCs. CONCLUSION: In summary, this study has shown that osteoblast-derived CM can dramatically enhance osteogenic differentiation of iPSC-MSCs toward osteoblasts. Results from this work will contribute to optimize the osteogenic induction conditions of iPSC-MSCs and will assist in the potential application of iPSC-MSCs for bone tissue engineering.
Alkaline Phosphatase ; Animals ; Bone and Bones ; Culture Media, Conditioned ; Induced Pluripotent Stem Cells ; Mesenchymal Stromal Cells ; Mice ; Osteoblasts ; Phenotype

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Proliferation Profile of Uterine Endometrial Stromal Cells during In Vitro Culture with Gonadotropins: Recombinant versus Urinary Follicle Stimulating Hormone

Yong Jin KIM ; Yoon Young KIM ; Da Young SONG ; Sang Hoon LEE ; Chan Woo PARK ; Hoon KIM ; Seung Yup KU

Tissue Engineering and Regenerative Medicine.2019;16(2):131-139. doi:10.1007/s13770-018-0156-4

BACKGROUND: Provision of optimal endometrial stromal cells is essential in uterine tissue engineering. Culture of these cells is significantly influenced by gonadotropin hormones. This investigation attempted to define the proliferation profiles of murine uterine endometrial stromal cells during in vitro culture with recombinant follicle stimulating hormone (rFSH), urinary follicle stimulating hormone (uFSH), and human chorionic gonadotropin (hCG). METHODS: Murine uterine endometrial stromal cells were collected from 8-week-old mice and cultured in vitro up to 72 h, with rFSH, uFSH, or hCG. Cell cycles were analyzed by BrdU assay, and cyclin D1 expression was evaluated according to dose and duration of gonadotropin treatment. RESULTS: BrdU assay showed a further inhibitory effect on murine uterine endometrial stromal cell proliferation when cultured with rFSH compared to uFSH, and a similar inhibitory proliferation profile when cultured with hCG at a specific range of concentrations. The expression of cyclin D1 of murine uterine endometrial stromal cells was down-regulated when cultured with rFSH, uFSH, or hCG, compared to control. CONCLUSIONS: FSH may inhibit the proliferation of murine uterine endometrial stromal cells during in vitro culture. rFSH may have more significant inhibitory effects on the proliferation of endometrial stromal cells than uFSH. Establishing an optimal endocrine milieu is necessary using more advanced combination of female hormones for in vitro culture of this type of cells.
Animals ; Bromodeoxyuridine ; Cell Cycle ; Chorionic Gonadotropin ; Cyclin D1 ; Female ; Follicle Stimulating Hormone ; Gonadotropins ; Humans ; In Vitro Techniques ; Mice ; Stromal Cells ; Tissue Engineering ; Uterus

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Efficient Production of Murine Uterine Damage Model

Yoon Young KIM ; Bo Bin CHOI ; Ji Won LIM ; Yong Jin KIM ; Sung Yob KIM ; Seung Yup KU

Tissue Engineering and Regenerative Medicine.2019;16(2):119-129. doi:10.1007/s13770-018-0149-3

BACKGROUND: Thin or damaged endometrium causes uterine factor-derived infertility resulting in a failure of embryonic implantation. Regeneration of endometrium is a major issue in gynecology and reproductive medicine. Various types of cells and scaffolds were studied to establish an effective therapeutic strategy. For this type of investigations, production of optimal animal models is indispensable. In this study, we tried to establish various murine uterine damage models and compared their features. METHODS: Three to ten-week-old C57BL/6 female mice were anesthetized using isoflurane. Chemical and mechanical methods using ethanol (EtOH) at 70 or 100% and copper scraper were compared to determine the most efficient condition. Damage of uterine tissue was induced either by vaginal or dorsal surgical approach. After 7-10 days, gross and microscopic morphology, safety and efficiency were compared among the groups. RESULTS: Both chemical and mechanical methods resulted in thinner endometrium and reduced number of glands. Gross morphology assessment revealed that the damaged regions of uteri showed various shapes including shrinkage or cystic dilatation of uterine horns. The duration of anesthesia significantly affected recovery after procedure. Uterine damage was most effectively induced by dorsal approach using 100% EtOH treatment compared to mechanical methods. CONCLUSION: Taken together, murine uterine damage models were most successfully established by chemical treatment. This production protocols could be applied further to larger animals such as non-human primate.
Anesthesia ; Animals ; Copper ; Dilatation ; Endometrium ; Ethanol ; Female ; Gynecology ; Horns ; Humans ; Infertility ; Isoflurane ; Mice ; Models, Animal ; Primates ; Regeneration ; Reproductive Medicine ; Uterus

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Stem Cell-Based Therapies for Liver Diseases: An Overview and Update

Jie WANG ; Meiyan SUN ; Wei LIU ; Yan LI ; Miao LI

Tissue Engineering and Regenerative Medicine.2019;16(2):107-118. doi:10.1007/s13770-019-00178-y

BACKGROUND: Liver disease is one of the top causes of death globally. Although liver transplantation is a very effective treatment strategy, the shortage of available donor organs, waiting list mortality, and high costs of surgery remain huge problems. Stem cells are undifferentiated cells that can differentiate into a variety of cell types. Scientists are exploring the possibilities of generating hepatocytes from stem cells as an alternative for the treatment of liver diseases. METHODS: In this review, we summarized the updated researches in the field of stem cell-based therapies for liver diseases as well as the current challenges and future expectations for a successful cell-based liver therapy. RESULTS: Several cell types have been investigated for liver regeneration, such as embryonic stem cells, induced pluripotent stem cells, liver stem cells, mesenchymal stem cells, and hematopoietic stem cells. In vitro and in vivo studies have demonstrated that stem cells are promising cell sources for the liver regeneration. CONCLUSION: Stem cell-based therapy could be a promising therapeutic method for patients with end-stage liver disease, which may alleviate the need for liver transplantation in the future.
Cause of Death ; Embryonic Stem Cells ; Hematopoietic Stem Cells ; Hepatocytes ; Humans ; In Vitro Techniques ; Induced Pluripotent Stem Cells ; Liver Diseases ; Liver Regeneration ; Liver Transplantation ; Liver ; Mesenchymal Stromal Cells ; Methods ; Mortality ; Stem Cells ; Tissue Donors ; Waiting Lists

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Repair of Osteochondral Defects in Rabbit Knee Using Menstrual Blood Stem Cells Encapsulated in Fibrin Glue: A Good Stem Cell Candidate for the Treatment of Osteochondral Defects

Manijeh KHANMOHAMMADI ; Hannaneh GOLSHAHI ; Zahra SAFFARIAN ; Samaneh MONTAZERI ; Somaye KHORASANI ; Somaieh KAZEMNEJAD

Tissue Engineering and Regenerative Medicine.2019;16(3):311-324. doi:10.1007/s13770-019-00189-9

BACKGROUND: In recent years, researchers discovered that menstrual blood-derived stem cells (MenSCs) have the potential to differentiate into a wide range of tissues including the chondrogenic lineage. In this study, we aimed to investigate the effect of MenSCs encapsulated in fibrin glue (FG) on healing of osteochondral defect in rabbit model. METHODS: We examined the effectiveness of MenSCs encapsulated in FG in comparison with FG alone in the repair of osteochondral defect (OCD) lesions of rabbit knees after 12 and 24 weeks. RESULTS: Macroscopical evaluation revealed that the effectiveness of MenSCs incorporation with FG is much higher than FG alone in repair of OCD defects. Indeed, histopathological evaluation of FG + MenSCs group at 12 weeks post-transplantation demonstrated that defects were filled with hyaline cartilage-like tissue with proper integration, high content of glycosaminoglycan and the existence of collagen fibers especially collagen type II, as well as by passing time (24 weeks post-transplantation), the most regenerated tissue in FG + MenSCs group was similar to hyaline cartilage with relatively good infill and integration. As the same with the result of 12 weeks post-implantation, the total point of microscopical examination in FG + MenSCs group was higher than other experimental groups, however, no significant difference was detected between groups at 24 weeks (p>0.05). CONCLUSION: In summary, MenSCs as unique stem cell population, is suitable for in vivo repair of OCD defects and promising for the future clinical application.
Collagen ; Collagen Type II ; Fibrin Tissue Adhesive ; Fibrin ; Hyalin ; Hyaline Cartilage ; Knee ; Stem Cells

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