Supplementation of TGF-Beta3 in low serum media promotes chondrogenesis of BMSCs

Tissue engineering has emerged as a new promising field that allow in vitro construction of whole transplantable tissue. Recently, bone marrow stem cells (BMSCs) fulfil the requirements as appropriate cell source that is renewable for cartilage tissue engineering since they were able to form hyaline-like cartilage in vitro and in vivo when cultured in media supplemented with specific growth factors. This study aimed to examine the potential of BMSCs chondrogenesis in vitro and in vivo. BMSCs were isolated from sheep and divided into a test group and control group. Control group was cultured in medium supplemented with 10% fetal bovine serum (FBS). Test group was cultured in medium supplemented with 1% FBS, 1% ITS, 5ng/mL TGF-ß3, 50ng/mL IGF-1, 40 ng/mL L-proline, 100 nM dexamethasone and 50 μg/mL ascorbic acid-2 phosphate. The experiment was carried out on athymic nude mice. Cell aggregates were formed in test group and indicated the early chondrogenesis in cell culture. Later, cells-fibrin constructs were made and implanted subcutaneously into nude mice for 5 wks, then explanted for histological examination and glycosaminoglycans (GAG) quantification. Test group constructs showed higher cartilage matrix synthesis as confirmed by Safranin O staining and GAG production. These results demonstrated the effectiveness of low serum media supplemented with TGF-ß3 in promoting chondrogenesis in BMSCs

Effects of PLGA nanofibre on osteoarthritic chondrocytes

Chondrocytes obtained from osteoarthritis (OA) joints has been recognized as an abnormal cell; however, it’s proven to have potential in supporting cartilage regeneration. We have isolated chondrocytes from OA joints (OAC) and expanded chondrocytes growth medium (CGM). The growth kinetic, immunophenotyping and cell multilineage differentiation were analyzed to confirm the OAC stemness. The optimal condition to developed PLGA nanofibre with ratio 50:50 were 20% concentration of PLGA, flow rate with 0.3 mL/h, 10 kv voltage and 10 cm distance from nozzle to the collector. The toxicity level, scanning electron microscopy (SEM) and q-PCR analysis was performed in the present study. OAC fulfills the minimal criteria to be known to have stem cell as the cell easily adheres to the culture plate, shows high expression (≥95%) for CD13, CD29, CD44, CD73 and CD90 and less expression (≤2%) for CD45 and HLA-DR and potentially induced to mesodermal multilineage, which is osteocytes, adipocytes and chondrocytes. Toxicity test showed no adverse effect of PLGA towards the cell. Based on the cell-PLGA nanofibre interaction, difference in fibre size will influence the proliferation of the cell. Nanofibres with 100 nm in size showed high proliferation of OAC and better gene and protein expression compared to monolayer culture. Thus, we concluded that OAC has the potential to be used in cartilage regeneration based on the presence of stem cell markers as similar to the human bone marrow. The cartilage regeneration will be more efficient if OAC cultured on 3D microenvironment as showed in the present study

Physicochemical and structural characterization of surface modified electrospun PMMA nanofibre

Although electrospun poly(methyl methacrylate) (PMMA) may mimic structural features of extracellular matrix, its highly hydrophobic nature causes reduced cell attachment. This study analysed the physicochemical and structural changes of the surface modified PMMA nanofiber. The electrospun PMMA nanofibers (PM) were surface-treated as follows: PM alone, collagen coated-PM (PM-C), UV-irradiated PM (PM-UV), collagen coated UV-irradiated PM (PM-C-UV) and collagen coated-PM crosslinked with genipin (PM-C-GEN). They were subjected to scanning electron microscopy, Fourier transform infrared (FTIR), cell attachment analysis, X-ray photoelectron spectroscopy (XPS), atomic force microscopy and X-ray powder diffraction (XRD). The surface roughness was lower in PM-C-UV group compared to others. Based on FTIR results, all expected functional group were present in all groups. XPS result showed that there are changes in the mass concentration of UV-treated surfaces and in the collagen coated surfaces. All PM groups showed amorphous nature through XRD. UV irradiation and collagen coating were shown to increase PM’s functional groups and modify its surface, which contributed to the increased attachment of cells onto the inert PM scaffold. As conclusion, collagen coated UV irradiated PMMA provided a better surface for cell to attach hence are suitable to be used further as scaffold for in vitro model

Long term effect of cryopreservation on primary human skin cells

Cryopreservation is essential for tissue engineering and regenerative medicine. This study was carried out to assess the effect of cryopreservation on skin cells and evaluate the performance of cells after 12 months of cryopreservation. Redundant skin tissue samples were obtained from surgery with consent from patients. The tissue was cleaned, processed and cultured until passage 3. Upon confluency, cells were trypsinised and total cell yield and viability were determined before and after being cryopreserved. Sterility and immunocytochemistry analysis for collagen type I (Col-1) and cytokeratin 14 (CK14) antibodies were also performed on cells cryopreserved for one, three, six and twelve months. There is no significant difference in growth rates for cryopreserved cells for 1 to 12 months, except for fibroblasts at 6 months. Cell viability for both keratinocytes and fibroblasts decreased with time (65%± 3.5% - 89%± 4.5%). Sterility testing showed no contamination after 12 months of cryopreservation. Immunocytochemistry analysis showed positive expression for CK14 (keratinocytes) and Col -1 (fibroblasts) after 12 months of cryopreservation. Morphologically, keratinocytes and fibroblasts were able to retain its phenotype. The loss in viability is consistent in all samples and possibly due to thermal-cycling effect. Immunocytochemistry and consistent cell growth analysis showed that keratinocytes and fibroblasts were able to retain their characteristics in cryopreservation condition. These preliminary findings show that primary skin cells can be stored via cryopreservation and still retain their characteristics. However, further investigations using longer periods of cryopreservation (24 months, 48 months) should be conducted

Stem cell based therapy retards the progression of osteoarthritis and promotes repair of meniscus injury of sheep model knee joint

ABSTRACT The aim of this study to determine if intra-articular injection of autologous bone marrow mesenchymal stem cells (ABMSCs) could repair surgically induced osteoarthritis in sheep model. Eighteen male healthy sheep (weighed 18-20kg) were divided into two test groups and one control group. The control groups were not different from the test groups with respect to age and weight, but the test animals underwent a bone marrow aspira- tion for cell preparation in the same time of osteoarthritis induction. ABMSCs were isolated from sheep bone marrow and divided into two groups, namely test group A; ABMSCs cultured in FD medium supplemented with 10% fetal bovine serum (FBS), Test group B; ABMSCs were cultured in FD medium supplimented with 1% FBS and 10 ng/ml TGFβ-3 for three weeks. OA was induced by complete excision of the medial meniscus and resection of the anterior cruciate ligament (ACL). Sheep were subjected to exercise for three weeks post OA induction. After 6 weeks post-operation, test groups received direct intra-articular injection of a single dose 10x106 cells suspended in basal medium into injured sheep knee joint. Con- trol animals received basal medium alone. Six weeks post- cel injection, the femoral condyle and the tibial plateau from test and control groups were removed,fixed,photographed, and assessed by two blinded evaluators based on ICRS grading system, decalcified. Specimens were sectioned into 5 µm and stained with H & E and Safranin O. The result demonstrated that Gross observation of femoral condyle and tibia plaetue of the operated knee joint had OA. The severe OA was clearly observed in in control group knee joints. Test group received intra-articular injection of ABMSCs alone showed moderate OA. Interestingly test group B that received intra-articularinjection of TGF-β3 induced ABMSCs showed mild OA. The histological examination showed clear evidence of articular cartilage and menicscus regeneration in test group B of sheep injured knee joint that received TGF-β3 induced ABMSCs when compared with other groups

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Down syndrome and cell therapy : a review

Cell therapy has been considered as an alternative treatment for many diseases, including Down syndrome (DS). However, this treatment remained debatable due to insufficient clinical data. Therefore, this study aims to identify and evaluate studies on the potential of cell therapy in improving the quality of life of DS patients. Relevant English articles and snowball sampling from Science Direct and PubMed (published until August 2019) on the effects of cell therapy on DS was retrieved. Only original articles on the effect of cell therapy in DS patients or Ts65Dn (trisomic) mice were selected. Two independent reviewers reviewed the articles with selective inclusion criteria using a standard data extraction form. Cell therapy showed no significant findings on the physical appearance, cognitive function, social, and behavior skills of DS patients. Interestingly, implantation of murine neural stem cell (mNSC) or murine neural progenitor cell (mNPC) showed better cell survival and response towards brain injury, decrease tau + granules and increase granules density in the dentate gyrus in the trisomic mice. mNSC/mNPC in mice brain was found to be able to migrate to the sites of the injury following chemokine signals and eventually provide neuroprotection and promote axonal growth. To conclude, mNSC/mNPC implantation could be considered as an alternative treatment for DS or DS with early onset of Alzheimer Disease (AD)

Laminin-Coated Poly(Methyl Methacrylate) (PMMA) Nanofiber Scaffold Facilitates the Enrichment of Skeletal Muscle Myoblast Population

Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate) (PMMA) nanofiber (PM) scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM) proteins, such as collagen or laminin, in the presence or absence of genipin. A mixed population of myoblasts and fibroblasts was isolated from human skeletal muscle tissues and cultured on plain surfaces, as well as coated and non-coated PM scaffolds. PMMA can produce smooth fibers with an average diameter of 360 ± 50 nm. Adsorption of collagen and laminin on PM scaffolds is significantly enhanced in the presence of genipin, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081 ± 0.0007 h−1) and migration (0.26 ± 0.04 μm/min), while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097 ± 0.0009 h−1) and migration (0.23 ± 0.03 μm/min). Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute

3D Culture of MSCs on a Gelatin Microsphere in a Dynamic Culture System Enhances Chondrogenesis

Recent advancement in cartilage tissue engineering has explored the potential of 3D culture to mimic the in vivo environment of human cartilaginous tissue. Three-dimensional culture using microspheres was described to play a role in driving the differentiation of mesenchymal stem cells to chondrocyte lineage. However, factors such as mechanical agitation on cell chondrogenesis during culture on the microspheres has yet to be elucidated. In this study, we compared the 2D and 3D culture of bone-marrow-derived mesenchymal stem cells (BMSCs) on gelatin microspheres (GMs) in terms of MSC stemness properties, immune-phenotype, multilineage differentiation properties, and proliferation rate. Then, to study the effect of mechanical agitation on chondrogenic differentiation in 3D culture, we cultured BMSCs on GM (BMSCs-GM) in either static or dynamic bioreactor system with two different mediums, i.e., F12: DMEM (1:1) + 10% FBS (FD) and chondrogenic induction medium (CIM). Our results show that BMSCs attached to the GM surface and remained viable in 3D culture. BMSCs-GM proliferated faster and displayed higher stemness properties than BMSCs on a tissue culture plate (BMSCs-TCP). GMs also enhanced the efficiency of in-vitro chondrogenesis of BMSCs, especially in a dynamic culture with higher cell proliferation, RNA expression, and protein expression compared to that in a static culture. To conclude, our results indicate that the 3D culture of BMSCs on gelatin microsphere was superior to 2D culture on a standard tissue culture plate. Furthermore, culturing BMSCs on GM in dynamic culture conditions enhanced their chondrogenic differentiation