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ABSTRACT: INTRODUCTION: Adipose tissue is an attractive and abundant source of multipotent stem cells. Human adipose stem cells (ASCs) have shown to have therapeutic relevancy in diverse clinical applications. Nevertheless, expansion of ASCs is often necessary prior to performing clinical studies. Standard in vitro cell culture techniques utilize animal-derived reagents which should be avoided in clinical use due to safety issues. Therefore, xeno- and serum-free (XF/SF) reagents are highly desirable for enhancing the safety and quality of the transplanted ASCs. METHODS: In the current study, animal component-free isolation and cell expansion protocols were developed for ASCs. StemPro(R) MSC SFM XF medium with either CELLstartTM CTSTM coating or Coating Matrix Kit were tested for their ability to support XF/SF growth. Basic stem cell characteristics such as immunophenotype (CD3, CD11a, CD14, CD19, CD34, CD45RO, CD54, CD73, CD80, CD86, CD90, CD105, HLA-DR), proliferation and differentiation potential were assessed in XF/SF conditions and compared with human serum (HS) or traditionally used fetal bovine serum (FBS) cultures. RESULTS: ASCs cultured in XF/SF conditions had significantly higher proliferation rate compared to HS/FBS cultures. Characteristic immunophenotype of ASCs was maintained in every condition; however cells expanded in XF/SF conditions showed significantly lower expression of CD54 (intercellular adhesion molecule 1, ICAM-1) at low passage number. Further, multilineage differentiation potential of ASCs was maintained in every culture condition. CONCLUSIONS: Our findings demonstrated that the novel XF/SF conditions maintained the basic stem cell features of ASCs and the animal-free workflow followed in this study has great potential in clinical cell therapies.
Stem Cell Research & Therapy 03/2013; 4(2):27. · 3.21 Impact Factor
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ABSTRACT: INTRODUCTION: Currently, human adipose stem cells (hASCs) are differentiated towards osteogenic lineages using culture medium supplemented with L-ascorbic acid 2-phosphate (AsA2-P), dexamethasone (Dex) and beta-glycerophosphate (beta-GP). Because this osteogenic medium (OM1) was initially generated for the differentiation of bone marrow-derived mesenchymal stem cells, the component concentrations may not be optimal for the differentiation of hASCs. After preliminary screening two efficient osteogenic media (OM2 and OM3) were chosen to be compared with the commonly used osteogenic medium (OM1). To further develop the culture conditions towards clinical usage, the osteo-inductive efficiencies of OM1, OM2 and OM3 were compared using human serum (HS)-based medium and a defined, xeno-free medium (RegES), with fetal bovine serum (FBS)-based medium serving as a control. METHODS: To compare the osteo-inductive efficiency of OM1, OM2 and OM3 in FBS-, HS- and RegES-medium, the osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, mineralization, and expression of osteogenic marker genes (runx2A, DLX5, collagen type I, osteocalcin, and ALP). RESULTS: In HS-medium, the ALP activity increased significantly by OM3, and mineralization was enhanced by both OM2 and OM3, which have high AsA2-P and low Dex concentrations. ALP activity and mineralization of hASCs was the weakest in FBS-medium, with no significant differences between the OM compositions due to donor variation. However, the qRT-PCR data demonstrated significant up-regulation of runx2A mRNA under osteogenic differentiation in FBS- and HS-medium, particularly by OM3 under FBS conditions. Further, the expression of DLX5 was greatly stimulated by OM1-3 on day 7 when compared to control. The regulation of collagen type I, ALP, and osteocalcin mRNA was modest under induction by OM1-3. The RegES medium was found to support the proliferation and osteogenic differentiation of hASCs, but the composition of the RegES medium hindered the comparison of OM1, OM2 and OM3. CONCLUSIONS: Serum conditions affect hASC proliferation and differentiation significantly. The ALP activity and mineralization was the weakest in FBS-medium, although osteogenic markers were up-regulated on mRNA level. When comparing the OM composition, the commonly used OM1 was least effective. Accordingly, higher concentration of AsA2-P and lower concentration of Dex, as in OM2 and OM3, should be used for the osteogenic differentiation of hASCs in vitro.
Stem Cell Research & Therapy 02/2013; 4(1):17. · 3.21 Impact Factor
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ABSTRACT: PURPOSE: Large mandibular resection defects historically have been treated using autogenous bone grafts and reconstruction plates. However, a major drawback of large autogenous bone grafts is donor-site morbidity. PATIENTS AND METHODS: This report describes the replacement of a 10-cm anterior mandibular ameloblastoma resection defect, reproducing the original anatomy of the chin, using a tissue-engineered construct consisting of β-tricalcium phosphate (β-TCP) granules, recombinant human bone morphogenetic protein-2 (BMP-2), and Good Manufacturing Practice-level autologous adipose stem cells (ASCs). Unlike prior reports, 1-step in situ bone formation was used without the need for an ectopic bone-formation step. The reconstructed defect was rehabilitated with a dental implant-supported overdenture. An additive manufactured medical skull model was used preoperatively to guide the prebending of patient-specific hardware, including a reconstruction plate and titanium mesh. A subcutaneous adipose tissue sample was harvested from the anterior abdominal wall of the patient before resection and simultaneous reconstruction of the parasymphysis. ASCs were isolated and expanded ex vivo over the next 3 weeks. The cell surface marker expression profile of ASCs was similar to previously reported results and ASCs were analyzed for osteogenic differentiation potential in vitro. The expanded cells were seeded onto a scaffold consisting of β-TCP and BMP-2 and the cell viability was evaluated. The construct was implanted into the parasymphyseal defect. RESULTS: Ten months after reconstruction, dental implants were inserted into the grafted site, allowing harvesting of bone cores. Histologic examination and in vitro analysis of cell viability and cell surface markers were performed and prosthodontic rehabilitation was completed. CONCLUSION: ASCs in combination with β-TCP and BMP-2 offer a promising construct for the treatment of large, challenging mandibular defects without the need for ectopic bone formation and allowing rehabilitation with dental implants.
Journal of oral and maxillofacial surgery: official journal of the American Association of Oral and Maxillofacial Surgeons 01/2013; · 1.58 Impact Factor
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ABSTRACT: An electrically conductive polypyrrole (PPy) doped with a bioactive agent is an emerging functional biomaterial for tissue engineering. We therefore used chondroitin sulfate (CS) doped PPy coating to modify initially electrically insulating polylactide resulting in novel osteogenic scaffolds. In situ chemical oxidative polymerization was used to obtain electrically conductive PPy coating on poly-96L/4D-lactide (PLA) non-woven scaffolds. The coated scaffolds were characterized and their electrical conductivity was evaluated in hydrolysis. The ability of the coated and conductive scaffolds to enhance proliferation and osteogenic differentiation of human adipose stem cells (hASC) under electrical stimulation in 3 dimensional (3D) geometry was compared to the non-coated PLA scaffolds. Electrical conductivity of PPy coated PLA scaffolds (PLA-PPy) was evident at the beginning of hydrolysis, but decreased during the first week of incubation due to de-doping. PLA-PPy scaffolds enhanced hASC proliferation significantly compared to the plain PLA scaffolds at 7 and 14 days. Furthermore, the alkaline phosphatase (ALP) activity of the hASCs was generally higher in PLA-PPy seeded scaffolds, but due to patient variation, no statistical significance could be determined. Electrical stimulation did not have a significant effect on hASCs. This study highlights the potential of novel PPy coated PLA scaffolds in bone tissue engineering.
Tissue Engineering Part A 11/2012; · 4.64 Impact Factor
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ABSTRACT: The performance of biodegradable knitted and rolled 3-dimensional (3D) polylactide-based 96/4 scaffolds modified with bioactive glass (BaG) 13-93, chitosan and both was compared with regard to the viability, proliferation and chondrogenic differentiation of rabbit adipose stem cells (ASCs). Scaffold porosities were determined by micro-computed tomography (μCT). Water absorption and degradation of scaffolds were studied during 28-day hydrolysis in Tris-buffer. Viability, number and differentiation of ASCs in PLA96/4 scaffolds were examined in vitro. The dimensions of the scaffolds were maintained during hydrolysis and mass loss was detected only in the BaG13-93 containing scaffolds. ASCs adhered and proliferated on each scaffold type. Cell aggregation and expression of chondral matrix components improved in all scaffold types in chondrogenic medium. Signs of hypertrophy were detected in the modified scaffolds but not in the plain PLA96/4 scaffold. Chondrogenic differentiation was most enhanced in the presence of chitosan. These findings indicate that the plain P scaffold provided a good 3D-matrix for ASC proliferation whereas the addition of chitosan to the PLA96/4 scaffold induced chondrogenic differentiation independent of the medium. Accordingly, a PLA96/4 scaffold modified by chitosan could provide a functional and bioactive basis for tissue-engineered chondral implants. Copyright © 2012 John Wiley & Sons, Ltd.
Journal of Tissue Engineering and Regenerative Medicine 10/2012; · 3.28 Impact Factor
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ABSTRACT: Different synthetic biomaterials such as polylactide (PLA), polycaprolactone and poly-l-lactide-co-ε-caprolactone (PLCL) have been studied for urothelial tissue engineering, with favourable results. The aim of this research was to further optimize the growth surface for human urothelial cells (hUCs) by comparing different PLCL-based membranes: smooth (s) and textured (t) PLCL and knitted PLA mesh with compression-moulded PLCL (cPLCL). The effects of topographical texturing on urothelial cell response and mechanical properties under hydrolysis were studied. The main finding was that both sPLCL and tPLCL supported hUC growth significantly better than cPLCL. Interestingly, tPLCL gave no significant advantage to hUC attachment or proliferation compared with sPLCL. However, during the 14 day assessment period, the majority of cells were viable and maintained phenotype on all the membranes studied. The material characterization exhibited potential mechanical characteristics of sPLCL and tPLCL for urothelial applications. Furthermore, the highest elongation of tPLCL supports the use of this kind of texturing. In conclusion, in light of our cell culture results and mechanical characterization, both sPLCL and tPLCL should be further studied for urothelial tissue engineering.
Journal of The Royal Society Interface 08/2012; 9(77):3444-54. · 4.40 Impact Factor
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ABSTRACT: Aims: Human adipose stem cells (hASCs) have been suggested as viable alternative for bone tissue engineering. However, the tissue response and osteogenic potential of BAG S53P4 or β-TCP granules has not been studied in vivo when seeded with hASCs and/or co-incubated with BMP-2 and thus, was evaluated in the current study.
Methods and results: Human ASCs were isolated, expanded and seeded on BAG and β-TCP in vitro and,
cell viability was assessed using Live/Dead staining. In a subcutaneous rodent implantation model, the cellular response and osteogenic potential of 1) plain, 2) hASC seeded, 3) BMP-2 co-incubated and 4) hASC seeded and BMP-2 co-incubated BAG and β-TCP granules were investigated using computed tomography and semi-quantitative histologic scores after 4 and 8 weeks. Live/Dead staining confirmed good cell viability on both biomaterials prior to implantation. Overall, implantation of both biomaterials resulted in formation of well-vascularized granulation tissue without excessive inflammation, fibrosis or adverse reactions independent on group assignment and time point evaluated and thus, suggesting safety for prospective applications. However, our results also indicate that β-TCP may temporarily stimulate foreign body giant cell formation after hASCs supplementation suggesting a resorptive response. Both biomaterials required supplementation of hASCs and/or BMP-2 to induce osteoblastic activity. However, BAG induced calcification exclusively when seeded with BMP-2 activated hASCs, whereas β-TCP required seeding with hASCs only.
Conclusion: BAG and β-TCP granules can be safely implanted subcutaneously, induce a different cellular
response and require hASC and/or BMP-2 supplementation to induce osteoblastic activity and calcification. A
combination of β-TCP and hASCs appeared to be a feasible way in enhancing osteoblastic activity resulting in early osteogenesis while minimizing safety and regulatory concerns in bone-tissue engineering.
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ABSTRACT: The current limitation in designing three-dimensional tissue models is the lack of adequate vascularization with mature and stable vessels. Adipose tissue is known to secrete several angiogenic factors, and human adipose stromal cells (hASC) are known to promote vessel growth, maturation and stabilization. In this study, hASC were induced to angiogenesis with growth factor-enriched medium either in monoculture or in coculture with human umbilical vein endothelial cells (HUVEC) and analyzed for vascular, pericytic and smooth muscle cell markers. hASC and HUVEC cocultures showed an accelerated proliferation rate and the cells self-assembled, independent of the cell passage number, into multilayered three-dimensional tubular networks. The networks of hASC and HUVEC expressed endothelial markers, a complete basement membrane and vessel-supporting cells with contractile properties. A hASC and green fluorescence protein-HUVEC-infection model revealed that cocultures consisted of a mosaic of von Willebrand factor-positive cells derived from both cell populations - hASC and HUVEC. hASC monoculture had passage- and donor-dependent ability to form tubular networks, with half of the cultures presenting tubule structures and basement membrane formation. Pericytic and smooth muscle cell markers were expressed in hASC monoculture even when tubules were absent. By combining the potential properties of hASC and features from the present angiogenesis assays, we generated a natural-like, xeno-free, prevascular-like network in vitro model with excellent reproducibility and minimal limitations in technical performance. This tubular network model is an excellent tool for studying cell interactions during vascular development, for chemical and drug testing and for developing natural-like, multilayered, vascularized, scaffold-free tissue models.
Cells Tissues Organs 06/2012; 196(5):385-97. · 2.20 Impact Factor
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ABSTRACT: Functional tissue engineering for bone augmentation requires the appropriate combination of biomaterials, mesenchymal stem cells, and specific differentiation factors. Therefore, we investigated the morphology, attachment, viability, and proliferation of human dental pulp stem cells cultured in xeno-free conditions in human serum medium seeded on β-tricalcium phosphate/poly(l-lactic acid/caprolactone) three-dimensional biomaterial scaffold. Additionally, osteogenic inducers dexamethasone and vitamin D(3) were compared to achieve osteogenic differentiation. Dental pulp stem cells cultured in human serum medium maintained their morphology; furthermore, cells attached, remained viable, and increased in cell number within the scaffold. Alkaline phosphatase staining showed the osteogenic potential of dental pulp stem cells under the influence of osteogenic medium containing vitamin D(3) or dexamethasone within the scaffolds. Maintenance of dental pulp stem cells for 14 days in osteogenic medium containing vitamin D(3) resulted in significant increase in osteogenic markers as shown at mRNA level in comparison to osteogenic medium containing dexamethasone. The results of this study show that osteogenic medium containing vitamin D(3) osteo-induced dental pulp stem cells cultured in human serum medium within β-tricalcium phosphate/poly(l-lactic acid/caprolactone) three-dimensional biomaterial, which could be directly translated clinically.
Journal of tissue engineering. 01/2012; 3(1):2041731412467998.
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Martin Waselau,
Mimmi Patrikoski,
Miia Juntunen,
Kasperi Kujala,
Minna Kääriäinen,
Hannu Kuokkanen,
George K Sándor,
Outi Vapaavuori,
Riitta Suuronen,
Bettina Mannerström,
Brigitte von Rechenberg, Susanna Miettinen
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ABSTRACT: The effects of bioactive glass S53P4 or beta-tricalcium phosphate; and bone morphogenetic proteins bone morphogenetic protein-2, bone morphogenetic protein-7, or bone morphogenetic protein-2 + 7 on osteogenic differentiation of human adipose stem cells were compared in control medium, osteogenic medium, and bone morphogenetic protein-supplemented osteogenic medium to assess suitability for bone tissue engineering. Cell amount was evaluated with qDNA measurements; osteogenic differentiation using marker gene expression, alkaline phosphate activity, and angiogenic potential was measured by vascular endothelial growth factor expression. As compared to beta-tricalcium phosphate, cell amount was significantly greater for bioactive glass in control medium after 7 days and in osteogenic medium after 14 days, and alkaline phosphate activity was always significantly greater for bioactive glass in control medium. However, alkaline phosphate activity increased for beta-tricalcium phosphate and decreased for bioactive glass granules in osteogenic medium. For both biomaterials, bone morphogenetic protein supplementation decreased cell amount and osteogenic differentiation of human adipose stem cells, and vascular endothelial growth factor expressions correlated with cell amounts. Effects of culture medium on human adipose stem cells are biomaterial dependent; bioactive glass in control medium enhanced osteogenic differentiation most effectively.
Journal of tissue engineering. 01/2012; 3(1):2041731412467789.
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ABSTRACT: The induction of adequate vascularization, a major challenge in tissue engineering, has been tried with numerous methods but with unsatisfactory results. Adipose tissue, an active endocrine organ with dense vasculature, secretes a wide number of angiogenic and adipogenic factors and seems an attractive source for these bioactive factors. We produced a novel cell-free extract from mature human adipose tissue (adipose tissue extract [ATE]) and analyzed the ability of this extract to induce angiogenesis and adipogenesis in vitro and studied the cytokine and growth factor composition of ATE with ELISA and cytokine array. We demonstrate that ATE, when added as cell culture supplement, effectively induced triglyceride accumulation in human adipose stem cells at concentrations from 200 μg/mL upward in less than a week and caused elevated levels of adipocyte differentiation markers (proliferator-activated receptor gamma and acyl-CoA-binding protein) when treated with at least 350 μg/mL of ATE. ATE induced angiogenesis from 450 μg/mL upward after a week in vitro. ATE contained numerous angiogenic and adipogenic factors, for example, vascular endothelial growth factor, basic fibroblast growth factor, interleukin-6, adiponectin, angiogenin, leptin, and insulin-like growth factor-I, as well as lower levels of a wide variety of other cytokines. We here present a novel cell-free angiogenesis- and adipogenesis-inducing agent that is cell-free and easy to produce, and its effect is dose dependent and its composition can be easily modified. Therefore, ATE is a promising novel agent to be used for angiogenesis induction to overcome the challenge of vascularization and for adipogenesis induction in a wide variety of tissue engineering applications in vitro and in vivo. ATE is also efficient for reproduction and modeling of natural adipogenesis in vitro for, for example, obesity and diabetes studies.
Tissue Engineering Part A 09/2011; 18(1-2):17-25. · 4.64 Impact Factor
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ABSTRACT: Adipose stem cells (ASCs) are an attractive and abundant stem cell source with therapeutic applicability in diverse fields for the repair and regeneration of acute and chronically damaged tissues. Importantly, unlike the human bone marrow stromal/stem stem cells (BMSCs) that are present at low frequency in the bone marrow, ASCs can be retrieved in high number from either liposuction aspirates or subcutaneous adipose tissue fragments and can easily be expanded in vitro. ASCs display properties similar to that observed in BMSCs and, upon induction, undergo at least osteogenic, chondrogenic, adipogenic and neurogenic, differentiation in vitro. Furthermore, ASCs have been shown to be immunoprivileged, prevent severe graft-versus-host disease in vitro and in vivo and to be genetically stable in long-term culture. They have also proven applicability in other functions, such as providing hematopoietic support and gene transfer. Due to these characteristics, ASCs have rapidly advanced into clinical trials for treatment of a broad range of conditions. As cell therapies are becoming more frequent, clinical laboratories following good manufacturing practices are needed. At the same time as laboratory processes become more extensive, the need for control in the processing laboratory grows consequently involving a greater risk of complications and possibly adverse events for the recipient. Therefore, the safety, reproducibility and quality of the stem cells must thoroughly be examined prior to extensive use in clinical applications. In this review, some of the aspects of examination on ASCs in vitro and the utilization of ASCs in clinical studies are discussed.
Stem cell reviews 06/2011; 7(2):269-91. · 5.08 Impact Factor
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Laura Tirkkonen,
Heidi Halonen,
Jari Hyttinen,
Hannu Kuokkanen,
Harri Sievänen,
Anna-Maija Koivisto,
Bettina Mannerström,
George K B Sándor,
Riitta Suuronen, Susanna Miettinen,
Suvi Haimi
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ABSTRACT: Mechanical stimulation is an essential factor affecting the metabolism of bone cells and their precursors. We hypothesized that vibration loading would stimulate differentiation of human adipose stem cells (hASCs) towards bone-forming cells and simultaneously inhibit differentiation towards fat tissue. We developed a vibration-loading device that produces 3g peak acceleration at frequencies of 50 and 100 Hz to cells cultured on well plates. hASCs were cultured using either basal medium (BM), osteogenic medium (OM) or adipogenic medium (AM), and subjected to vibration loading for 3 h d(-1) for 1, 7 and 14 day. Osteogenesis, i.e. differentiation of hASCs towards bone-forming cells, was analysed using markers such as alkaline phosphatase (ALP) activity, collagen production and mineralization. Both 50 and 100 Hz vibration frequencies induced significantly increased ALP activity and collagen production of hASCs compared with the static control at 14 day in OM. A similar trend was detected for mineralization, but the increase was not statistically significant. Furthermore, vibration loading inhibited adipocyte differentiation of hASCs. Vibration did not affect cell number or viability. These findings suggest that osteogenic culture conditions amplify the stimulatory effect of vibration loading on differentiation of hASCs towards bone-forming cells.
Journal of The Royal Society Interface 05/2011; 8(65):1736-47. · 4.40 Impact Factor
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ABSTRACT: There is no optimal method for reconstruction of large calvarial defects. Because of the limitations of autologous bone grafts and alloplastic materials, new methods for performing cranioplasties are needed.
To create autologous bone to repair cranial defects.
We performed a cranioplasty procedure with this new method in 4 patients who had large calvarial defects of different etiologies. We used autologous adipose-derived stem cells seeded in beta-tricalcium phosphate granules. For 2 patients, we used a bilaminate technique with resorbable mesh.
During follow-up, there were no clinically relevant postoperative complications. The computed tomography scans revealed satisfactory outcome in ossification, and in the clinical examinations, the outcomes were good. The cranioplasty was measured in Hounsfield units from each computed tomography scan. The Hounsfield units increased gradually to equal the value of bone.
The combination of scaffold material such as beta-tricalcium phosphate and autologous adipose-derived stem cells constitutes a promising model for reconstruction of human large cranial defects. The success of these clinical cases paves way for further studies and clinical applications to turn this method into a reliable treatment regimen.
Neurosurgery 02/2011; 68(6):1535-40. · 2.79 Impact Factor
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Janek Frantzén,
Aliisa Pälli,
Esa Kotilainen,
Harri Heino,
Bettina Mannerström,
Heini Huhtala,
Hannu Kuokkanen,
George K Sándor,
Kari Leino,
Matias Röyttä,
Riitta Parkkola,
Riitta Suuronen, Susanna Miettinen,
Hannu T Aro,
Suvi Haimi
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ABSTRACT: A poly-70L/30DL-lactide (PLA70)-β-tricalcium phosphate (β-TCP) composite implant reinforced by continuous PLA-96L/4D-lactide (PLA96) fibers was designed for in vivo spinal fusion. The pilot study was performed with four sheep, using titanium cage implants as controls. The composite implants failed to direct bone growth as desired, whereas the bone contact and the proper integration were evident with controls 6 months after implantation. Therefore, the PLA70/β-TCP composite matrix material was further analyzed in the in vitro experiment by human and ovine adipose stem cells (hASCs and oASCs). The composites proved to be biocompatible as confirmed by live/dead assay. The proliferation rate of oASCs was higher than that of hASCs at all times during the 28 d culture period. Furthermore, the composites had only a minor osteogenic effect on oASCs, whereas the hASC osteogenesis on PLA70/β-TCP composites was evident. In conclusion, the composite implant material can be applied with hASCs for tissue engineering but not be evaluated in vivo with sheep.
International Journal of Biomaterials 01/2011; 2011:109638.
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ABSTRACT: This study was designed to investigate the potential merits of the combined use of bone morphogenetic protein (BMP)-2 or BMP-6 and osteogenic supplements (OS) [dexamethasone, ascorbic acid (AA), and β-glycerophosphate] on osteogenic differentiation of periodontal ligament cells (PDLCs). Osteogenic differentiation was evaluated by quantitative alkaline phosphatase (ALP) assay, alizarin red staining, quantitative calcium assay, and the qRT-PCR analysis for the expression of collagen type I, runt-related transcription factor-2, osteopontin (OPN), and osteocalcin in PDLCs. Culture with BMP-2 or BMP-6+AA increased ALP activity of PDLCs, suggesting their osteo-inductive effects. However, longer duration of culture showed neither of the BMPs induced in vitro mineralization. In contrast, OS were able to increase ALP activity and OPN expressions, and also induced in vitro mineralization. The mineralization ability was not enhanced by the addition of BMP-2 or BMP-6. These findings suggest that the addition of BMP-2 or BMP-6 to OS may not enhance an osteogenic differentiation of hPDLCs.
Growth factors (Chur, Switzerland) 12/2010; 28(6):437-46. · 2.47 Impact Factor
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ABSTRACT: The reconstructive surgery of urothelial defects, such as severe hypospadias is susceptible to complications. The major problem is the lack of suitable grafting materials. Therefore, finding alternative treatments such as reconstruction of urethra using tissue engineering is essential. The aim of this study was to compare the effects of naturally derived acellular human amniotic membrane (hAM) to synthetic poly-L-lactide-co-ε-caprolactone (PLCL) on human urothelial cell (hUC) viability, proliferation and urothelial differentiation level. The viability of cells was evaluated using live/dead staining and the proliferation was studied using WST-1 measurement. Cytokeratin (CK)7/8 and CK19 were used to confirm that the hUCs maintained their phenotype on different biomaterials. On the PLCL, the cell number significantly increased during the culturing period, in contrast to the hAM, where hUC proliferation was the weakest at 7 and 14 days. In addition, the majority of cells were viable and maintained their phenotype when cultured on PLCL and cell culture plastic, whereas on the hAM, the viability of hUCs decreased with time and the cells did not maintain their phenotype. The PLCL membranes supported the hUC proliferation significantly more than the hAM. These results revealed the significant potential of PLCL membranes in urothelial tissue engineering applications.
Journal of The Royal Society Interface 11/2010; 8(58):671-7. · 4.40 Impact Factor
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ABSTRACT: Vitamin D(3) metabolites regulate the bone metabolism and 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)) is known to play an important role in teeth mineralization. However, little is known about the potential of vitamin D as an osteogenic inducer in human dental pulp (hDPCs) and dental follicle cells (hDFCs) in vitro. Therefore, we investigated the effects of vitamin D(3) metabolites 1α,25(OH)(2)D(3) and 25-hydroxyvitamin D(3) (25OHD(3)) on proliferation and osteogenic differentiation of hDPCs and hDFCs in vitro. We also examined whether vitamin D(3) metabolic enzymes were regulated in hDFCs and hDPCs. Cell proliferation was decreased by both metabolites in hDPCs and hDFCs. Vitamin D(3) metabolites increased ALP activity and induced mineralization when osteogenic supplements (OS; l-ascorbic acid-2-phosphate+β-glycerophosphate) were added, though the expression of osteocalcin (OC) and osteopontin (OPN) were regulated without the addition of OS. CYP24 and CYP27B1 expressions were upregulated by vitamin D(3) metabolites and 25OHD(3) was converted into 1α,25(OH)(2)D(3) in the culture medium. These results confirm that 1α,25(OH)(2)D(3) (10 and 100 nM) and 25OHD(3) (500 nM) can be used as osteogenic inducers synergistically with osteogenic supplements for differentiation of hDPCs and hDFCs. Furthermore, our findings strengthen our knowledge about the role of hDPCs and hDFCs as vitamin D(3) target cells.
The Journal of steroid biochemistry and molecular biology 10/2010; 122(4):133-41. · 2.66 Impact Factor
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ABSTRACT: Porous polylactide/beta-tricalcium phosphate (PLA/beta-TCP) composite scaffolds were fabricated by freeze-drying. The aim of this study was to characterize these graded porous composite scaffolds in two different PLA concentrations (2 and 3 wt%). Also, three different beta-TCP ratios (5, 10 and 20 wt%) were used to study the effect of beta-TCP on the properties of the polymer. The characterization was carried out by determining the pH, weight change, component ratios, thermal stability, inherent viscosity and microstructure of the scaffolds in 26 weeks of hydrolysis. This study indicated that no considerable change was noticed in the structure of the scaffolds when the beta-TCP filler was added. Also, the amount of beta-TCP did not affect the pore size or the pore distribution in the scaffolds. We observed that the fabrication method improved the thermal stability of the samples. Our results suggest that, from the structural point of view, these scaffolds could have potential for the treatment of osteochondral defects in tissue engineering applications. The porous bottom surface of the scaffold and the increased osteogenic differentiation potential achieved with beta-TCP particles may encourage the growth of bone cells. In addition, the dense surface skin of the scaffold may inhibit the ingrowth of osteoblasts and bone tissue, while simultaneously encouraging the ingrowth of chondrocytes.
Journal of Tissue Engineering and Regenerative Medicine 07/2010; 4(5):366-73. · 3.28 Impact Factor
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ABSTRACT: In preclinical studies, human adipose stem cells (ASCs) have been shown to have therapeutic applicability, but standard expansion methods for clinical applications remain yet to be established. ASCs are typically expanded in the medium containing fetal bovine serum (FBS). However, sera and other animal-derived culture reagents stage safety issues in clinical therapy, including possible infections and severe immune reactions. By expanding ASCs in the medium containing human serum (HS), the problem can be eliminated. To define how allogeneic HS (alloHS) performs in ASC expansion compared to FBS, a comparative in vitro study in both serum supplements was performed. The choice of serum had a significant effect on ASCs. First, to reach cell proliferation levels comparable with 10% FBS, at least 15% alloHS was required. Second, while genes of the cell cycle pathway were overexpressed in alloHS, genes of the bone morphogenetic protein receptor-mediated signaling on the transforming growth factor beta signaling pathway regulating, for example, osteoblast differentiation, were overexpressed in FBS. The result was further supported by differentiation analysis, where early osteogenic differentiation was significantly enhanced in FBS. The data presented here underscore the importance of thorough investigation of ASCs for utilization in cell therapies. This study is a step forward in the understanding of these potential cells.
Tissue Engineering Part A 02/2010; 16(7):2281-94. · 4.64 Impact Factor
