Sheep Mesenchymal Stem Cells Research Articles

P-788 Analysis of tissue regeneration after transplantation of bioengineered uterine grafts in a sheep model of uterine injury

Abstract Study question Can we regenerate a damaged uterus using decellularized (further repopulated with stem cells or not) uterine scaffolds in a sheep model? Summary answer Bioengineered uterine grafts can promote the regeneration of a damaged uterus, but this process is highly dependent on the basal immune status of the recipient. What is known already Uterus bioengineering aims to develop scaffolds to repair defective uterine tissue that may cause infertility. The remaining extracellular matrix after uterus decellularization has frequently been used as a scaffold material for preclinical treatment applications. So far, multiple in vivo studies using rodents have shown successful restoration of uterine tissue and even fertility when small grafts were used. Furthermore, this pro-regenerative effect is enhanced when repopulating the scaffolds with mesenchymal stem cells (MSCs). However, studies using larger animal models and larger scaffolds are still missing. Therefore, we investigated the therapeutic potential of transplanting these scaffolds in the sheep model. Study design, size, duration All experiments were performed in university facilities, including the sheep animal work. Uterine scaffold donor sheep (from a local abattoir) and recipient sheep (n = 10) were of the same mixed sheep breed (Swedish fin-ull and Dutch texel mixed breed) and age (8-12 months old). Recipient sheep were transplanted with uterine decellularized (either recellularized or not) grafts that were maintained for 6 weeks. Participants/materials, setting, methods We implemented our previously established decellularization protocol (2% sodium deoxycholate) for sheep uterine scaffold production, generating 3x2cm grafts; half of these were recellularized with sheep MSCs. Decellularized (DC; n = 7) and recellularized (RC; n = 8) grafts were transplanted as replacements for native uterine tissue. After six weeks, histological, immunohistochemistry (CD31, CD45), and gene expression (progesterone/estrogen receptors, HOXA10, VEGF, vWF, TNFA, IFNG) analysis were conducted. Simultaneously, we used cell sorting to monitor the systemic immune response over time. Main results and the role of chance Angiogenesis was increased in the RC group as more new blood vessels (CD31+ cells) were evidenced (P=0.0120). VEGF and vWF gene expression also indicated the presence of blood vessels in the grafts but without differences among the groups. Regarding the immune response, CD45 staining revealed leukocyte recruitment into both transplanted grafts, with an increased significance in the DC group (P=0.0071) compared to the RC (P=0.0181). Furthermore, the pro-inflammatory genes TNFA and IFNG expression remained invariable compared to the native tissue. Finally, hormone receptor gene levels were comparable to those in the native tissue, with HOXA10 presenting higher values in the RC group compared to the DC (P=0.0548). So, the RC group showed signs of better uterine regeneration, probably due to the MSC’s anti-inflammatory action. Still, differences were subtle between groups. So, we blindly analyzed the histology of all grafts and detected substantially but also poorly regenerated grafts in both DC and RC groups. The analysis of the systemic immune response likely answers this, as when segregating individuals in substantially and poorly regenerated grafts, there was a significant difference in the percentage of activated CD4+ T-cells, being higher (P<0.0001) in those with poorly regenerated grafts already at day 0. Limitations, reasons for caution Due to the lack of available sheep antibodies, we did not study specific subtypes of leukocytes by immunohistochemistry as we did for the cell sorting analysis. Also, despite experienced group members performing the blinded histological analysis of the grafts to detect substantially/poorly regenerated grafts, this method requires refinement. Wider implications of the findings Basal immune system status should be considered before transplanting uterine grafts, as inflammation seems crucial in tissue regeneration. Trial registration number Not applicable

Osteogenic Potential of Sheep Mesenchymal Stem Cells Preconditioned with BMP-2 and FGF-2 and Seeded on an nHAP-Coated PCL/HAP/β-TCP Scaffold.

Mesenchymal stem cells (MSCs) attract interest in regenerative medicine for their potential application in bone regeneration. However, direct transplantation of cells into damaged tissue is not efficient enough to regenerate large bone defects. This problem could be solved with a biocompatible scaffold. Consequently, bone tissue engineering constructs based on biomaterial scaffolds, MSCs, and osteogenic cytokines are promising tools for bone regeneration. The aim of this study was to evaluate the effect of FGF-2 and BMP-2 on the osteogenic potential of ovine bone marrow-derived MSCs seeded onto an nHAP-coated PCL/HAP/β-TCP scaffold in vitro and its in vivo biocompatibility in a sheep model. In vitro analysis revealed that cells preconditioned with FGF-2 and BMP-2 showed a better capacity to adhere and proliferate on the scaffold than untreated cells. BM-MSCs cultured in an osteogenic medium supplemented with FGF-2 and BMP-2 had the highest osteogenic differentiation potential, as assessed based on Alizarin Red S staining and ALP activity. qRT-PCR analysis showed increased expression of osteogenic marker genes in FGF-2- and BMP-2-treated BM-MSCs. Our pilot in vivo research showed that the implantation of an nHAP-coated PCL/HAP/β-TCP scaffold with BM-MSCs preconditioned with FGF-2 and BMP-2 did not have an adverse effect in the sheep mandibular region and induced bone regeneration. The biocompatibility of the implanted scaffold-BM-MSC construct with sheep tissues was confirmed by the expression of early (collagen type I) and late (osteocalcin) osteogenic proteins and a lack of an elevated level of proinflammatory cytokines. These findings suggest that FGF-2 and BMP-2 enhance the osteogenic differentiation potential of MSCs grown on a scaffold, and that such a tissue engineering construct may be used to regenerate large bone defects.

Polycaprolactone–carboxymethyl cellulose composites for manufacturing porous scaffolds by material extrusion

Polycaprolactone–carboxymethyl cellulose composites have been obtained and used to print porous structures by material extrusion. The materials used contained 0, 2 and 5% w/w of the carboxymethyl cellulose additive. These structures have been analyzed in terms of their morphology (including the evaluation of their porosity), mechanical properties under compression load and cell affinity. Cell affinity has been evaluated by culturing sheep mesenchymal stem cells and analyzing their viability by the Alamar Blue® assay at days 1, 3, 6 and 8. The results show that composites samples have similar values of porosity and apparent density than pure polycaprolactone ones. However, samples containing 5% w/w of carboxymethyl cellulose have micropores on the filaments due to a hindered deposition process. This characteristic affects the mechanical properties of the structures, so these ones have a mean compression modulus significantly lower than pure polycaprolactone scaffolds. However, the samples containing 2% w/w of carboxymethyl cellulose show no significant difference with the pure polycaprolactone ones in terms of their mechanical properties. Moreover, the presence of 2% w/w of additive improves cell proliferation on the surface of the porous structures. As complementary information, the flow properties of the composite materials were studied and the power law equations at 210 °C obtained, as this temperature was the 3D printing temperature. These equations can be useful for simulation and designing purposes of other manufacturing processes.

Optimizing combination of vascular endothelial growth factor and mesenchymal stem cells on ectopic bone formation in SCID mice.

Insufficient blood supply may limit bone regeneration in bone defects. Vascular endothelial growth factor (VEGF) promotes angiogenesis by increasing endothelial migration. This outcome, however, could depend on time of application. Sheep mesenchymal stem cells (MSCs) in severe combined immunodeficient (SCID) mice were used in this study to evaluate optimal time points for VEGF stimulation to increase bone formation. Twenty-eight SCID (NOD.CB17-Prkdcscid /J) mice had hydroxyapatite granules seeded with 5 × 105 MSCs inserted subcutaneous. Pellets released VEGF on days 1-7, days 1-14, days 1-21, days 1-42, days 7-14, and days 21-42. After 8 weeks, the implant-bone-blocks were harvested, paraffin embedded, sectioned, and stained with both hematoxylin and eosin (HE) and immunohistochemistry for human vimentin (hVim) staining. Blood samples were collected for determination of bone-related biomarkers in serum. The groups with 5 × 105 MSCs and VEGF stimulation on days 1-14 and days 1-21 showed more bone formation when compared to the control group of 5 × 105 MSCs alone (p < 0.01). Serum biomarkers had no significant values. The hVim staining confirmed the ovine origin of the observed ectopic bone formation. Optimal bone formation of MSCs was reached when stimulating with VEGF during the first 14 or 21 days after surgery. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3326-3332, 2017.

Combining electrospinning and cell sheet technology for the development of a multiscale tissue engineered ligament construct (TELC).

Ligament tissue rupture is a common sport injury. Although current treatment modalities can achieve appropriate reconstruction of the damaged ligament, they present significant drawbacks, mostly related to reduced tissue availability and pain associated with tissue harvesting. Stem cell based tissue regeneration combined with electrospun scaffolds represents a novel treatment method for torn ligaments. In this study, a low fiber density polycaprolactone (PCL) electrospun mesh and sheep mesenchymal stem cells (sMSCs) were used to develop tissue engineered ligament construct (TELC) in vitro. The assembly of the TELC was based on the spontaneous capacity of the cells to organize themselves into a cell sheet once seeded onto the electrospun mesh. The cell sheet matured over 4 weeks and strongly integrated with the low fiber density electrospun mesh which was subsequently processed into a ligament-like bundle and braided with two other bundles to develop the final construct. Live/dead assay revealed that the handling of the construct through the various phases of assembly did not cause significant difference in viability compared to the control. Mechanical evaluation demonstrated that the incorporation of the cell sheet into the braided construct resulted in significantly modifying the mechanical behavior. A stress/displacement J-curve was observed for the TELC that was similar to native ligament, whereas this particular feature was not observed in the non-cellularized specimens. The regenerative potential of the TELC was evaluated ectopically in immunocompromized rats, compared to non cellularized electrospun fiber mesh and this demonstrated that the TELC was well colonized by host cells and that a significant remodelling of the implanted construct was observed. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 399-409, 2018.

Perivascular Mesenchymal Stem Cells in Sheep: Characterization and Autologous Transplantation in a Model of Articular Cartilage Repair.

Previous research has indicated that purified perivascular stem cells (PSCs) have increased chondrogenic potential compared to conventional mesenchymal stem cells (MSCs) derived in culture. This study aimed to develop an autologous large animal model for PSC transplantation and to specifically determine if implanted cells are retained in articular cartilage defects. Immunohistochemistry and fluorescence-activated cell sorting were used to ascertain the reactivity of anti-human and anti-ovine antibodies, which were combined and used to identify and isolate pericytes (CD34-CD45-CD146+) and adventitial cells (CD34+CD45-CD146-). The purified cells demonstrated osteogenic, adipogenic, and chondrogenic potential in culture. Autologous ovine PSCs (oPSCs) were isolated, cultured, and efficiently transfected using a green fluorescence protein (GFP) encoding lentivirus. The cells were implanted into articular cartilage defects on the medial femoral condyle using hydrogel and collagen membranes. Four weeks following implantation, the condyle was explanted and confocal laser scanning microscopy demonstrated the presence of oPSCs in the defect repaired with the hydrogel. These data suggest the testability in a large animal of native MSC autologous grafting, thus avoiding possible biases associated with xenotransplantation. Such a setting will be used in priority for indications in orthopedics, at first to model articular cartilage repair.

Bone tissue engineering using polyetherketoneketone scaffolds combined with autologous mesenchymal stem cells in a sheep calvarial defect model.

Polyetherketoneketone (PEKK) a high performance thermoplastic polymer that is FDA-approved for cranio- and maxillo-facial as well as spineal surgery. We studied the viability, growth and osteogenic differentiation of bone marrow-derived human and sheep mesenchymal stem cells (MSC) in combination with a 3D scaffold made of PEKK using different cell-based assays. To investigate if autologous MSC, either undifferentiated or osteogenically pre-differentiated, augmented bone formation after implantation, we implanted cell-seeded 3D PEKK scaffolds into calvarial defects in sheep for 12 weeks. The volume and quality of newly formed bone were investigated using micro-computer tomography (micro-CT) and histological stainings. Our results show that the 3D PEKK scaffolds were cyto- and bio-compatible. They allowed for adherence, growth and osteogenic differentiation of human and ovine MSC. However, bone healing seemed unaffected by whether the scaffolds were seeded with MSC. Considerable amounts of newly formed bone were found in all PEKK treated groups, but a fibrous capsule was formed around the implants regardless of cell seeding with MSC.

Characterization of mesenchymal stem cells in sheep naturally infected with scrapie.

Mesenchymal stem cells (MSCs) can be infected with prions and have been proposed as in vitro cell-based models for prion replication. In addition, autologous MSCs are of interest for cell therapy in neurodegenerative diseases. To the best of our knowledge, the effect of prion diseases on the characteristics of these cells has never been investigated. Here, we analysed the properties of MSCs obtained from bone marrow (BM-MSCs) and peripheral blood (PB-MSCs) of sheep naturally infected with scrapie — a large mammal model for the study of prion diseases. After three passages of expansion, MSCs derived from scrapie animals displayed similar adipogenic, chondrogenic and osteogenic differentiation ability as cells from healthy controls, although a subtle decrease in the proliferation potential was observed. Exceptionally, mesenchymal markers such as CD29 were significantly upregulated at the transcript level compared with controls. Scrapie MSCs were able to transdifferentiate into neuron-like cells, but displayed lower levels of neurogenic markers at basal conditions, which could limit this potential .The expression levels of cellular prion protein (PrPC) were highly variable between cultures, and no significant differences were observed between control and scrapie-derived MSCs. However, during neurogenic differentiation the expression of PrPC was upregulated in MSCs. This characteristic could be useful for developing in vitro models for prion replication. Despite the infectivity reported for MSCs obtained from scrapie-infected mice and Creutzfeldt–Jakob disease patients, protein misfolding cyclic amplification did not detect PrPSc in BM- or PB-MSCs from scrapie-infected sheep, which limits their use for in vivo diagnosis for scrapie.

Custom-made novel biomimetic composite scaffolds for the bone regenerative medicine

The aim of this pilot study was to test a new customized biomimetic composite scaffolds to restore bone defects using CAD–CAM technology. The new biomaterial was synthesized using biomimetic mineralized collagen, and poly (ɛ-caprolactone) (PCL). The mold used in scaffolding, the surgical guide, and the bony plate used during the surgery, were rapid prototyped (CAD–CAM technology). Our in vitro results showed that sheep mesenchymal stem cells (MSC) seeding efficiency was very high (>94%). Furthermore, cells can adhere, grow and maintain cell viability up to 7 days of in vitro cultivation on the scaffold as revealed by SEM and confocal laser scanning microscope analysis. These results are indicative of good scaffold biocompatibility. The use of the sheep experimental model allowed exploring the efficacy of the customized bony plate used to give extra mechanical support to the scaffold, the potential of CAD–CAM technology in customization of the scaffold, and the in vivo biomimetic properties of the composite biomaterial.

Do progenitor cells from different tissue have the same phenotype?

The purpose of this work was to validate isolation methods for sheep mesenchymal stem cells (MSC) from different sources and to explore the hypothesis that MSC exhibit markers of the same phenotype independent from tissue source. Cells derived from ovine bone marrow, synovial membrane and adipose tissue were characterized using the following markers: CD44, CD45, CD11b and MHC-I. The isolated MSC were cultivated, went through osteogenic, chondrogenic and adipogenic differentiation, and were characterized by flow cytometry using mouse anti-ovine CD44, CD45 and MHC-I monoclonal antibody (mAb), and mouse anti-bovine CD11b mAb. Ovine MSC from all three sources differentiated under chondorgenic, osteogenic and adipogenic conditions. Also, MSC from the three tissues were found to express CD44 and MHC-I but lack of CD11b and CD45. The results obtained revealed that our isolation methods for the different tissues tested are valid and that MSC from the three sources studied have same immunophenotic characteristics.

Differentiation potential and GFP labeling of sheep bone marrow‐derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are an important cell population in the bone marrow microenvironment. MSCs have the capacity to differentiate in vitro into several mesenchymal tissues including bone, cartilage, fat, tendon, muscle, and marrow stroma. This study was designed to isolate, expand, and characterize the differentiation ability of sheep bone marrow-derived MSCs and to demonstrate the possibility to permanently express a reporter gene. Bone marrow was collected from the iliac crest and mononuclear cells were separated by density gradient centrifugation. Sheep MSCs cell lines were stable characterized as CD44+ and CD34- and then transfected with a green fluorescent protein (GFP) reporter gene. The GFP expression was maintained in about half (46.6%) of cloned blastocysts produced by nuclear transfer of GFP+ sheep MSCs, suggesting the possibility to establish multipotent embryonic cells' lines carrying the fluorescent tag for comparative studies on the differentiation capacity of adult stem cells (MSCs) versus embryonic stem cells. We found that sheep MSCs under appropriate culture conditions could be induced to differentiate into adipocytes, chondrocytes, and osteoblast lineages. Our results confirm the plasticity of sheep MSCs and establish the foundation for the development of a pre-clinical sheep model to test the efficiency and safety of cell replacement therapy.

Mesenchymal stem cell culture in convection-enhanced hollow fibre membrane bioreactors for bone tissue engineering

Preparation of tissue engineered bone constructs to repair large size defects is limited by the difficult supply of oxygen and nutrients to cells deep inside the constructs. Hollow fibre membrane bioreactors (HFMBs) with medium flowing in membrane lumen have structural and functional analogy with cortical bone. In fact, membranes resemble the Haversian canals and provide for a distributed and delocalized source of nutrients and oxygen to surrounding cells. HFMBs where oxygen and nutrients diffuse to cells have been proposed for bone tissue engineering. HFMBs may also be so operated as to promote spontaneous Starling flows to enhance nutrients transfer to cells. In this study, the effect of low and high Starling flows was investigated on sheep mesenchymal stem cells (shMSC) cultured in the extracapillary space of HFMBs with respect to cell distribution, proliferation and early differentiation. After 12 days of culture, at low Starling flows cells formed thin layers around the membranes uniformly along the bioreactor. At high Starling flows, cells proliferated well and formed thick multilayer aggregates filling the space among membranes at the bioreactor outlet. These results make it possible to postulate the use of HFMBs for obtaining tissue engineered constructs for the repair of large bone defects.

Directly auto‐transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

Bone tissue engineering approaches increasingly focus on the use of mesenchymal stem cells (MSC). In most animal transplantation models MSC are isolated and expanded before auto cell transplantation which might be critical for clinical application in the future. Hence this study compares the potential of directly auto-transplanted versus in vitro expanded MSC with or without bone morphogenetic protein-2 (BMP-2) to induce bone formation in a large volume ceramic bone substitute in the sheep model. MSC were isolated from bone marrow aspirates and directly auto-transplanted or expanded in vitro and characterized using fluorescence activated cell sorting (FACS) and RT-PCR analysis before subcutaneous implantation in combination with BMP-2 and β-tricalcium phosphate/hydroxyapatite (β-TCP/HA) granules. Constructs were explanted after 1 to 12 weeks followed by histological and RT-PCR evaluation. Sheep MSC were CD29+, CD44+ and CD166+ after selection by Ficoll gradient centrifugation, while directly auto-transplanted MSC-populations expressed CD29 and CD166 at lower levels. Both, directly auto-transplanted and expanded MSC, were constantly proliferating and had a decreasing apoptosis over time in vivo. Directly auto-transplanted MSC led to de novo bone formation in a heterotopic sheep model using a β-TCP/HA matrix comparable to the application of 60 μg/ml BMP-2 only or implantation of expanded MSC. Bone matrix proteins were up-regulated in constructs following direct auto-transplantation and in expanded MSC as well as in BMP-2 constructs. Up-regulation was detected using immunohistology methods and RT-PCR. Dense vascularization was demonstrated by CD31 immunohistology staining in all three groups. Ectopic bone could be generated using directly auto-transplanted or expanded MSC with β-TCP/HA granules alone. Hence BMP-2 stimulation might become dispensable in the future, thus providing an attractive, clinically feasible approach to bone tissue engineering.

Matrix-Associated Implantation of Predifferentiated Mesenchymal Stem Cells Versus Articular Chondrocytes

Background: The use of predifferentiated mesenchymal stem cells (MSC) leads to better histological results compared with undifferentiated MSC in sheep. This raises the need for a longer term follow-up study and comparison with a clinically established method. Hypothesis: We hypothesized that chondrogenic in vitro predifferentiation of autologous MSC embedded in a collagen I hydrogel leads to better structural repair of a chronic osteochondral defect in an ovine stifle joint after 1 year. We further hypothesized that resulting histological results would be comparable with those of chondrocyte-seeded matrix-associated autologous chondrocyte transplantation (MACT). Study Design: Controlled laboratory study. Methods: Predifferentiation period of ovine MSC within collagen gel in vitro was defined by assessment of several cellular and molecular biological parameters. For the animal study, 2 osteochondral lesions (7-mm diameter) were created at the medial femoral condyles of the hind legs in 9 sheep. Implantation of MSC gels was performed 6 weeks after defect creation. Thirty-six defects were divided into 4 treatment groups: (1) chondrogenically predifferentiated MSC gels (pre-MSC gels), (2) undifferentiated MSC gels (un-MSC gels), (3) MACT gels, and (4) untreated controls (UC). Histological, immunohistochemical, and radiological evaluations followed after 12 months. Results: After 12 months in vivo, pre-MSC gels showed significantly better histological outcome compared with un-MSC gels and UC. Compared with MACT gels, the overall scores were higher for O’Driscoll and International Cartilage Repair Society (ICRS). The repair tissue of the pre-MSC group showed immunohistochemical detection of interzonal collagen type II staining. Radiological evaluation supported superior bonding of pre-MSC gels to perilesional native cartilage. Compared with previous work by our group, no degradation of the repair tissue between 6 and 12 months in vivo, particularly in pre-MSC gels, was observed. Conclusion: Repair of chronic osteochondral defects with collagen hydrogels composed of chondrogenically predifferentiated MSC shows no signs of degradation after 1 year in vivo. In addition, pre-MSC gels lead to partially superior histological results compared with articular chondrocytes. Clinical Relevance: The results suggest an encouraging method for future treatment of focal osteochondral defects without donor site morbidity by harvesting articular chondrocytes.

Cell Distribution in a Scaffold with Random Architectures under the Influence of Fluid Dynamics

Fluid dynamic environment and scaffold architectures have an important influence on cell growth and distribution inside the scaffold. A porous cylindrical scaffold with a central channel is seeded with the sheep mesenchymal stem cells (MSCs) in this study. Then the cell seeded scaffold is continuously perfused with alpha-MEM medium by a peristaltic pump for 7, 14, and 28 days. Histological study shows that the cell proliferation rates are different throughout the whole scaffolds. The different cell coverage is shown in various positions of the scaffold. A computational fluid dynamics (CFD) modeling is used to simulate the flow conditions within perfused cell-seeded scaffolds to give insight into the mechanisms of these cell growth phenomena. Relating the simulation results to perfusion experiments, the even fluid velocity (approximately 0.26-0.64 mm/s) and shear stress (approximately 0.0029-0.027 Pa) are found to correspond to increased cell proliferation within the cell-scaffold constructs. This method exhibits novel capabilities to compare results obtained for different perfusion rates or different scaffold microarchitectures and may allow specific fluid velocities and shear stresses to be determined that optimize the perfusion flow rate, porous scaffold architecture, and distribution of in vitro tissue growth.

The growth of stem cells within β-TCP scaffolds in a fluid-dynamic environment

A three-dimensional dynamic perfusion system was developed to provide mass transport and nutrient supply to permit the cell proliferation during the long-term culture inside a β-tricalcium phosphate (β-TCP) scaffold. Also the flow field throughout the scaffold was studied. The porous cylindrical scaffold with a central channel was seeded with the sheep mesenchymal stem cells (MSCs). Then the cell-seeded scaffolds were continuously perfused with the complete α-MEM medium by a peristaltic pump for 7, 14 and 28 days, respectively. Histological study showed that the cell proliferation rates were different throughout the whole scaffolds and the different cell coverage was shown in different positions of the scaffold. Unoccupied spaces were found in many macropores. A computational fluid dynamics (CFD) modeling was used to simulate the flow conditions within perfused cell-seeded scaffolds to give an insight into the mechanisms of these cell growth phenomena. Relating the simulation results to perfusion experiments, the even fluid velocity (approximately 0.52 mm/s) and shear stress (approximately 0.0055 Pa) were found to correspond to increased cell proliferation within the cell–scaffold constructs. Flow speeds were between 0.25 and 0.75 mm/s and shear stresses were between 0.003 and 0.008 Pa in approximately 75% of the regions. This method exhibits novel capabilities to compare the results obtained for different perfusion rates or different scaffold microarchitectures. It may allow specific fluid velocities and shear stresses to be determined to optimize the perfusion flow rate, porous scaffold architecture and distribution of in vitro tissue growth.

Adult Stem Cells: Perspectives for Therapeutic Applications

The use of adult stem cells in tissue regeneration appears to be a powerful research tool, due to the intrinsic characteristics of these cells, i.e., self-renewal and unlimited capacity for proliferation. In particular, mesenchymal stem cells (MSCs) obtained from bone marrow or peripheral blood can be easily isolated, cultivated, propagated and can be differentiated into several specialized cell types thanks to their plasticity. Among these cells, MSCs can evolve into cardiac cell lineages. Since heart damage leads to the irreversible loss of cardiac function, cell transplantation could be a potential therapy for heart injury. Our laboratory has focused on the purification and expansion of rat and sheep MSCs, their differentiation into cardiomyocytes and their characterisation. Numerous results indicate that MSCs could be promising for therapy, however we need to better understand the biology of stem cells to improve methods for delivery and/or pharmacological activation. These techniques can indeed track engrafted cells and systems to guarantee their safe use.

Three-Dimensional Flow Perfusion Culture System for Stem Cell Proliferation Inside the Critical-Size β-Tricalcium Phosphate Scaffold

A 3-dimensional flow perfusion system has been created in our laboratory to provide continuous and homogeneous nutrient supply inside the critical-size beta-tricalcium phosphate (beta-TCP) scaffold and permit cell proliferation during long-term incubation. The critical-size porous cylindrical scaffold (14 mm in diameter, 30 mm in length) with a central tunnel was impregnated with sheep mesenchymal stem cells. In the flow perfusion group, the hybrid scaffolds were continuously perfused with complete alpha-minimum essential medium via a peristaltic pump for 7, 14, and 28 days. In the static culture group, the hybrid composites were immersed in the medium without perfusion for 14 and 28 days. The daily glucose consumption was much higher in the flow perfusion group than in the static group (p < 0.001). In the flow perfusion group, glucose consumption increased dramatically in the first 14 days, and the increase slowed in the last 14 days. In the static group, the increase occurred only in the first 14 days. Cell viability via MTT colorimetry increased with time, which coincided with the results of glucose consumption. Histological study showed that the cells proliferated through the whole scaffolds under the flow perfusion culture. While under the static culture, the cells survived and proliferated only inside the first to third rows of the macropores under the scaffold surface. The cell quantity increased with time under flow perfusion culture. The results suggest that flow perfusion culture is superior to static culture for mesenchymal stem cell proliferation in the critical-size porous scaffold. This perfusion culture system permits a constant nutrition supply into the center of a large-scale scaffold for at least 4 weeks. Determination of D-glucose in the culture medium is a noninvasive way to survey cell proliferation in this system.