Bio-Medical Materials and Engineering

Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been increasing interest in the study of undifferentiated progenitors that have ability to proliferate and differentiate in different tissues. Different stem cells (SC) with different potential can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult stem cells provide a more interesting approach to clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue or MSC of Wharton Jelly, which have limited potential, are of interest for clinical applications in regenerative medicine because they are easily separated and prepared and no ethical problems are involved in their use.

During the last 10 years, these multipotent cells have generated considerable interest and in particular have been shown to escape allogeneic immune response and be capable of immunomodulatory activity. These properties may be of a great interest for regenerative medicine. Different clinical applications are under study (cardiac insufficiency, atherosclerosis, stroke, bone, cartilage, diabetes, ophthalmology, urology, liver, organ's reconstruction…).

INTRODUCTION: Stem cells for autologous and allogenic transplantation are obtained from several sources including bone marrow, peripheral blood or cord blood. Accurate enumeration of viable CD34+ hematopoietic stem cells (HSC) is routinely used in clinical settings, especially to monitor progenitor cell mobilization and apheresis. The number of viable CD34+ HSC has also been shown to be the most critical factor in haematopoietic engraftment. The International Society for Cellular Therapy actually recommends the use of single-platform flow cytometry system using 7-AAD as a viability dye.

AIM: In a way to move routine analysis from a BD FACSCalibur^TM instrument to a BD FACSCanto^TM II, according to ISO 15189 standard guidelines, we define laboratory performance data of the BD^TM Stem Cell Enumeration (SCE) kit on a CE-IVD system including a BD FACSCanto II flow cytometer and the BD FACSCanto^TM Clinical Software. InterQC^TM software, a real time internet laboratory QC management system developed by Vitro^TM and distributed by Becton Dickinson^TM, was also tested to monitor daily QC data, to define the internal laboratory statistics and to compare them to external laboratories.

METHODS: Precision was evaluated with BD^TM Stem Cell Control (high and low) results and the InterQC software, an internet laboratory QC management system by Vitro. This last one drew Levey–Jennings curves and generated numeral statistical parameters allowing detection of potential changes in the system performances as well as interlaboratory comparisons. Repeatability, linearity and lower limits of detection were obtained with routine samples from different origins. Agreement evaluation between BD FACSCanto II system versus BD FACSCalibur system was tested on fresh peripheral blood, freeze-thawed apheresis, fresh bone marrow and fresh cord blood samples.

RESULTS: Instrument's measure and staining repeatability clearly evidenced acceptable variability on the different samples tested. Intra- and inter-laboratory CV in CD34+ cell absolute count are consistent and reproducible. Linearity analysis, established between 2 and 329 cells/μl showed a linear relation between expected counts and measured counts (R²=0.97). Linear regression and Bland–Altman representations showed an excellent correlation on samples from different sources between the two systems and allowed the transfer of routine analysis from BD FACSCalibur to BD FACSCanto II.

CONCLUSIONS: The BD SCE kit provides an accurate measure of the CD34 HSC, and can be used in daily routine to optimize the enumeration of hematopoietic CD34+ stem cells by flow cytometry. Moreover, the InterQC system seems to be a very useful tool for laboratory daily quality monitoring and thus for accreditation.

Under physiological conditions, there is a production of limited range of free radicals. However, when the cellular antioxidant defence systems, overwhelm and fail to reverse back the free radicals to their normal basal levels, there is a creation of a condition of redox disequilibrium termed “oxidative stress”, which is implicated in a very wide spectrum of genetic, metabolic, and cellular responses. The excess of free radicals can, cause unfavourable molecular alterations to biomolecules through oxidation of lipids, proteins, RNA and DNA, that can in turn lead to mutagenesis, carcinogenesis, and aging. Mesenchymal stem cells (MSCs) have been proven to be a promising source of cells for regenerative medicine, and to be useful in the treatment of pathologies in which tissue damage is linked to oxidative stress. Moreover, MSCs appeared to efficiently manage oxidative stress and to be more resistant to oxidative insult than normal somatic cells, making them an interesting and testable model for the role of oxidative stress in the aging process. In addition, aging is accompanied by a progressive decline in stem cell function, resulting in less effective tissue homeostasis and repair. Also, there is an obvious link between intracellular reactive oxygen species levels and cellular senescence. To date, few studies have investigated the promotion of aging by oxidative stress on human MSCs, and the mechanism by which oxidative stress induce stem cell aging is poorly understood. In this context, the aim of this review is to gain insight the current knowledge about the molecular mechanisms of aging-induced oxidative stress in human MSCs.

A series of composite films were prepared from glycerol-plasticized starch and zein by intensive mixing and hot press. The structure and physical properties of the starch/zein (SZ) composite films were characterized by scanning electron microscope (SEM), optical microscopy and water contract angle testing. The hemocompatibility and cytocompatibility of SZ films were evaluated by plasma recalcification time, hemolysis assay and cell culture experiment. SEM and optical observation showed that starch and zein domains can be differed in the films and in a two phase separation status. Glycerol affects the surface hydrophilicity/hydrophobicity of the films. The hemocompatibility and cytocompatibility evaluation showed that SZ composites are anticoagulant materials with no hemolysis and low cytotoxicity. The SZ composites maybe have potentials for applications as biomaterials.

OBJECTIVE: To evaluate the in vitro cytocompatibility of cellulose/soy protein isolate composite membranes (CSM) with Schwann cells and in vivo toxicity to animals.

METHODS: A series of cellulose/soy protein isolate composite membranes (CSM) were prepared by blending, solution casting and coagulation process. The cytocompatibility of the CSM to Schwann cells were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by direct cells culture of Schwann cells on the surfaces of the CSM, respectively. The in vivo toxicity of the CSM to animals were also evaluated by acute toxicity testing, skin sensitization testing, pyrogen testing and intracutaneous stimulation testing, respectively, according to the ISO 10993 standard.

RESULTS: The MTT assay showed that the cell viability of Schwann cells cultured in extracts from the CSM was higher than that from the neat cellulose membrane without containing SPI component. The direct cells culture indicated that the Schwann cells could attach and grow well on the surface of the CSM and the incorporation of SPI into cellulose contributed to improvement of cell adhesion and proliferation. The evaluations of in vivo biological safety suggested that the CSM showed no acute toxicity, no skin sensitization and no intracutaneous stimulation to the experimental animals.

CONCLUSION: The CSM had in vitro cytocompatibility with Schwann cells and biological safety to animals, suggesting potential for the applications as nerve conduit for the repair of nerve defect.

The scaffolds prepared from the tissue decellularization conserve the porous 3-D structure and provide an optimal matrix for the tissue regeneration. Since decade, the enzymatic digestion, chemical reagent treatment and mechanical actions such as eversion and abrasion have been used to remove the cells from the intact matrix. In this study, we optimized an enzymatic method to decellularize the umbilical artery to construct a 3-D porous scaffold which is suitable for the culture of mesenchymal stem cells (MSCs). The scaffold maintained the interconnected porous structure. It remained the similar high water content 95.3±1% compared to 94.9±0.6% in the intact umbilical artery (p>0.05). The decellularization process decreased the stress from 0.24±0.05 mPa to 0.15±0.06 mPa (p<0.05). However the decellularization did not change the strain of the artery (45±15% vs. 53±10%, p>0.05). When the scaffold was transplanted to the subcutaneous tissue in the wild type mice, there were less T cells appeared in the surrounding tissue which meant the decreased the immunogenicity by decellularization. This scaffold also supported the adhesion and proliferation of the MSCs. In this study, we constructed a biological compatible porous scaffold from the decellularized umbilical artery which may provide a suitable scaffold for cell-matrix interaction studies and for tissue engineering.

Because many of patients with peripheral arterial disease are not eligible for direct or conventional revascularization procedures, because stem cell therapy is being investigated as to its possible role in the treatment of limb ischemia, there is a need to evaluate this treatment and his true application. On the basis of experimental data, preliminary clinical studies have established the safety and feasibility of stem cells implantation in case of critical limb ischemia. Forthcoming large studies, especially randomized placebo controlled double blind studies, related to the optimal cell type, dosage, administration route, will consolidate this evidence and establish mid and long term effectiveness.

Designing unique nanostructured biomimetic materials is a new challenge in modern regenerative medicine. In order to develop functional substitutes for damaged organs or tissues, several methods have been used to create implants able to regenerate robust and durable bone. Electrospinning produces nonwoven scaffolds based on polymer nanofibers mimicking the fibrillar organization of bone extracellular matrix. Here, we describe a biomimetic 3D thick nanofibrous scaffold obtained by electrospinning of the biodegradable, bioresorbable and FDA-approved polymer, poly(ε-caprolactone). Such scaffold presents a thickness reaching one centimeter. We report here the demonstration that the designed nanostructured implant is able to induce in vivo bone regeneration.

OBJECTIVES: Chondrocytes hypertrophy is a physiological process observed in endochondral ossification during development until adolescence in human. It can also be observed during pathophysiological conditions such as osteoarthritis. Hypertrophic chondrocytes synthesise collagen X and express matrix metalloproteinase 13 and alkaline phosphatase. The cellular models available to study this process are either not convenient, they might lead to a rapid dedifferentiation of chondrocytes, or they are far from the physiological conditions. The objective of this study was to design an user-friendly 2D-primary cell culture of young articular chondrocytes of rat able to follow the terminal differentiation process.

EXPERIMENTAL DESIGN: After confluence, chondrocytes were cultured according to 4 differentiation protocols. Protocol 1 contained DMEM/F12 supplemented with 10% foetal bovine serum (FBS) and 2 μg/ml insulin. Protocol 2 contained alpha-MEM supplemented with 5% FBS and 2 μg/ml insulin. Protocol 3 contained 2% FBS and 2 μg/ml insulin. Protocol 4 contained DMEM/F12 supplemented with 2% FBS in absence or in presence of 2 μg/ml insulin and 37.5 μg/ml ascorbate. The cell morphology was observed by phase-contrast microscopy and the expression of markers specific of mature and hypertrophic chondrocytes were assessed by RT-qPCR.

RESULTS: The effect of a decrease in nutrient quality of the culture medium after confluence was tested using protocols 1, 2 and 3. Protocol 1 did not allow the maintenance of chondrocyte phenotype more than one week, because cells became fibroblastic. A decrease in Sox9 mRNA expression, in collagen II/collagen I and in aggrecan/versican mRNA ratios was also found with protocol 1. Protocol 3 was the best when compared with protocols 1 and 2. It allowed chondrocytes to adopt a hypertrophic morphology. Cells also expressed the collagen X specific hypertrophic marker, and presented an increase in collagen II/I and aggrecan/versican ratios after 15 days of culture post-confluence. The effect of the insulin/ascorbate supplementation was studied using protocol 4. The insulin/ascorbate supplementation allowed an earlier chondrocytes conversion to terminal differentiation with a prolonged effect till 3 weeks post-confluence, compared to control without insulin/ascorbate. Finally, the profile of chondrocyte differentiation was checked during 5 successive sub-cultures. Only the first passage could be used to study hypertrophy.

CONCLUSION: A convenient protocol to study chondrocyte hypertrophy is proposed. Protocol 4 offers the possibility to study this differentiation phenotype which is crucial for the development of articular diseases such as osteoarthritis. Our model could also be used in tissue engineering for cartilage repair strategies in which hypertrophic differentiation of chondrocyte should be avoided.

INTRODUCTION: Few studies have tried to discriminate a differential behavior between osteoarthritic (OA) osteoblasts (Obs). Based on osteocalcin level, we aimed, in the present study, to evaluate the capacity of OA Obs for producing molecules of the Wnt/β-catenin signaling pathway.

METHODS: Human primary OA Obs (n=11) were exposed or not to 50 nM of 1,25 dihydroxyvitamin D₃ (VitD₃) for 24 h. Osteocalcin (OCN), TGF-β1, Dickkopf-related protein 2 (DKK2), R-spondin 2 (Rspo2), Wnt5b, and low density lipoprotein related-receptor 1 (LRP1) were evaluated by real time RT-PCR.

RESULTS: All samples responded to VitD₃ as validated by the increase in OCN expression. However two populations of Obs were discriminated; one called “high responders” whose OCN stimulation was higher than 100 fold (mean 881 fold, p<0.01, n=5) and the second one whose stimulation was inferior to 100 fold (mean 47 fold, p<0.01, n=6), namely “low responders”. In fact, high responders have a weaker basal expression of OCN. With regards to these two cell populations and in absence of VitD₃ challenge, the expression level of TGF-β1 (15 fold, p<0.001), DKK2 (2.5 fold, p<0.002) and Wnt5b (5.5 fold, p<0.003) was higher in “high responders”, meanwhile Rspo2 and LRP1 expression was unchanged. VitD₃ exacerbated this pattern but corrected OCN expression and favored Wnt agonist expression.

CONCLUSION: We identified 2 populations of OA Obs according to the OCN expression under the control of VitD₃. In addition under basal conditions, these 2 populations expressed differently TGF-β1, Wnt5b, DKK2, suggesting a heterogeneous differentiation and phenotype in Obs among OA patients.

X-linked Hypohidrotic Ectodermal Dysplasia (XLHED) is associated to a large spectrum of ectodermal and extra-ectodermal symptoms, especially craniofacial bone morphological, structural and metabolic anomalies. This skeletal phenotype described in affected patients and in the Ta mutant mouse model leads to craniofacial dysmorphies, endosseous implants and jaw bone grafts complications. Bone tissue bioengineering based on the use of PCL synthetic nanofibrous membrane and BMP nanoreservoirs appears as an original and promising approach to prevent such complications in the context of dysfunctional bone. Use of osteoblasts or stem cells seeded biomembranes appears as another strategy developed on the Tabby (Ta) model of XLHED. The Ta mouse experimental model is used to study the jaw bone response during the post-operative period after bone lesion and placement of synthetic PCL membrane functionalized with nanoreservoirs embedding different BMPs dimers or seeded with living cells.

Chitosan has been used as scaffolds with various methods of fabrication including expensive commercial available ones for tissue engineering. The objective of this study is to assemble our novel method of chitosan scaffold fabrication in economical and uncomplicated way that suitable for dental pulp stem cell (DPSC) and stem cells of human exfoliated deciduous teeth (SHED). Chitosan scaffolds (2% and 3%) were fabricated in an uncomplicated procedure, including centrifugation and freeze-drying steps. The chitosan scaffolds were compared and the pore size, swelling and degradation were assessed. In addition, the cytocompatibility was assessed of chitosan scaffolds seeded with DPSC and SHED. The pore size of 2% and 3% chitosan scaffolds were similar being 188.71±51.90 μm and 195.30±67.21 μm, respectively. Swelling ratios of 3% chitosan scaffolds were significantly lower than those of 2% chitosan scaffolds. Dimension of scaffolds changed in first 5 minutes. After that, those scaffolds could maintain their dimension. Chitosan scaffolds degraded as from day 7. No differences were found between 2% and 3% chitosan scaffolds. The scaffolds were shown to be non-toxic and to promote DPSCs and SHED growth. The viability of DPSCs and SHED on 2% scaffolds proved to be higher than that of the 3% scaffold group. This study suggested that chitosan scaffolds fabricated with our novel method were suitable for the growth and survival of DPSC and SHED.

The shortage of organ resource has been limiting the application of liver transplantation. Bioartificial liver construction is increasingly focused as a replacement treatment. To product a bioartificial liver, three elements must be considered: seeding cells, scaffold and bioreactor. Recent studies have shown that several methods can successfully differentiate MSC (mesenchymal stem cells) derived from Wharton's jelly into hepatocyte, such as stimulating MSC by cytokines and growth factors, direct and indirect co-culture MSC with hepatocytes, or promote MSC differentiation by 3-dimensional matrix. In some cases, differentiation of MSC into hepatocytes can also be an alternative approach for whole organ transplantation in treatment of acute and chronic liver diseases. In this review, the characterization of MSC from Wharton's jelly, their potential of application in liver tissue engineering on base of decellularized scaffold, their status of banking and their preclinical work performed will be discussed.

OBJECTIVE: The stem cell based therapy is a potential alternative to liver transplantation. The aim of the study is to investigate the hepatocytic differentiation ability of human umbilical cord derived mesenchymal stem cells (HUC-MSCs) in vitro.

METHODS: The HUC-MSCs were isolated from Wharton's jelly of human umbilical cord. The cells were identified by assessing the stem cell markers. The HUC-MSCs were characterized by multipotency of differentiation. We modified the hepatogenic differentiation protocol and examine the function of differentiated cell by Periodic acid-Schiff (PAS) staining and Low-Density Lipoprotein (LDL) uptake. The protein expressions of Total protein (TP), Albumin (ALB), Globulin (GLB), Urea (BUN) and α-fetoprotein (AFP) were also detected.

RESULTS: The cells of the hepatogenic differentiation group showed the function of hepatocyte. Protein expressions of TP, ALB, GLB BUN and AFP improve that the HUC-MSCs are able to different into the functional hepatogenic-like cell.

CONCLUSION: The above founding indicated that the HUC-MSCs are able to different into the functional hepatogenic-like cell by present method.

OBJECTIVES: In present study, we plan to produce a decellularization protocol from rat liver to generate a three-dimensional whole organ scaffold.

METHODS: A combination of 1% SDS and 1% tritonX-100 were used orderly to decellularize rat livers. After about 6 h of interactive antegrade/retrograde perfusion, a decellularized whole translucent liver scaffold with integrated blood vessel networks was generated. The decellularized livers are charactered by light microscopy, scanning electron microscopy, and biochemical analysis (DNA quantification) for preservation of the three-dimension of extracellular matrix architecture.

RESULTS: The decellularization protocol was verified by observation of the whole translucent liver organ with intact vascular trees under macroscopy, in conjunction with the hematoxylin–eosin staining that showed no cells or nuclear material remained. Additionally, the Masson's stain indicted that the extracellular proteins were well kept and scanning electron microscopy (SEM) revealed a preserved decellularized matrix architecture. Compared to normal livers, DNA in the decellularized livers was quantified less than 10% at the same mass.

CONCLUSIONS: The current method of decellularization protocol was feasible, simple and quick, and was verified by an absence of residual cells. The decellularized extracellular matrix had preserved integrate vascular network and a three-dimensional architecture.

Bone marrow mesenchymal stem cells (BMSCs) and myeloid-derived suppressor cells (MDSCs) can be mobilized from bone marrow (BM) into blood stream and home in tumor stroma, where they either help or hinder tumor growth. The issue of whether BMSCs could affect MDSCs in ascitogenous hepatoma BALB/c mice, thus influencing their functional activity, remains unclear. In this study, we demonstrated that after transfusion into ascitogenous hepatoma BALB/c mice, the homing fraction of BMSCs in BM was 2%–5% in 24–72 h and the percentage of Gr-1⁺CD11b⁺ MDSCs was downregulated in peripheral blood (PB) and BM. Meanwhile, IFN-γ⁺ T lymphocytes in PB increased. As a result of such immunoregulation, BMSCs treatment caused a delayed tumor growth and a prolonged survival in H22 ascitogenous hepatoma model. Because the proliferation of H22 cells was not affected by in vitro coculture with BMSCs, this observation is likely due to a systemic suppressive effect on the host MDSCs. We also demonstrated that BMSCs inhibited the induction and proliferation of MDSCs from hematopoietic stem cells (HSCs) in an in vitro tumor conditioned medium. Thus, our findings show for the first time that BMSCs are potentially inhibitor during MDSCs induction and proliferation and that when injected intravenously into tumor bearing mice they might be effective antitumor agents suitable for cancer therapy.

The dried root of Angelica sinensis is widely used in Chinese traditional medicine for its beneficial effects against several diseases, including osteoarthritis. In order to understand the mechanism of action, two main components of the plant, the phytochemical, sodium ferulate, and a polysaccharidic fraction have been tested on osteoarthritis animal models or in human chondrocytes stimulated by the pro-inflammatory cytokine, Interleukine-1β. The results showed that sodium ferulate exhibited marked anti-inflammatory and anti-apoptotic properties by inhibiting the TNF/TNFR signal transduction pathway. On the other hand, the polysaccharidic fraction which contains a mixture of various carbohydrates was found to promote proteoglycan biosynthesis in cartilage matrix by stimulating the activity of the UDP-glycosyltransferases that synthesize the chondroitin sulfate chains of aggrecans. It is suggested that the combined action of sodium ferulate and polysaccharidic fraction would prevent cartilage destruction in osteoarthritis and favor cartilage repair.

BACKGROUND: Non-union of long bones is still a current problem in traumatology. Although corticocancellous bone autograft remains the usual procedure for the treatment of non-union, innovative therapies such as, percutaneous autologous concentrated bone marrow grafting (PABMG), are now appearing.

MATERIAL AND METHODS: Over a period of 8 years, 45 non-union of long bones were treated by PABMG in the Department of Orthopaedic and Traumatologic Surgery (University Hospital of Nancy, France): 26 tibiae, 16 femurs, 3 humeri. Efficiency was evaluated by clinical criteria: full weight-bearing without pain, absence of motion at non-union site, and radiological criteria: healing of 3 corticales out of 4.

RESULTS: Eighteen out of 28 non-unions at the tibia were healed (69%), 10 at the femur (63%), but none was noticed at the humerus. Some pejorative prognosis factors were noted such as: tobacco, alcohol abuse, diabetes and history of infection at the fracture site. An earlier grafting improved the success rate. The number of CFU-F (Colony Forming Unit Fibroblastic) affected the healing time more than the healing rate.

CONCLUSION: The procedure, even though a little invasive, enables the healing of non-union in two out of three cases with less morbidity than conventional procedures. This procedure fits perfectly into the therapeutic arsenal of non-union.

Recent advances of fundamental research on the in vitro generation of red blood cells (RBCs) from hematopoietic stem cells in the laboratory open new possibilities of the utilization of cultured RBCs in transfusion medicine. We study the economic challenge of the setup and development of the mass industrial production of RBCs in mature transfusion organizations. We argue that: (i) RBC manufacturing could be set up and developed in the short-medium run for the treatment of the small proportion of transfused patients who have a rare blood type or are alloimmunized against blood antigens; (ii) manufactured RBCs could substitute for donated RBCs in the long run if the physical productivity of RBC engineering technology approaches that of bone marrow.