Repopulation of decellularised articular cartilage by laser-based matrix engraving.

BACKGROUND: In spite of advances in the treatment of cartilage defects using cell and scaffold-based therapeutic strategies, the long-term outcome is still not satisfying since clinical scores decline years after treatment. Scaffold materials currently used in clinical settings have shown limitations in providing suitable biomechanical properties and an authentic and protective environment for regenerative cells. To tackle this problem, we developed a scaffold material based on decellularised human articular cartilage. METHODS: Human articular cartilage matrix was engraved using a CO2 laser and treated for decellularisation and glycosaminoglycan removal. Characterisation of the resulting scaffold was performed via mechanical testing, DNA and GAG quantification and in vitro cultivation with adipose-derived stromal cells (ASC). Cell vitality, adhesion and chondrogenic differentiation were assessed. An ectopic, unloaded mouse model was used for the assessment of the in vivo performance of the scaffold in combination with ASC and human as well as bovine chondrocytes. The novel scaffold was compared to a commercial collagen type I/III scaffold. FINDINGS: Crossed line engravings of the matrix allowed for a most regular and ubiquitous distribution of cells and chemical as well as enzymatic matrix treatment was performed to increase cell adhesion. The biomechanical characteristics of this novel scaffold that we term CartiScaff were found to be superior to those of commercially available materials. Neo-tissue was integrated excellently into the scaffold matrix and new collagen fibres were guided by the laser incisions towards a vertical alignment, a typical feature of native cartilage important for nutrition and biomechanics. In an ectopic, unloaded in vivo model, chondrocytes and mesenchymal stromal cells differentiated within the incisions despite the lack of growth factors and load, indicating a strong chondrogenic microenvironment within the scaffold incisions. Cells, most noticeably bone marrow-derived cells, were able to repopulate the empty chondrocyte lacunae inside the scaffold matrix. INTERPRETATION: Due to the better load-bearing, its chondrogenic effect and the ability to guide matrix-deposition, CartiScaff is a promising biomaterial to accelerate rehabilitation and to improve long term clinical success of cartilage defect treatment. FUNDING: Austrian Research Promotion Agency FFG ("CartiScaff" #842455), Lorenz Böhler Fonds (16/13), City of Vienna Competence Team Project Signaltissue (MA23, #18-08).

Stromal vascular fraction cells as biologic coating of mesh for hernia repair.

BACKGROUND: The interest in non-manipulated cells originating from adipose tissue has raised tremendously in the field of tissue engineering and regenerative medicine. The resulting stromal vascular fraction (SVF) cells have been successfully used in numerous clinical applications. The aim of this experimental work is, first to combine a macroporous synthetic mesh with SVF isolated using a mechanical disruption process, and to assess the effect of those cells on the early healing phase of hernia. METHODS: Human SVF cells combined with fibrin were used to coat commercial titanized polypropylene meshes. In vitro, viability and growth of the SVF cells were assessed using live/dead staining and scanning electron microscopy. The influence of SVF cells on abdominal wall hernia healing was conducted on immunodeficient rats, with a focus on short-term vascularization and fibrogenesis. RESULTS: Macroporous meshes were easily coated with SVF using a fibrin gel as temporary carrier. The in vitro experiments showed that the whole process including the isolation of human SVF cells and their coating on PP meshes did not impact on the SVF cells' viability and on their capacity to attach and to proliferate. In vivo, the SVF cells were well tolerated by the animals, and coating mesh with SVF resulted in a decrease degree of vascularity compared to control group at day 21. CONCLUSIONS: The utilization of SVF-coated mesh influences the level of angiogenesis during the early onset of tissue healing. Further long-term animal experiments are needed to confirm that this effect correlates with a more robust mesh integration compared to non-SVF-coated mesh.

Adipose-tissue-derived therapeutic cells in their natural environment as an autologous cell therapy strategy: the microtissue-stromal vascular fraction.

The prerequisite for a successful clinical use of autologous adipose-tissue-derived cells is the highest possible regenerative potential of the applied cell population, the stromal vascular fraction (SVF). Current isolation methods depend on high enzyme concentration, lysis buffer, long incubation steps and mechanical stress, resulting in single cell dissociation. The aim of the study was to limit cell manipulation and obtain a derivative comprising therapeutic cells (microtissue-SVF) without dissociation from their natural extracellular matrix, by employing a gentle good manufacturing practice (GMP)-grade isolation. The microtissue-SVF yielded larger numbers of viable cells as compared to the improved standard-SVF, both with low enzyme concentration and minimal dead cell content. It comprised stromal tissue compounds (collagen, glycosaminoglycans, fibroblasts), capillaries and vessel structures (CD31+, smooth muscle actin+). A broad range of cell types was identified by surface-marker characterisation, including mesenchymal, haematopoietic, pericytic, blood and lymphatic vascular and epithelial cells. Subpopulations such as supra-adventitial adipose-derived stromal/stem cells and endothelial progenitor cells were significantly more abundant in the microtissue-SVF, corroborated by significantly higher potency for angiogenic tube-like structure formation in vitro. The microtissue-SVF showed the characteristic phenotype and tri-lineage mesenchymal differentiation potential in vitro and an immunomodulatory and pro-angiogenic secretome. In vivo implantation of the microtissue-SVF combined with fat demonstrated successful graft integration in nude mice. The present study demonstrated a fast and gentle isolation by minor manipulation of liposuction material, achieving a therapeutically relevant cell population with high vascularisation potential and immunomodulatory properties still embedded in a fraction of its original matrix.

Repopulation of an auricular cartilage scaffold, AuriScaff, perforated with an enzyme combination.

Biomaterials currently in use for articular cartilage regeneration do not mimic the composition or architecture of hyaline cartilage, leading to the formation of repair tissue with inferior characteristics. In this study we demonstrate the use of "AuriScaff", an enzymatically perforated bovine auricular cartilage scaffold, as a novel biomaterial for repopulation with regenerative cells and for the formation of high-quality hyaline cartilage. AuriScaff features a traversing channel network, generated by selective depletion of elastic fibers, enabling uniform repopulation with therapeutic cells. The complex collagen type II matrix is left intact, as observed by immunohistochemistry, SEM and TEM. The compressive modulus is diminished, but three times higher than in the clinically used collagen type I/III scaffold that served as control. Seeding tests with human articular chondrocytes (hAC) alone and in co-culture with human adipose-derived stromal/stem cells (ASC) confirmed that the network enabled cell migration throughout the scaffold. It also guides collagen alignment along the channels and, due to the generally traverse channel alignment, newly deposited cartilage matrix corresponds with the orientation of collagen within articular cartilage. In an osteochondral plug model, AuriScaff filled the complete defect with compact collagen type II matrix and enabled chondrogenic differentiation inside the channels. Using adult articular chondrocytes from bovine origin (bAC), filling of even deep defects with high-quality hyaline-like cartilage was achieved after 6 weeks in vivo. With its composition and spatial organization, AuriScaff provides an optimal chondrogenic environment for therapeutic cells to treat cartilage defects and is expected to improve long-term outcome by channel-guided repopulation followed by matrix deposition and alignment. STATEMENT OF SIGNIFICANCE: After two decades of tissue engineering for cartilage regeneration, there is still no optimal strategy available to overcome problems such as inconsistent clinical outcome, early and late graft failures. Especially large defects are dependent on biomaterials and their scaffolding, guiding and protective function. Considering the currently used biomaterials, structure and mechanical properties appear to be insufficient to fulfill this task. The novel scaffold developed within this study is the first approach enabling the use of dense cartilage matrix, repopulate it via channels and provide the cells with a compact collagen type II environment. Due to its density, it also provides better mechanical properties than materials currently used in clinics. We therefore think, that the auricular cartilage scaffold (AuriScaff) has a high potential to improve future cartilage regeneration approaches.

Photobiomodulation of freshly isolated human adipose tissue-derived stromal vascular fraction cells by pulsed light-emitting diodes for direct clinical application.

A highly interesting source for adult stem cells is adipose tissue, from which the stromal vascular fraction (SVF)-a heterogeneous cell population including the adipose-derived stromal/stem cells-can be obtained. To enhance the regenerative potential of freshly isolated SVF cells, low-level light therapy (LLLT) was used. The effects of pulsed blue (475 nm), green (516 nm), and red (635 nm) light from light-emitting diodes applied on freshly isolated SVF were analysed regarding cell phenotype, cell number, viability, adenosine triphosphate content, cytotoxicity, and proliferation but also osteogenic, adipogenic, and proangiogenic differentiation potential. The colony-forming unit fibroblast assay revealed a significantly increased colony size after LLLT with red light compared with untreated cells, whereas the frequency of colony-forming cells was not affected. LLLT with green and red light resulted in a stronger capacity to form vascular tubes by SVF when cultured within 3D fibrin matrices compared with untreated cells, which was corroborated by increased number and length of the single tubes and a significantly higher concentration of vascular endothelial growth factor. Our study showed beneficial effects after LLLT on the vascularization potential and proliferation capacity of SVF cells. Therefore, LLLT using pulsed light-emitting diode light might represent a new approach for activation of freshly isolated SVF cells for direct clinical application.

Extracorporeal shock wave therapy in situ - novel approach to obtain an activated fat graft.

One of the mainstays of facial rejuvenation strategies is volume restoration, which can be achieved by autologous fat grafting. In our novel approach, we treated the adipose tissue harvest site with extracorporeal shock wave therapy (ESWT) in order to improve the quality of the regenerative cells in situ. The latter was demonstrated by characterizing the cells of the stromal vascular fraction (SVF) in the harvested liposuction material regarding cell yield, adenosine triphosphate (ATP) content, proliferative capacity, surface marker profile, differentiation potential and secretory protein profile. Although the SVF cell yield was only slightly enhanced, viability and ATP concentration of freshly isolated cells as well as proliferation doublings after 3 weeks in culture were significantly increased in the ESWT compared with the untreated group. Likewise, cells expressing mesenchymal and endothelial/pericytic markers were significantly elevated concomitant with an improved differentiation capacity towards the adipogenic lineage and enhancement in specific angiogenic proteins. Hence, in situ ESWT might be applied in the future to promote cell fitness, adipogenesis and angiogenesis within the fat graft for successful facial rejuvenation strategies with potential long-term graft survival.

In vitro adipogenesis of adipose-derived stem cells in 3D fibrin matrix of low component concentration.

This study evaluated the suitability of adipose-derived stem cells (ASCs) combined with fibrin matrix of variable composition for adipose tissue-equivalent formation in vitro. Therefore, undifferentiated ASCs were embedded in fibrin clots composed of 2 IU/ml thrombin and fibrinogen of varying concentrations (6.25, 12.5 and 25 mg/ml) and kept under control or adipogenic conditions. Fibrin-cell composites were evaluated by scanning electron microscopy, live/dead staining, lactate-dehydrogenase (LDH) assay, quantitative PCR for the adipogenic markers fatty acid binding protein 4 (FABP4), peroxisome proliferative activated receptor-γ (PPARγ) and leptin, leptin ELISA and oil red O staining. Cells were found homogeneously distributed throughout the clot. Their number increased to day 7 (up to 3.62-fold median) and decreased thereafter until day 28. The proliferation was unaffected by fibrinogen concentration in the control. Adipogenic conditions generally yielded higher cell numbers, which were in addition increasing with increasing fibrinogen concentrations. FABP4, PPARγ and leptin mRNA expression was strongly upregulated by adipogenic medium, which was confirmed by the levels of leptin secretion and lipid vesicles formation demonstrated by oil red O staining. When embedded in 25 mg/ml fibrinogen clots, ASCs showed the highest expression levels of FABP4 (up to 629.0-fold), PPARγ (up to 1.6-fold) and leptin (up to 57.9-fold), corroborated by significantly elevated leptin secretion (median 33.29 ng/ml) on day 14. Constructs composed of fibrin matrix of low component concentrations-allowing homogeneous cell distribution-with ASCs should represent a suitable strategy for adipose tissue formation in vivo.

Meeting report of the first conference of the International Placenta Stem Cell Society (IPLASS).

The International Placenta Stem Cell Society (IPLASS) was founded in June 2010. Its goal is to serve as a network for advancing research and clinical applications of stem/progenitor cells isolated from human term placental tissues, including the amnio-chorionic fetal membranes and Wharton's jelly. The commitment of the Society to champion placenta as a stem cell source was realized with the inaugural meeting of IPLASS held in Brescia, Italy, in October 2010. Officially designated as an EMBO-endorsed scientific activity, international experts in the field gathered for a 3-day meeting, which commenced with "Meet with the experts" sessions, IPLASS member and board meetings, and welcome remarks by Dr. Ornella Parolini, President of IPLASS. The evening's highlight was a keynote plenary lecture by Dr. Diana Bianchi. The subsequent scientific program consisted of morning and afternoon oral and poster presentations, followed by social events. Both provided many opportunities for intellectual exchange among the 120 multi-national participants. This allowed a methodical and deliberate evaluation of the status of placental cells in research in regenerative and reparative medicine. The meeting concluded with Dr. Parolini summarizing the meeting's highlights. This further prepared the fertile ground on which to build the promising potential of placental cell research. The second IPLASS meeting will take place in September 2012 in Vienna, Austria. This meeting report summarizes the thought-provoking lectures delivered at the first meeting of IPLASS.

Isolation of pig bone marrow mesenchymal stem cells suitable for one-step procedures in chondrogenic regeneration.

Large animals such as pigs are good models for skeletal tissue engineering, since they provide physical forces similar to those of humans. Porcine bone marrow mesenchymal stem cells (BMSCs) have shown regenerative capacity similar to those of human BMSCs and can therefore be preclinically applied in settings corresponding to autologous transplantation in patients. Aiming at a one-step procedure for cartilage regeneration with autologous BMSCs, three straightforward isolation methods for BMSCs of Göttingen minipigs were compared. For this purpose, the BMSC fraction was enriched by red blood cell (RBC) lysis, dextran sedimentation or density gradient centrifugation. Isolated BMSCs were evaluated with regard to cell yield, proliferation capacity, phenotype and ability to differentiate to the chondrogenic lineage. Highest cell yields determined at the time of subcultivation were obtained using RBC lysis. In comparison, dextran sedimentation was less efficient but superior to density gradient centrifugation, which yielded significantly lower cell numbers than RBC lysis. The evaluated isolation methods resulted in cultures with equal proliferative capacity, with constant population doubling times of 50-55 h for at least 100 days (approximating to 40 cumulative population doublings) in vitro. Chondrogenic differentiation in micromass pellet cultures was evaluated by glycosaminoglycan quantification, histological staining with Alcian blue and safranin O and immunohistochemical analysis for collagen type II. These evaluations demonstrated that all three isolation methods yielded cells capable of generating cartilaginous tissue in vitro. According to our data, RBC lysis can be used to efficiently isolate porcine BMSCs in a short time frame which would allow for intraoperative one-step procedures in preclinical cartilage regeneration studies.

How chondrogenic are human umbilical cord matrix cells? A comparison to adipose-derived stem cells.

The umbilical cord matrix as well as liposuction material have been demonstrated to contain cells capable of differentiating towards the mesodermal lineage. High availability and low donor site morbidity appear promising for the use of human umbilical cord matrix cells (HUCMs) and adipose-derived stem cells (ASCs) in cell-based therapies. In the present study we focused on cartilage regeneration and compared HUCMs and ASCs regarding their potential to differentiate towards the chondrogenic lineage. Cells were isolated by explantation culture or enzymatic digestion, phenotypically characterized by flow cytometry and differentiated as 3D micromass pellets for up to 35 days. Under tested conditions, ASCs demonstrated significantly higher glycosaminoglycan synthesis compared to HUCMs. qRT-PCR data gave evidence that chondrogenic genes are expressed by both ASCs and HUCMs. However, higher expression levels of ASCs suggest that this cell type has higher potential for differentiation towards a cartilage-like phenotype than HUCMs. In conclusion, both cell types, HUCMs and ASCs, are easily available, possess typical properties of mesenchymal stem cells and are thus promising for cell-based therapies. However, in terms of cartilage regeneration, ASCs might be more suitable than HUCMs.

Impact of human amniotic membrane preparation on release of angiogenic factors.

Preserved amniotic membrane (AM) has been used in the field of ophthalmology and wound care due to its bacteriostatic, antiphlogistic, protease-inhibiting, re-epithelialization, wound-protecting and scar formation-reducing properties. Typically, AM is applied after banking in a glycerol-preserved or freeze-dried state. Cell viabilities in different forms of preparation vary substantially, which in consequence may also be reflected in the amount and type of growth factors released from the preserved material. Therefore, we characterized the angiogenic factor (AF) profile released from different AM preparations. For this, medium was conditioned with non-preserved, glycerol- and cryo-preserved AM for 48 h, which was screened for AFs using a protein array system. In parallel, the preparations were tested for cell viability. Non-preserved as well as cryo-preserved AM maintained viabilities at 106.5 +/- 23.9% and 21.9 +/- 23.3%, respectively, whereas glycerol-preserved AM was found to be non-viable. Of the 20 investigated factors, high levels of angiogenin, GRO, IL-6/8, TIMP-1/2 and MCP-1 and low levels of EGF, IFNgamma, IGF-1, leptin, RANTES, TGFbeta1 and thrombopoietin were identified to be secreted from non-preserved AM. Cryo-preserved AM secreted high levels of IL-8, intermediate levels of GRO and TIMP-1/2 but only low levels of angiogenin, IFNgamma, IL-6 and MCP-1 and no detectable EGF, IGF-1, leptin, RANTES, TGFbeta1 and thrombopoietin. After banking in glycerol, AM releases only minute amounts of TIMP-1/2. Along with viability, the AF profile of amniotic membrane largely depends on the preparation method applied for banking. This should be considered for selection of an AM product for a specific clinical application.

Mechanical strain using 2D and 3D bioreactors induces osteogenesis: implications for bone tissue engineering.

Fracture healing is a complicated process involving many growth factors, cells, and physical forces. In cases, where natural healing is not able, efforts have to be undertaken to improve healing. For this purpose, tissue engineering may be an option. In order to stimulate cells to form a bone tissue several factors are needed: cells, scaffold, and growth factors. Stem cells derived from bone marrow or adipose tissues are the most useful in this regard. The differentiation of the cells can be accelerated using mechanical stimulation. The first part of this chapter describes the influence of longitudinal strain application. The second part uses a sophisticated approach with stem cells on a newly developed biomaterial (Sponceram) in a rotating bed bioreactor with the administration of bone morphogenetic protein-2. It is shown that such an approach is able to produce bone tissue constructs. This may lead to production of larger constructs that can be used in clinical applications.

Phenotypic shift of human amniotic epithelial cells in culture is associated with reduced osteogenic differentiation in vitro.

BACKGROUND: Amniotic membrane is a highly promising cell source for tissue engineering. Being part of the placenta, this tissue is abundantly available. It can be processed easily to yield large amounts of epithelial and mesenchymal cells that have shown broad differentiation potential. For tissue-engineering purposes, cells may be applied either directly after isolation from the tissue or after a period of in vitro expansion to obtain higher cell numbers. In order to investigate the advantages and drawbacks of these strategies we compared freshly isolated and cultivated human amniotic epithelial cells (hAEC) regarding their surface antigen (Ag) expression profile and osteogenic differentiation capacity. METHODS: Expression of surface Ag that are characteristic for mesenchymal stromal and embryonic stem cells was analyzed by flow cytometry. Different protocols for osteogenic and adipogenic differentiation were compared. RESULTS: We have demonstrated that expression of surface Ag changes dramatically during cultivation of hAEC. While not or only weakly expressed on primary isolates, the mesenchymal markers CD13, CD44, CD49e, CD54, CD90 and CD105 are strongly up-regulated during in vitro propagation. In contrast, expression of the embryonic markers TRA-1-60 and TRA-1-81, but not SSEA-4, rapidly decreases upon cultivation. This phenotypic shift is associated with a reduction in osteogenic differentiation. DISCUSSION: Our results suggest that phenotypic alterations of hAEC during in vitro cultivation might be responsible for a functional reduction of the differentiation potential, which has to be considered for the potential application of these cells in regenerative medicine.

A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1.

We have established a panel of human monoclonal antibodies against human immunodeficiency virus type 1 (HIV-1). The antibodies 2F5 and 2G12 have been identified to be two of the most potently in vitro neutralizing antibodies against HIV-1. Here we report on a further antibody, 4E10, of similar in vitro neutralizing potency. 4E10 binds to a novel epitope C terminal of the ELDKWA sequence recognized by 2F5, which has been so far the only described broadly neutralizing anti-gp41 antibody. Both 4E10 and 2F5 bind only weakly to infected cells compared with gp120-specific 2G12 and polyclonal anti-HIV-1 immunoglobulin (HIVIG), but show potent in vitro neutralizing properties. 4E10 neutralizes potently not only tissue culture-adapted strains but also primary isolates of different clades, including A, B, C, D, and E. Viruses that were found to be resistant to 2F5 were neutralized by 4E10 and vice versa; none of the tested isolates was resistant to both anti-gp41 antibodies. This confirms that the region recognized by 2F5 and 4E10 is essential for viral infectivity and may be important for vaccine design. Moreover, our results suggest that 4E10 should be further investigated for passive anti-HIV immunotherapy.