Enhanced early chondrogenesis in articular defects following arthroscopic mesenchymal stem cell implantation in an equine model

Mesenchymal stem cells (MSCs) provide an important source of pluripotent cells for musculoskeletal tissue repair. This study examined the impact of MSC implantation on cartilage healing characteristics in a large animal model. Twelve full-thickness 15-mm cartilage lesions in the femoropatellar articulations of six young mature horses were repaired by injection of a self-polymerizing autogenous fibrin vehicle containing mesenchymal stem cells, or autogenous fibrin alone in control joints. Arthroscopic second look and defect biopsy was obtained at 30 days, and all animals were euthanized 8 months after repair. Cartilage repair tissue and surrounding cartilage were assessed by histology, histochemistry, collagen type I and type II immunohistochemistry, collagen type II in situ hybridization, and matrix biochemical assays. Arthroscopic scores for MSC-implanted defects were significantly improved at the 30-day arthroscopic assessment. Biopsy showed MSC-implanted defects contained increased fibrous tissue with several defects containing predominantly type II collagen. Long-term assessment revealed repair tissue filled grafted and control lesions at 8 months, with no significant difference between stem cell-treated and control defects. Collagen type II and proteoglycan content in MSC-implanted and control defects were similar. Mesenchymal stem cell grafts improved the early healing response, but did not significantly enhance the long-term histologic appearance or biochemical composition of full-thickness cartilage lesions.

Sensitivity of osteoblasts, fibroblasts, bone marrow cells, and dendritic cells to 5-aminolevulinic acid based photodynamic therapy

INTRODUCTION: Photodynamic therapy with 5-aminolevulinic acid (5-ALA-PDT) exerts cell type specific effects on target cells. Since chondrocytes were found to be more resistant than osteoblasts to 5-ALA-PDT, the pre-treatment of osteochondral grafts with 5-ALA-PDT may represent a means to devitalize the osseous portion while maintaining functional cartilage. The present study was designed to determine the effects of 5-ALA-PDT in vitro on cell populations residing in skeletal tissues. METHODS: Osteoblasts, fibroblasts, bone marrow cells, and dendritic cells were incubated with 0.5 mM 5-ALA for 4 h. Protoporphyrin IX (PpIX) accumulation and after exposure to light cellular functions were assessed for up to 6 days. RESULTS: Accumulation of PpIX reached a plateau at 0.5 mM in osteoblasts, fibroblasts, and dendritic cells, and at 2.0 mM in bone marrow cells. At 0.5 mM 5-ALA, similar responses to illumination were observed in all cells with a survival rate of less than 12% at a light dose of 20 J/cm(2). The function of osteoblasts (proliferation, levels of mRNA encoding collagen type I, alkaline phosphatase activity) and fibroblasts (proliferation, levels of mRNAs encoding collagens type I and III) was not affected, when the cells were treated with 5-ALA and light doses of < or =10 J/cm(2). Paralleling the reduction of viable cells after 5-ALA-PDT, the capacity of dendritic cells to stimulate T cells in a mixed leukocyte reaction decreased to 4+/-2% at 20 J/cm(2). CONCLUSION: The investigated cell types were sensitive to 5-ALA-PDT and the residual cell debris did not elicit an allogenic response. These findings, together with the resistance of chondrocytes to 5-ALA-PDT, encourage the further investigation of this protocol in the pretreatment of osteochondral allografts.

Effect of reduced oxygen tension and long-term mechanical stimulation on chondrocyte-polymer constructs

We have investigated the influence of long-term confined dynamic compression and surface motion under low oxygen tension on tissue-engineered cell-scaffold constructs. Porous polyurethane scaffolds (8 mm x 4 mm) were seeded with bovine articular chondrocytes and cultured under normoxic (21% O(2)) or hypoxic (5% O(2)) conditions for up to 4 weeks. By means of our joint-simulating bioreactor, cyclic axial compression (10-20%; 0.5 Hz) was applied for 1 h daily with a ceramic ball, which simultaneously oscillated over the construct surface (+/-25 degrees; 0.5 Hz). Culture under reduced oxygen tension resulted in an increase in mRNA levels of type II collagen and aggrecan, whereas the expression of type I collagen was down-regulated at early time points. A higher glycosaminoglycan content was found in hypoxic than in normoxic constructs. Immunohistochemical analysis showed more intense type II and weaker type I collagen staining in hypoxic than in normoxic cultures. Type II collagen gene expression was slightly elevated after short-term loading, whereas aggrecan mRNA levels were not influenced by the applied mechanical stimuli. Of importance, the combination of loading and low oxygen tension resulted in a further down-regulation of collagen type I mRNA expression, contributing to the stabilization of the chondrocytic phenotype. Histological results confirmed the beneficial effect of mechanical loading on chondrocyte matrix synthesis. Thus, mechanical stimulation combined with low oxygen tension is an effective tool for modulating the chondrocytic phenotype and should be considered when chondrocytes or mesenchymal stem cells are cultured and differentiated with the aim of generating cartilage-like tissue in vitro.

Adherence to host extracellular matrix and serum components by Enterococcus faecium isolates of diverse origin.

Enterococcus faecium has emerged as an important cause of nosocomial infections over the last two decades. We recently demonstrated collagen type I (CI) as a common adherence target for some E. faecium isolates and a significant correlation was found to exist between acm-mediated CI adherence and clinical origin. Here, we evaluated 60 diverse E. faecium isolates for their adherence to up to 15 immobilized host extracellular matrix and serum components. Adherence phenotypes were most commonly observed to fibronectin (Fn) (20% of the 60 isolates), fibrinogen (17%) and laminin (Ln) (13%), while only one or two of the isolates adhered to collagen type V (CV), transferrin or lactoferrin and none to the other host components tested. Adherence to Fn and Ln was almost exclusively restricted to clinical isolates, especially the endocarditis-enriched nosocomial genogroup clonal complex 17 (CC17). Thus, the ability to adhere to Fn and Ln, in addition to CI, may have contributed to the emergence and adaptation of E. faecium, in particular CC17, as a nosocomial pathogen.

Ligand-signaled upregulation of Enterococcus faecalis ace transcription, a mechanism for modulating host-E. faecalis interaction.

Enterococcus faecalis, the third most frequent cause of bacterial endocarditis, appears to be equipped with diverse surface-associated proteins showing structural-fold similarity to the immunoglobulin-fold family of staphylococcal adhesins. Among the putative E. faecalis surface proteins, the previously characterized adhesin Ace, which shows specific binding to collagen and laminin, was detectable in surface protein preparations only after growth at 46 degrees C, mirroring the finding that adherence was observed in 46 degrees C, but not 37 degrees C, grown E. faecalis cultures. To elucidate the influence of different growth and host parameters on ace expression, we investigated ace expression using E. faecalis OG1RF grown in routine laboratory media (brain heart infusion) and found that ace mRNA levels were low in all growth phases. However, quantitative reverse transcription-PCR showed 18-fold-higher ace mRNA amounts in cells grown in the presence of collagen type IV compared to the controls. Similarly, a marked increase was observed when cells were either grown in the presence of collagen type I or serum but not in the presence of fibrinogen or bovine serum albumin. The production of Ace after growth in the presence of collagen type IV was demonstrated by immunofluorescence microscopy, mirroring the increased ace mRNA levels. Furthermore, increased Ace expression correlated with increased collagen and laminin adhesion. Collagen-induced Ace expression was also seen in three of three other E. faecalis strains of diverse origins tested, and thus it appears to be a common phenomenon. The observation of host matrix signal-induced adherence of E. faecalis may have important implications on our understanding of this opportunistic pathogen.

Attenuation of fibrosis in vitro and in vivo with SPARC siRNA.

INTRODUCTION: SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs. METHODS: In in vitro studies, skin fibroblasts obtained from a Tgfbr1 knock-in mouse (TBR1CA; Cre-ER) were transfected with SPARC siRNA. Gene and protein expressions of the Col1a2 and the Ctgf were examined by real-time RT-PCR and Western blotting, respectively. In in vivo studies, C57BL/6 mice were induced for skin and lung fibrosis by bleomycin and followed by SPARC siRNA treatment through subcutaneous injection and intratracheal instillation, respectively. The pathological changes of skin and lungs were assessed by hematoxylin and eosin and Masson's trichrome stains. The expression changes of collagen in the tissues were assessed by real-time RT-PCR and non-crosslinked fibrillar collagen content assays. RESULTS: SPARC siRNA significantly reduced gene and protein expression of collagen type 1 in fibroblasts obtained from the TBR1CA; Cre-ER mouse that was induced for constitutively active TGF-beta receptor I. Skin and lung fibrosis induced by bleomycin was markedly reduced by treatment with SPARC siRNA. The anti-fibrotic effect of SPARC siRNA in vivo was accompanied by an inhibition of Ctgf expression in these same tissues. CONCLUSIONS: Specific inhibition of SPARC effectively reduced fibrotic changes in vitro and in vivo. SPARC inhibition may represent a potential therapeutic approach to fibrotic diseases.

The development and characterization of two types of chronic responses in irradiated mouse colon

The hypothesis to be tested is that there are two distinct types of chronic responses in irradiated normal tissues, each resulting from damage to different cell populations in the tissue. The first is a sequala of chronic epithelial depletion in which the tissue's integrity cannot be maintained, i.e. a "consequential" chronic response. The other response is due to cell loss in the connective tissue and/or vascular stroma, i.e. a "primary" chronic response. The purpose of this study was to test the hypothesis in the murine colon by first, establishing a model of each chronic response and then, by determining whether the responses differed in timing of expression, histology, and expression of specific collagen types. The model of late damage used was colonic obstructions/strictures induced by a single dose of 27 Gy ("consequential" response) and two equal doses of 14.75 Gy (t = 10 days) ("primary" response). "Consequential" lesions appeared as early as 5 weeks after 27 Gy and were characterized by a deep mucosal ulceration and a thickened fibrotic serosa containing excessive accumulations of collagen types I and III. Both types were commingled in the scar at the base of the ulcer. Fibroblasts were synthesizing pro-collagen types I and III mRNA 10 weeks prior to measurable increases in collagen. A significant decrease in the ratio of collagen types I:III was associated with the "consequential" response at 4-5 months post-irradiation. The "primary" response, on the other hand, did not appear until 40 weeks after the split dose even though the total dose delivered was approximately the same as that for the "consequential" response. The "primary" response was characterized with an intact mucosa and a thickened fibrotic submucosa which contained excessive amounts of only collagen type I. An increased number of fibroblasts were synthesizing pro-collagen type I mRNA nearly 25 weeks before collagen type I levels were increased. The "primary" response lesion had a significantly elevated collagen type I:III ratio at 10-13 months post-irradiation. These data show a clear difference between the two chronic response and suggest that not all chronic responses share a common pathogenesis, but depend on the cell population in the tissue that is damaged. ^

Role of tumor–host interactions in interstitial diffusion of macromolecules: Cranial vs. subcutaneous tumors

The large size of many novel therapeutics impairs their transport through the tumor extracellular matrix and thus limits their therapeutic effectiveness. We propose that extracellular matrix composition, structure, and distribution determine the transport properties in tumors. Furthermore, because the characteristics of the extracellular matrix largely depend on the tumor–host interactions, we postulate that diffusion of macromolecules will vary with tumor type as well as anatomical location. Diffusion coefficients of macromolecules and liposomes in tumors growing in cranial windows (CWs) and dorsal chambers (DCs) were measured by fluorescence recovery after photobleaching. For the same tumor types, diffusion of large molecules was significantly faster in CW than in DC tumors. The greater diffusional hindrance in DC tumors was correlated with higher levels of collagen type I and its organization into fibrils. For molecules with diameters comparable to the interfibrillar space the diffusion was 5- to 10-fold slower in DC than in CW tumors. The slower diffusion in DC tumors was associated with a higher density of host stromal cells that synthesize and organize collagen type I. Our results point to the necessity of developing site-specific drug carriers to improve the delivery of molecular medicine to solid tumors.

Produção e caracterização de biomateriais acelulares bioativos obtidos a partir da decelularização de placentas

A bioengenharia de tecidos baseia-se no uso de moléculas bioativas, células-tronco e biomateriais para reparação de tecidos e/ou órgãos. Biomateriais podem ser classificados de acordo com sua origem em sintéticos ou biológicos. Biomateriais biológicos podem ser produzidos por decelularização, que visa a remoção de células da matriz extracelular (MEC), a qual deve manter sua integridade química e física. Placentas são órgãos de grande interesse na bioengenharia de tecidos visto que são descartadas após o parto e possuem grande volume de matriz extracelular. Métodos de decelularização podem ser classificados em químicos, físicos e enzimáticos. Todos conhecidamente causam alterações na MEC, sendo que a associação deles é comumente utilizada. Este trabalho comparou diferentes protocolos e estabeleceu um método mais favorável para a decelularização de placentas caninas, visando a produção de um biomaterial para futuras aplicações clínicas. Inicialmente ambas as porções - materna e fetal - das placentas foram submetidas à 10 protocolos, que avaliaram variáveis como concentração e tempo de incubação em detergentes, diferentes gradientes de temperatura e a influência da perfusão versus imersão das soluções, na MEC remanescente. Com base na transparência do tecido e na ausência de núcleo celular em cortes histológicos, dois protocolos foram selecionados (I e II). Além dos critérios já mencionados, ambos os protocolos foram comparados quanto à quantidade de DNA remanescente na MEC decelularizada e à permanência e distribuição de algumas das proteínas da matriz. O detergente SDS foi o mais eficaz na remoção de células, embora não tenha sido suficiente para promover uma decelularização tecidual completa. O congelamento prévio das placentas requereu um maior tempo de incubação posterior das amostras nos distintos detergentes. Ambos métodos de perfusão e imersão foram eficazes na remoção das células, embora grande concentração de proteínas do citoesqueleto tenham permanecido retidas na matriz. As amostras processadas pelo protocolo I (SDS 1%, 5mM EDTA + 50mM TRIS + 0,5% antibiótico, e Triton X-100 1%) apresentaram maior preservação da organização estrutural da MEC quando comparadas àquelas processadas de acordo com o protocolo II (que diferiu do anterior pela utilização de solução contendo 0,05% tripsina ao invés de 50mM TRIS), esse último método entretanto foi o que melhor removeu as células das placentas, conforme observado em lâminas histológicas e demonstrado pela menor concentração de DNA. Tanto as porções materna quanto fetal submetidas à ambos protocolos, mantiveram as proteínas laminina, fibronectina e colágeno tipo I. O colágeno tipo III foi observado somente na porção fetal. Conclui-se que o protocolo II foi o mais eficaz no processo de decelularização de placentas caninas tendo promovido a remoção do conteúdo celular e diminuição da concentração de DNA na MEC remanescente. No entanto é necessário otimizar o tempo de incubação das placentas em soluções enzimáticas visando maior conservação do arranjo da matriz decelularizada. A análise da capacidade da MEC decelularizada por tal método para ser utilizada em bioengenharia de tecidos ainda deve ser avaliada in vitro e in vivo

Associação de infecção por Streptococcus Bovis e o carcinoma colorrectal

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Direct electrospinning of 3D auricle-shaped scaffolds for tissue engineering applications.

Thirty-two poly(ε)caprolactone (PCL) scaffolds have been produced by electrospinning directly into an auricle-shaped mould and seeded with articular chondrocytes harvested from bovine ankle joints. After seeding, the auricle shaped constructs were cultured in vitro and analysed at days 1, 7, 14 and 21 for regional differences in total DNA, glycosaminoglycan (GAG) and collagen (COL) content as well as the expression of aggrecan (AGG), collagen type I and type II (COL1/2) and matrix metalloproteinase 3 and 13 (MMP3/13). Stress-relaxation indentation testing was performed to investigate regional mechanical properties of the electrospun constructs. Electrospinning into a conductive mould yielded stable 3D constructs both initially and for the whole in vitro culture period, with an equilibrium modulus in the MPa range. Rapid cell proliferation and COL accumulation was observed until week 3. Quantitative real time PCR analysis showed an initial increase in AGG, no change in COL2, a persistent increase in COL1, and only a slight decrease initially for MMP3. Electrospinning of fibrous scaffolds directly into an auricle-shape represents a promising option for auricular tissue engineering, as it can reduce the steps needed to achieve an implantable structure.

A study of the growth and differentiation of the human adenocarcinoma of the colon cell line HT-29

The HT-29 human colon adenocarcinoma cell line, like many epithelial cells, displays an undifferentiated phenotype when cultured on plastic substrata. Biochemical markers of differentiation, such as brush border associated enzymes and carcinoembryonic antigen were expressed at very low levels. The differentiation-inducing effects of the culture of HT-29 cells on collagen type I gels were evaluated, and were assessed by morphological appearance, brush border associated enzyme activities and the secretion of CEA. The effect that this more physiological environment had on their chemosensitivity to a panel of chemotherapeutic agents was determined, so as to indicate whether this system could be used to improve the selectivity of screening for novel anticancer agents. Initial studies were performed on HT-29 cells derived from cells seeded directly from plastic substrata onto the collagen gels (designated Non-PPC gels). Their time of exposure to the collagen was limited to the time course of a single experiment and the results suggested that a longer, more permanent exposure might produce a more pronounced differentiation. HT-29 cells were then passaged continuously on collagen gels for a minimum of 10 passages prior to experimentation (designated PPC gels). The same parameters were measured, and compared to those for the cells grown on plastic and on the non-passaged collagen gels (Non-PPC) from the original studies. Permanently passaged cells displayed a similar degree of morphological differentiation as the non-passaged cells, with both culture conditions resulting in a more pronounced differentiation than that achieved by culture on plastic. It was noted that the morphological differentiation observed was very heterogeneous, a situation also seen in xenografted tumours in vivo. The activity of alkaline phosphatase and the production of CEA was higher in the cells passaged on collagen (PPC) than the cells cultured on non-passaged collagen gel (Non-PPC) and plastic. The biochemical determination of aminopeptidase activity showed that collagen gel culture enhanced the activity in both non-passaged and passaged HT-29 cells above that of the cells cultured on plastic. However, immunocytochemical localization of aminopeptidase and sucrase-isomaltase of samples of cells grown on the various substrata for 7, 14, 21 and 28 days showed a reduction in both enzymes in the cells grown on collagen gels when compared to cells grown on plastic. The reason for the discrepancy between the two assays for aminopeptidase is at this stage unexplained. Although, there was evidence to suggest that the culture of HT-29 cells on collagen gels was capable of inducing morphological and biochemical markers of enterocytic differentiation, there were no differences in the chemosensitivity of the different cell groups to a panel of anticancer agents. Preliminary studies suggested that the ability of the cells to polarize by their culture on porous filter chambers without any exogenous ECM was sufficient to enhance HT-29 differentiation and the onset of differentiation was probably correlated with the production of ECM by the cells themselves.

Mesenchymal stem cell-conditioned medium accelerates skin wound healing:an in vitro study of fibroblast and keratinocyte scratch assays

We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-beta1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

Human intervertebral disc aggrecan inhibits endothelial cell adhesion and cell migration in vitro

STUDY DESIGN: The effect of human intervertebral disc aggrecan on endothelial cell growth was examined using cell culture assays. OBJECTIVE: To determine the response of endothelial cells to human intervertebral disc aggrecan, and whether the amount and type of aggrecan present in the intervertebral disc may be implicated in disc vascularization. SUMMARY OF BACKGROUND DATA: Intervertebral disc degeneration has been associated with a loss of proteoglycan, and the ingrowth of blood vessels and nerves. Neovascularization is a common feature also of disc herniation. Intervertebral disc aggrecan is inhibitory to sensory nerve growth, but the effects of disc aggrecan on endothelial cell growth are not known. METHODS: Aggrecan monomers were isolated separately from the anulus fibrosus and nucleus pulposus of human lumbar intervertebral discs, and characterized to determine the amount and type of sulfated glycosaminoglycan side chains present. The effects of these aggrecan isolates on the cellular adhesion and migration of the human endothelial cell lines, HMEC-1 and EAhy-926, were examined in vitro. RESULTS: Homogenous substrata of disc aggrecan inhibited endothelial cell adhesion and cell spreading in a concentration dependent manner. In substrata choice assays, endothelial cells seeded onto collagen type I migrated over the collagen until they encountered substrata of disc aggrecan, where they either stopped migrating, retreated onto the collagen, or, more commonly, changed direction to align along the collagen-aggrecan border. The inhibitory effect of aggrecan on endothelial cell migration was concentration dependent, and reduced by enzymatic treatment of the aggrecan monomers with a combination of chondroitinase ABC and keratinase/keratinase II. Anulus fibrosus aggrecan was more inhibitory to endothelial cell adhesion than nucleus pulposus aggrecan. However, this difference did not relate to the extent to which the different aggrecan isolates were charged, as determined by colorimetric assay with 1,9-dimethylmethylene blue, or to marked differences in the distribution of chondroitin sulfated and keratan sulfated side chains. CONCLUSIONS: Human intervertebral disc aggrecan is inhibitory to endothelial cell migration, and this inhibitory effect appears to depend, in part, on the presence of glycosaminoglycan side chains on the aggrecan monomer.

Human mesenchymal stem cells stimulate EaHy926 endothelial cell migration:combined proteomic and in vitro analysis of the influence of donor-donor variability

Mesenchymal stem cells (MSCs) stimulate angiogenesis within a wound environment and this effect is mediated through paracrine interactions with the endothelial cells present. Here we report that human MSC-conditioned medium (n=3 donors) significantly increased EaHy-926 endothelial cell adhesion and cell migration, but that this stimulatory effect was markedly donor-dependent. MALDI-TOF/TOF mass spectrometry demonstrated that whilst collagen type I and fibronectin were secreted by all of the MSC cultures, the small leucine rich proteoglycan, decorin was secreted only by the MSC culture that was least effective upon EaHy-926 cells. These individual extracellular matrix components were then tested as culture substrata. EaHy-926 cell adherence was greatest on fibronectin-coated surfaces with least adherence on decorin-coated surfaces. Scratch wound assays were used to examine cell migration. EaHy-926 cell scratch wound closure was quickest on substrates of fibronectin and slowest on decorin. However, EaHy-926 cell migration was stimulated by the addition of MSC-conditioned medium irrespective of the types of culture substrates. These data suggest that whilst the MSC secretome may generally be considered angiogenic, the composition of the secretome is variable and this variation probably contributes to donor-donor differences in activity. Hence, screening and optimizing MSC secretomes will improve the clinical effectiveness of pro-angiogenic MSC-based therapies.