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Biomedical subjects

M Heberer

Publications and source records attributed to M Heberer.

At least 19 recordsLinked to original sources
PubMed2006

Cartilage tissue engineering by expanded goat articular chondrocytes.

In this study we investigated whether expanded goat chondrocytes have the capacity to generate cartilaginous tissues with biochemical and biomechanical properties improving with time in culture. Goat chondrocytes were expanded in monolayer with or without combinations of FGF-2, TGF-beta1, and PDGFbb, and the postexpansion chondrogenic capacity assessed in pellet cultures. Expanded chondrocytes were also cultured for up to 6 weeks in HYAFF-M nonwoven meshes or Polyactive foams, and the resulting cartilaginous tissues were assessed histologically, biochemically, and biomechanically. Supplementation of the expansion medium with FGF-2 increased the proliferation rate of goat chondrocytes and enhanced their postexpansion chondrogenic capacity. FGF-2-expanded chondrocytes seeded in HYAFF-M or Polyactive scaffolds formed cartilaginous tissues with wet weight, glycosaminoglycan, and collagen content, increasing from 2 days to 6 weeks culture (up to respectively 2-, 8-, and 41-fold). Equilibrium and dynamic stiffness measured in HYAFF M-based constructs also increased with time, up to, respectively, 1.3- and 16-fold. This study demonstrates the feasibility to engineer goat cartilaginous tissues at different stages of development by varying culture time, and thus opens the possibility to test the effect of maturation stage of engineered cartilage on the outcome of cartilage repair in orthotopic goat models.

Animals↗
PubMed2005

Structural characterization and reliable biomechanical assessment of integrative cartilage repair.

Structural and functional characterization of integrative cartilage repair in controlled model systems can play a key role in the development of innovative strategies to improve the long-term outcome of many cartilage repair procedures. In this work, we first developed a method to reproducibly generate geometrically defined disk/ring cartilage composites and to remove outgrown fibrous layers which can encapsulate cartilaginous tissues during culture. We then used the model system to test the hypothesis that such fibrous layers lead to an overestimation of biomechanical parameters of integration at the disk/ring interface. Transmission electron microscopy images of the composites after 6 weeks of culture indicated that collagen fibrils in the fibrous tissue layer were well integrated into the collagen network of the cartilage disk and ring, whereas molecular bridging between opposing disk/ring cartilage surfaces was less pronounced and restricted to regions with narrow interfacial regions (< 2 microm). Stress-strain profiles generated from mechanical push-out tests for composites with the layers removed displayed a single and distinct peak, whereas profiles for composites with the layers left intact consisted of multiple superimposed peaks. As compared to composites with removed layers, composites with intact layers had significantly higher adhesive strengths (161+/-9 vs. 71+/-11 kPa) and adhesion energies (15.0+/-0.7 vs. 2.7+/-0.4 mJ/mm2). By combining structural and functional analyses, we demonstrated that the outgrowing tissue formed during in vitro culture of cartilaginous specimens should be eliminated in order to reliably quantify biomechanical parameters related to integrative cartilage repair.

Adhesiveness↗
PubMed2004

Chondrogenesis of expanded adult human articular chondrocytes is enhanced by specific prostaglandins.

OBJECTIVE: To investigate the effects of the cyclooxygenase-2 (cox-2)-dependent prostaglandins D(2) (PGD(2)), E(2) (PGE(2)) and F(2)alpha (PGF(2)alpha) on the redifferentiation and cartilage matrix production of dedifferentiated articular chondrocytes. METHODS: Human articular chondrocytes from three adult donors were dedifferentiated by monolayer expansion and induced to redifferentiate by culture as 3D pellets in a defined serum-free medium containing TGF-beta(1) and dexamethasone, without or with further supplementation with PGD(2), PGE(2) or PGF(2)alpha. After 2 weeks, pellets were assessed histologically, immunohistochemically, biochemically and by real-time quantitative reverse transcriptase-polymerase chain reaction. RESULTS: All three PGs, but predominantly PGE(2), reduced the staining intensity of pellets for collagen type I, whereas PGD(2) and PGF(2)alpha increased the staining intensity of pellets for collagen type II and glycosaminoglycans (GAG). The GAG/DNA content of pellets was not affected by PGE(2) but was increased 1.5- and 2.1-fold by PGD(2) and PGF(2)alpha respectively. PGE(2) reduced the expression of collagen type I mRNA (9.0-fold), whereas PGD(2) and PGF(2)alpha increased the mRNA expression of collagen type II (6.2- and 4.1-fold respectively) and aggrecan (29.8- and 10.7-fold respectively). CONCLUSION: In contrast to PGE(2), PGD(2) and PGF(2)alpha enhanced chondrogenic differentiation and hyaline cartilage matrix deposition by expanded human articular chondrocytes, and could thus be used to improve in vitro or in vivo cartilage regeneration approaches based on these cells.

Adult↗
PubMed2004

Encapsulation into sterically stabilised liposomes enhances the immunogenicity of melanoma-associated Melan-A/MART-1 epitopes.

Tumour-associated antigens (TAA)-specific vaccination requires highly immunogenic reagents capable of inducing cytotoxic T cells (CTL). Soluble peptides are currently used in clinical applications despite an acknowledged poor immunogenicity. Encapsulation into liposomes has been suggested to improve the immunogenicity of discrete antigen formulations. We comparatively evaluated the capacity of HLA-A2.1 restricted Melan-A/MART-1 epitopes in soluble form (S) or following inclusion into sterically stabilised liposomes (SSL) to be recognised by specific CTL, to stimulate their proliferation and to induce them in healthy donors' peripheral blood mononuclear cells (PBMC), as well as in melanoma-derived tumour-infiltrating lymphocytes (TIL). HLA-A2.1(+), Melan-A/MART-1-NA-8 melanoma cells served as targets of specific CTL in 51Cr release assays upon pulsing by untreated or human plasma-treated soluble or SSL-encapsulated Melan-A/MART-1 27-35 (M27-35) or 26-35 (M26-35) epitopes. These reagents were also used to stimulate CTL proliferation, measured as 3H-thymidine incorporation, in the presence of immature dendritic cells (iDC), as antigen-presenting cells (APC). Induction of specific CTL upon stimulation with soluble or SSL-encapsulated peptides was attempted in healthy donors' PBMC or melanoma-derived TIL, and monitored by 51Cr release assays and tetramer staining. Na-8 cells pulsing with SSL M27-35 resulted in a five-fold more effective killing by specific CTL as compared with equal amounts of S M27-35. Encapsulation into SSL also provided a partial (50%) protection of M27-35 from plasma hydrolysis. No specific advantages regarding M26-35 were detectable in these assays. However, at low epitope concentrations (</=100 ng ml(-1)), SSL M26-35 was significantly more effective in inducing CTL proliferation than S M26-35, in the presence of iDC, as APC. Preincubation with iDC for 6 h virtually abolished the capacity of S M26-35 to stimulate specific CTL proliferation, but only partially affected that of SSL M26-35. Most importantly, SSL M26-35 was able to enhance the induction of specific CTL in healthy donors PBMC and in melanoma-derived TIL as compared to S M26-35. Taken together, our data indicate that encapsulation of TAA epitopes into SSL results in effective immunogenic formulations suitable for clinical use in active specific tumour immunotherapy.

Antigens, Neoplasm↗
PubMed2004

[Engineering and characterization of functional osteochondral replacement tissue].

Extensive osteochondral lesions require repair of the cartilage and underlying bone. We generated osteochondral repair tissue by tissue engineering. Standardized defects, 7 x 5 x 5 mm, were created in femoropatellar grooves of adult rabbits. Engineered cartilage, generated in vitro starting from chondrocytes and a biodegradable scaffold, was implanted using Collagraft as subchondral support. Cell-free implants, defects left empty, and unoperated knee joints served as controls. Explants were characterized morphologically and mechanically. Engineered cartilage implants were superior to cell-free implants and to natural healing of empty defects with respect to the histologic score and Young's modulus of the 6-month repair tissue. These data suggest that engineered cartilage can provide primary stability for the treatment of critical osteochondral defects.

Bone Remodeling↗
PubMed2003

Phase I/II clinical trial of a nonreplicative vaccinia virus expressing multiple HLA-A0201-restricted tumor-associated epitopes and costimulatory molecules in metastatic melanoma patients.

We performed a phase I/II clinical trial in metastatic melanoma patients with an ultraviolet (UV)-inactivated nonreplicating recombinant vaccinia virus enabling the expression, from a single construct, of endoplasmic reticulum-targeted HLA-A0201-restricted Melan-A/MART-1(27-35), gp100(280-288), and tyrosinase(1-9) epitopes, together with CD80 and CD86 costimulatory proteins. Corresponding soluble peptides were used to boost responses and granulocyte-macrophage colony-stimulating factor was used as systemic adjuvant. Safety and immunogenicity, as monitored with in vitro-restimulated peripheral blood mononuclear cells by cytotoxic T lymphocyte precursor (CTLp) frequency analysis and tetramer staining, were specifically addressed. Of 20 patients entering the protocol, 2 had to withdraw because of rapidly progressing disease. Immune responses were evaluated in 18 patients (stage III, n = 5; stage IV, n = 13) and increases in specific CTLp frequencies were observed in 15. In 16 patients responsiveness against all 3 antigens could be analyzed: 7 (43%), including all stage III cases, showed evidence of induction of CTLs specific for the three epitopes, and 2 (12%) and 4 (25%), respectively, showed reactivity against two or one tumor-associated antigen. In three stage IV patients no specific CTL reactivity could be induced. Increases in CTLp frequency were detected mostly after viral vaccine injections. However, in a majority of patients final CTLp levels were comparable to initial levels. Tetramer characterization of Melan-A/MART-1(27-35)-specific CTLs during the protocol also suggested preferential expansion after recombinant virus administration. Vector-specific humoral responses, frequently undetectable in stage IV patients, did not appear to prevent tumor-associated antigen-specific CTL induction. Aside from a single occurrence of transient grade 3 leukopenia, no major clinical toxicity was reported. Seventeen of 18 patients completed the 3-month trial (one patient died before the last delayed-type hypersensitivity test). Three displayed regression of individual metastases, seven had stable disease, and progressive disease was observed in seven patients. This is the first report on the administration of a UV-inactivated recombinant vaccinia virus coexpressing five transgenes in cancer patients. The results described here, in terms of safety and immunogenicity, support the use of this reagent in active specific immunotherapy.

Adult↗
PubMed2003

Oscillating perfusion of cell suspensions through three-dimensional scaffolds enhances cell seeding efficiency and uniformity.

We developed a bioreactor for automated cell seeding of three-dimensional scaffolds by continuous perfusion of a cell suspension through the scaffold pores in oscillating directions. Using quantitative biochemical and image analysis techniques, we then evaluated the efficiency and uniformity of perfusion seeding of Polyactive foams as compared to conventional static and spinner flask methods. Finally, we assessed the efficacy of the perfusion seeding technique for different scaffolds and cell types. Perfusion seeding of chondrocytes into Polyactive foams resulted in "viable cell seeding efficiencies," defined as the percentages of initially loaded cells that were seeded and remained viable, that were significantly higher (75 +/- 6%) than those by static (57% +/- 5%) and spinner flask seeding (55% +/- 8%). In addition, as compared to static and spinner flask methods, cells seeded by perfusion were respectively 2.6-fold and 3.8-fold more uniformly distributed and formed more homogeneously sized cell clusters. Chondrocytes seeded by perfusion into Hyaff-11 nonwoven meshes were 26% and 63%, respectively, more uniformly distributed than following static and spinner flask seeding. Bone marrow stromal cells seeded by perfusion into ChronOS porous ceramics were homogeneously distributed throughout the scaffold volume, while following the static method, cells were found only near the top surface of the ceramic. In summary, we demonstrated that our cell seeding perfusion bioreactor generated constructs with remarkably uniform cell distributions at high efficiencies, and was effective for a variety of scaffolds and different mesenchymal cell types.

Animals↗
PubMed2003

Dynamic compression of cartilage constructs engineered from expanded human articular chondrocytes.

Recent works have shown that mechanical loading can alter the metabolic activity of chondrocytes cultured in 3D scaffolds. In this study we determined whether the stage of development of engineered cartilaginous constructs (expanded adult human articular chondrocytes/Polyactive foams) regulates the effect of dynamic compression on glycosaminoglycan (GAG) metabolism. Construct maturation depended on the culture time (3-14 days) and the donor (4 individuals). When dynamic compression was subsequently applied for 3 days, changes in GAG synthesized, accumulated, and released were significantly positively correlated to the GAG content of the constructs prior to loading, and resulted in stimulation of GAG formation only in the most developed tissues. Conversely, none of these changes were correlated with the expression of collagen type II mRNA, indicating that the response of chondrocytes to dynamic compression does not depend directly upon the stage of cell differentiation, but rather on the extracellular matrix surrounding the cells.

Cartilage, Articular↗
PubMed2003

[Swiss surgery: quo vadis? Reader and market analysis for strategic positioning of a specialty journal].

UNLABELLED: Scientific journals currently face challenges including cost pressures caused by economic constraints, increasing rivalry among competitors, limited market potential of non-english speaking journals, increasing medical specialization with resulting market fragmentation, and internet-based competition. We therefore analyzed strategic opportunities of the journal Swiss Surgery on the basis of customer surveys and of a market analysis. RESULTS: Swiss surgeons expressed their interest in the continuation of the journal but also indicated their support for changes in its concept and for an increased use of electronic media. An international market analysis points-out the difficulties of national, non-english speaking journals in gaining impact points and in attracting authors and readers of scientific medical articles. Therefore, a journal such as Swiss Surgery should identify and use publication niches. RECOMMENDATION: The demand for a concept addressing surgical training including continuous postgraduate education was confirmed by the customers of Swiss Surgery. A corresponding offer does not presently exist in the area and could become the new focus of the journal. This change of concept may have a number of consequences: A journal focusing on surgical training and education should use the results of readers' surveys rather than impact point assignment to evaluate quality. The journal should increasingly use electronic services including data bases, pictures, videos and closed user groups to supplement the print version. At short term, however, the printed version should be continued and not be substituted by the electronic version in order to maintain the established brand "Swiss Surgery".

Attitude of Health Personnel↗
PubMed2003

Development and validation of a bioreactor for physical stimulation of engineered cartilage.

A bioreactor has been developed to apply different regimes of physical stimulation to tissue specimens under highly controlled conditions. The computer-controlled device exposes specimens to compressive deformation at various strains and frequencies, measures the load applied to each sample and allows simultaneous medium stirring at different velocities. Validation tests confirmed the accuracy of the system in (i) its displacement (errors averaged 0.072+/-0.051 microm), and in (ii) setting the contact with the samples utilizing micrometer screws coupled to plungers (errors averaged 1.74+/-0.36% for samples of 1.60-3.18 mm thickness), thus ensuring accurate compressive deformation. The developed bioreactor, which represents an advance in the technology for physical stimulation of tissue specimens, is currently used to apply compressive deformation and hydrodynamic forces to human chondrocytes cultured in biodegradable polymer scaffolds, with the goals of (i) engineering functional grafts for the repair of cartilage defects (ii).

Bioreactors↗
PubMed2002

[Internet communication between family physicians and the university hospital].

The potential of electronic communication in medicine is assessed based on an analysis of a pilot project pertaining to internet based communication among referring and hospital physicians. Advantages of electronic data exchange in medicine pertain to speed and capacity for data transfer, availability of data and data integration, ultimately enabling consistent medical case management. Quality requirements of electronic communication of medical data are related to safety, availability, data integration, potential for case management and system qualities. Medical efficiency can be increased by use of electronic communication only if complex functions beyond the substitution of conventional mail by e-mail are implemented and an exhaustive use of the technology can be achieved.

Case Management↗
PubMed2001

Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro.

Adult human articular chondrocytes were expanded in a medium with 10% serum (CTR) or further supplemented with different mitogens (i.e., EGF, PDGFbb, FGF-2, TGF beta 1, or FGF-2/TGF beta 1). Cells were then induced to redifferentiate in 3D pellets using serum-supplemented medium (SSM), serum-free medium (SFM), or SFM supplemented with factors inducing differentiation of chondroprogenitor cells (i.e., TGF beta 1 and/or dexamethasone). All factors tested during expansion enhanced chondrocyte proliferation and dedifferentiation, as assessed by the mRNA ratios of collagen type II to type I (CII/CI) and aggrecan to versican (Agg/Ver), using real-time PCR. FGF-2/TGF beta 1-expanded chondrocytes displayed the lowest doubling times, CII/CI and Agg/Ver ratios, averaging, respectively, 50, 0.2 and 15% of CTR-expanded cells. Redifferentiation in pellets was more efficient in SFM than SSM only for EGF-, PDGFbb- or FGF-2-expanded chondrocytes. Upon supplementation of SFM with TGF beta and dexamethasone (SFM TD), CII/CI ratios decreased 4.4-fold for EGF- and PDGFbb-expanded chondrocytes, but increased 96-fold for FGF-2/TGF beta 1-expanded cells. Chondrocytes expanded with FGF-2/TGF beta 1 and redifferentiated in SFM TD expressed the largest mRNA amounts of CII and aggrecan and generated cartilaginous tissues with the highest accumulation of glycosaminoglycans and collagen type II. Our results provide evidence that growth factors during chondrocyte expansion not only influence cell proliferation and differentiation, but also the cell potential to redifferentiate and respond to regulatory molecules upon transfer into a 3D environment.

Adult↗
PubMed2001

Expression of MAGE tumour-associated antigens is inversely correlated with tumour differentiation in invasive ductal breast cancers: an immunohistochemical study.

MAGE (Melanoma antigen E) family gene products encompass tumour-associated antigens (TAAs) recognised by human leukocyte antigen (HLA)-restricted specific T-cells. Agents inducing DNA demethylation, an event typically detectable in cellular de-differentiation processes, were shown to induce the expression of MAGE genes. By using a monoclonal antibody specific for MAGE family gene products, we have studied the expression of these TAAs in a group of 144 patients with invasive ductal breast cancers. Immunohistochemical data were correlated with tumour differentiation, lymphatic vessel invasion, oestrogen receptor expression, intratumoural necrosis, lymphocytic infiltration, perineural invasion, tumour microcalcifications and axillary lymph node metastases. MAGE immunoreactivity was undetectable in non-neoplastic cells. In poorly differentiated cancers positive staining was observed in 30/63 cases (47.6%) as compared with 13/51 (25.4%) and 5/30 (16.6%) in moderately and well-differentiated tumours, respectively (P<0.05). In addition, MAGE immunoreactivity was significantly correlated with lymphatic vessel invasion and intratumoural necrosis. Moreover, a significant inverse relationship with oestrogen receptor expression was also observed. However, no significant correlation could be established between MAGE immunoreactivity and defined phenotypic characteristics of tumour infiltrating lymphocytes, including expression of CD3, CD4, CD8, CD20 or granzyme B. Thus, expression of MAGE family gene products in invasive ductal breast cancers appears to be associated with poorly differentiated histological phenotypes. These data support the concept of specific immunotherapy in highly aggressive forms of breast neoplasms. Furthermore, they suggest that MAGE immunoreactivity could represent a tumour marker of potential prognostic relevance.

Antibodies, Monoclonal↗
PubMed2001

Modulation of dendritic cell phenotype and mobility by tumor cells in vitro.

To gain new insights into the functional interaction between DC and neoplastic cells, we have analyzed the effects of melanoma and colorectal cancer lines on the chemotaxis and the phenotype of monocyte-derived DC in vitro. Both types of tumor cells displayed effective chemoattractive capacity towards immature, but not mature DC. Furthermore, conditioned medium of discrete melanoma lines induced upregulation of CD80, CD86, MHC class I, and MHC class II molecules on immature DC. However, de novo expression of E-cadherin and strong upregulation of CD15 could also be detected in the absence of CD83 expression. Melanoma-conditioned DC exhibited an increased adhesion capacity to a melanoma cell line in vitro and did not migrate in response to SLC chemokine. Tumor-infiltrating CD15(+) cells displaying DC morphology could also be detected by immunohistochemistry in the original tumor specimens from which discrete melanoma cell lines under investigation were derived. Colorectal cancer cell lines, although able to chemoattract immature DC, were apparently unable to modulate their phenotype. Altogether our results suggest that tumor cells can attract immature DC in vitro and, eventually, modulate their phenotype. As a result, DC mobility could be severely impaired.

Animals↗
PubMed2001

Immunogenicity of nonreplicating recombinant vaccinia expressing HLA-A201 targeted or complete MART-1/Melan-A antigen.

The effect on immunogenicity of different tumor T cell epitope formulations was evaluated in vitro using nonreplicating recombinant vaccinia vector expressing two forms of the melanoma-associated MART-1/Melan-A antigen. The first recombinant virus expressed a minigene encoding a fusion product between an endoplasmic reticulum (ER)-targeting signal and the HLA-A201 binding 27-35 peptide. The second viral construct encoded the complete MART-1/Melan-A protein. The capacity of HLA-A201 cells infected with either viral construct to generate and to stimulate MART-1/Melan-A 27-35 specific cytotoxic T-lymphocytes (CTL), was comparatively characterized. The results obtained here with a tumor antigen confirmed the capacity of vaccinia virus-encoded ER-minigene to generate a very strong antigenic signal. In cytotoxicity assays, recognition of target cells infected with high amounts of both recombinant viruses with activated specific CTL clones, resulted in similar lytic activity. With regard to calcium mobilization, TCR down-regulation, IFN-gamma release, and T cell proliferation assays, the targeted epitope elicited 10- to 1000-fold stronger responses. Remarkably, the immunogenic difference between the two formulations, in their respective capacity to generate CTL from naive HLA-A2 peripheral blood mononuclear cells in vitro as measured by tetramer detection, was lower (2- to 3-fold). Recombinant vectors expressing complete antigens have demonstrated their capacity to generate specific responses and such vaccines might take advantage of a broader potential of presentation. However, as demonstrated here for the HLA-A201-restricted MART-1/Melan-A immunodominant epitope, nonreplicative vaccinia virus expressing ER-targeted minigenes appear to represent a significantly more immunogenic epitope vaccine formulation.

Antigen Presentation↗
PubMed2001

Quantitative analysis of gene expression in human articular cartilage from normal and osteoarthritic joints.

OBJECTIVE: To quantify the expression of genes encoding extracellular matrix (ECM) proteins in human cartilage from normal and osteoarthritic (OA) joints. DESIGN: Human cartilage samples were classified as control (CTR) or OA according to clinical evaluation and assessed histologically and biochemically to confirm the diagnosis. mRNAs encoding collagen types I, II and X, aggrecan, versican, osteopontin and osteocalcin were quantified by real-time reverse transcription-PCR assays and normalized to a reference mRNA (GAPDH). RESULTS: RNA from native cartilage could be reproducibly and efficiently amplified by real-time PCR only if isolated using purification membranes. Primers and fluorescent probes for real-time PCR, endowed with comparable (<6% difference from GAPDH) and high (>91%) amplification efficiencies, were designed and validated for the selected ECM genes. The expression of most genes under investigation displayed large variations and was not significantly different in CTR and OA cartilage. Only osteopontin mRNA levels were significantly higher in OA than CTR specimens. mRNA ratios of collagen type II to I and of aggrecan to versican, defined as indexes of chondrocyte differentiation, were less variable within each population than the single genes and markedly higher (27.0 and 7.6-fold, respectively) in CTR than OA cartilage, with high statistical significance (P = 0.00013 and P = 0.00007, respectively). CONCLUSIONS: Our results provide evidence that gene patterns related to chondrocyte differentiation discriminate between CTR and OA human cartilage with higher sensitivity than single ECM genes. The method described here has the potential to improve understanding of the progression of OA and could become a valuable diagnostic tool.

Adult↗