Specific inductive potential of a novel nanocomposite biomimetic biomaterial for osteochondral tissue regeneration.

Osteochondral lesions require treatment to restore the biology and functionality of the joint. A novel nanostructured biomimetic gradient scaffold was developed to mimic the biochemical and biophysical properties of the different layers of native osteochondral structure. The present results show that the scaffold presents important physicochemical characteristics and can support the growth and differentiation of mesenchymal stromal cells (h-MSCs), which adhere and penetrate into the cartilaginous and bony layers. H-MSCs grown in chondrogenic or osteogenic medium decreased their proliferation during days 14-52 on both scaffold layers and in medium without inducing factors used as controls. Both chondrogenic and osteogenic differentiation of h-MSCs occurred from day 28 and were increased on day 52, but not in the control medium. Safranin O staining and collagen type II and proteoglycans immunostaining confirmed that chondrogenic differentiation was specifically induced only in the cartilaginous layer. Conversely, von Kossa staining, osteocalcin and osteopontin immunostaining confirmed that osteogenic differentiation occurred on both layers. This study shows the specific potential of each layer of the biomimetic scaffold to induce chondrogenic or osteogenic differentiation of h-MSCs. These processes depended mainly on the media used but not the biomaterial itself, suggesting that the local milieu is fundamental for guiding cell differentiation. Copyright © 2013 John Wiley & Sons, Ltd.

Long-term in vivo experimental investigations on magnesium doped hydroxyapatite bone substitutes.

Despite several efforts to find suitable alternatives to autologous bone, no bone substitute currently available provides the same characteristics and properties. Nevertheless, among the wide range of materials proposed as bone substitutes, calcium phosphate materials represent the most promising category and the present study is aimed at improving the knowledge on non-stoichiometric magnesium-doped hydroxyapatite substitutes (Mg-HA), tested in two different formulations: Mg-HA Putty and Mg-HA Granules. These bone substitutes were implanted bilaterally into iliac crest bone defects in healthy sheep and comparative histological, histomorphometric, microhardness and ultrastructural assessments were performed 9, 12, 18 and 24 months after surgery to elucidate bone tissue apposition, mineralization and material degradation in vivo. The results confirmed that the biomimetic bone substitutes provide a histocompatible and osteoconductive structural support, during the bone formation process, and give essential information about the in vivo resorption process and biological behavior of biomimetic bone substitutes.

Effects of different crosslinking conditions on the chemical-physical properties of a novel bio-inspired composite scaffold stabilised with 1,4-butanediol diglycidyl ether (BDDGE).

Serious cartilage lesions (Outerbridge III, IV) may be successfully treated with a three-layered gradient scaffold made by magnesium-doped hydroxyapatite and type I collagen, manufactured through a bio-inspired process and stabilised by a reactive bis-epoxy (1,4-butanediol diglycidyl ether, BDDGE). Each layer was analysed to elucidate the effects of crosslinking variables (concentration, temperature and pH). The chemical stabilisation led to an homogeneous and aligned collagenous matrix: the fibrous structures switched to a laminar foils-based arrangement and organic phases acquired an highly coordinated 3D-organization. These morphological features were strongly evident when crosslinking occurred in alkaline solution, with BDDGE concentration of at least 1 wt%. The optimised crosslinking conditions did not affect the apatite nano-crystals nucleated into self-assembling collagen fibres. The present work allowed to demonstrate that acting on BDDGE reaction parameters might be an useful tool to control the chemical-physical properties of bio-inspired scaffold suitable to heal wide osteochondral defects, even through arthroscopic procedure.

Chemical-physical properties and in vitro cell culturing of a novel biphasic bio-mimetic scaffold for osteo-chondral tissue regeneration.

The requirements for a successful regeneration of an osteo-chondral defect could effectively be met by using a bi-layered composite scaffold, able to support proliferation and differentiation of mesenchymal stem cells, while providing a biochemical environment promoting the formations of the two distinct tissues. The novel strategy here presented consists of developing a bio-mimetic scaffolds obtained by the combination of two integrated organic compounds (type I collagen and chitosan) with or without bioactive Mg-doped hydroxyapatite (Mg-HA) nanocrystals, depending on the specific layer, reproducing cartilaginous or subchondral bone tissue. An innovative patented methodology for scaffolds production, called - pH-dependent 3-phasic assembling -, allowed to development of a highly homogenous and chemically stable scaffold, presenting a very good integration among all three components, as confirmed by extensive SEM and thermogravimetric analyses. A preliminary in vitro evaluation was also carried out by seeding bi-layered scaffold with human bone marrow stromal cells (h-MSCs), by giving particular emphasis to cell viability and distribution at day 0, 7 and 14. Cells were viable and uniformly colonized the whole scaffold until day 14, indicating that the scaffold contributed to the maintenance of cell behaviour.

[Tissue engineering applications: cartilage lesions repair by the use of autologous chondrocytes]. / Applicazioni dell'ingegneria tissutale: riparazione di lesioni cartilaginee con condrociti autologhi.

Promising new therapies based on tissue engineering have been recently developed for cartilage repair. The association of biomaterials with autologous chondrocytes expanded in vitro can represent a useful tool to regenerate this tissue. The scaffolds utilised in such therapeutical applications should provide a pre-formed three-dimensional shape, prevent cells from floating out of the defect, have sufficient mechanical strength, facilitate uniform spread of cells and stimulate the phenotype of transplanted cells. Hyaff-11 is a hyaluronic-acid based biodegradable polymer, that has been shown to provide successful cell carrier for tissue-engineered repair. From our findings we can state that human chondrocytes seeded on Hyaff-11 are able to maintain in vitro the characteristic of differentiated cells, expressing and producing collagen type II and aggrecan which are the main markers of cartilage phenotype, down-regulating collagen type I. Moreover, it seems to be a useful scaffold for cartilage repair both in animal models and clinical trials in humans, favouring the formation of a hyaline-like tissue. In the light of these data, we can hypothesise, for the future, the use of autologous chondrocyte transplantation together with gene therapy as a treatment for rheumatic diseases such as osteoarthritis.

Transplantation of chondrocytes seeded on a hyaluronan derivative (hyaff-11) into cartilage defects in rabbits.

Different methods have been used to improve chondrocyte transplantation for the repair of articular cartilage defects. Several groups of biomaterials have been proposed as support for in vitro cell growth and for in vivo implantation. Here. we describe a new approach investigating the healing of rabbit cartilage by means of autologous chondrocytes seeded on a hyaluronan derivative referred to as Hyaff-11. Full thickness defects were created bilaterally in the weight-bearing surface of the medial femoral condyle of both femora of New Zealand male rabbits. The wounds were then repaired using both chondrocytes seeded on the biomaterial and biomaterial alone. Controls were similarly treated but received either no treatment or implants of the delivery substance. Histologic samples from in and around the defect sites were examined 1, 3 and 6 months after surgery and were scored from 0 to 16. Statistically significant differences in the quality of the regenerated tissue were found between the grafts carried out with biomaterial carrying chondrocyte cells compared to the biomaterial alone or controls. This study demonstrates the efficacy of this hyaluronan-based scaffold for autologous chondrocytes transplantation.

Binding of vancomycin group antibiotics to D-alanine and D-lactate presenting self-assembled monolayers.

Peptides terminating in -Lys-D-Ala-D-Ala, -Lys-D-Ala-L-Ala and -Lys-D-Ala-D-Lactate were covalently coupled via an N-terminal aminohexanoic acid linker to a self-assembled monolayer of HS(CH2)15CO2H on a thin gold film. Binding of the glycopeptide antibiotics vancomycin and chloroeremomycin to these surfaces was then measured using a surface plasmon resonance biosensor. Both antibiotics bound with micromolar affinity to the D-Ala-terminating surface and with millimolar affinity to the D-Lactate-terminating surface. Increasing density of these covalently attached peptides on the surface had no effect on the resultant affinities of either antibiotic for the surface. In contrast, when the lipid-anchored peptide N-alpha-docosanoyl-epsilon-acetyl-Lys-D-Ala-D-Ala was inserted into a supported lipid monolayer, the affinity of the strongly dimerizing antibiotic chloroeremomycin for the surface showed a dependence on ligand density. This was not the case with the weakly dimerizing antibiotic vancomycin. The lipid monolayer surface, which is a more realistic model of the surface of a bacterium, was thus better suited for the study of the cooperative binding interactions that occur between dimeric glycopeptide antibiotics and surface-bound ligands.

Inhibition of ErbB-2 mitogenic and transforming activity by RALT, a mitogen-induced signal transducer which binds to the ErbB-2 kinase domain.

The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen employing the ErbB-2 juxtamembrane and kinase domains as bait. This interaction was reproduced in vitro with a glutathione S-transferase fusion protein spanning positions 282 to 395 of the 459-residue gene 33 protein. Activation of ErbB-2 catalytic function was required for ErbB-2-gene 33 physical interaction in living cells, whereas ErbB-2 autophosphorylation was dispensable. Expression of gene 33 protein was absent in growth-arrested NIH 3T3 fibroblasts but was induced within 60 to 90 min of serum stimulation or activation of the ErbB-2 kinase and decreased sharply upon entry into S phase. New differentiation factor stimulation of mitogen-deprived mammary epithelial cells also caused accumulation of gene 33 protein, which could be found in a complex with ErbB-2. Overexpression of gene 33 protein in mouse fibroblasts inhibited (i) cell proliferation driven by ErbB-2 but not by serum, (ii) cell transformation induced by ErbB-2 but not by Ras or Src, and (iii) sustained activation of ERK 1 and 2 by ErbB-2 but not by serum. The gene 33 protein may convey inhibitory signals downstream to ErbB-2 by virtue of its association with SH3-containing proteins, including GRB-2, which was found to associate with gene 33 protein in living cells. These data indicate that the gene 33 protein is a feedback inhibitor of ErbB-2 mitogenic function and a suppressor of ErbB-2 oncogenic activity. We propose that the gene 33 protein be renamed with the acronym RALT (receptor-associated late transducer).

Development of autologous T lymphocytes in two males with X-linked severe combined immune deficiency: molecular and cellular characterization.

We report on two patients affected with severe combined immune deficiency (SCID) with an unusual immunological phenotype and a substantial number of autologous, poorly functioning T cells. Distinct mutations identified at the IL2RG locus in the two patients impaired IL-2-mediated signaling but affected T-cell lymphopoiesis differently, resulting in generation of a polyclonal or oligoclonal T-cell repertoire. These observations add to the growing complexity of the immunological spectrum of SCID in humans and indicate the need for detailed immunological and molecular investigations in atypical cases.

Of genes and phenotypes: the immunological and molecular spectrum of combined immune deficiency. Defects of the gamma(c)-JAK3 signaling pathway as a model.

Cytokines play a major role in lymphoid development. Defects of the common gamma chain (gamma(c)) or of the JAK3 protein in humans have been shown to result in a severe combined immune deficiency (SCID), with a profound defect in T and natural killer (NK)-cell development, whereas B-cell generation is apparently unaffected (T-B+NK-SCID). While extensive molecular and biochemical analysis of these patients has been instrumental in understanding better the biological properties of the gamma(c) and JAK3 protein, an unexpected phenotypic heterogeneity of gamma(c) and JAK3 deficiency has emerged, indicating the need for appropriate and extensive investigations even in patients with atypical presentations. At the same time, characterization of the defects has been instrumental in the development of novel therapeutic approaches, from in utero hematopoietic stem cell transplantation to gene therapy.

Prenatal diagnosis of JAK3 deficient SCID.

The JAK3 gene, encoding a tyrosine kinase functionally coupled to cytokine receptors which share the common gamma chain, has been identified as the defective gene for autosomal recessive severe combined immunodeficiency (SCID). Thus, specific mutational diagnosis has become possible. We screened all exons with a combined single strand conformational polymorphism and hetero-duplex formation assay followed by sequence analysis to identify specific mutations in two families. This assay was used on chorionic villus sampling derived DNA in two fetuses from two unrelated families, where we found mutations in both parents. We were able to exclude the mutations in both fetuses by the 12th week of gestation. The described method for first-trimester prenatal diagnosis of autosomal recessive T-B+SCID provides a valid tool to aid in genetic counselling and possibly prenatal therapy in this disease.

Prenatal molecular diagnosis of Wiskott-Aldrich syndrome by direct mutation analysis.

We have performed prenatal diagnosis for Wiskott Aldrich syndrome (WAS) in two unrelated families by direct gene analysis. Using a combined non-radioactive analysis of single-strand conformational polymorphism (SSCP) and heteroduplex formation (HD), followed by automated sequencing, we studied DNA from chorionic villus sampling (CVS), allowing the diagnosis of one affected and one healthy male at the 12th week of gestation.

Defective actin polymerization in EBV-transformed B-cell lines from patients with the Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive disorder characterized by eczema, thrombocytopenia, and immunodeficiency. An allelic variant of the disease is characterized by isolated thrombocytopenia (XLT). The gene responsible for WAS/XLT (WASP) encodes for a 502 amino acid protein (WASP) that is possibly involved in actin binding and cytoskeleton organization. The expression of WASP and the distribution of F-actin and alpha-actinin (which binds to and stabilizes actin filaments) have been analysed in lymphoblastoid cell lines from six patients with WAS and one with XLT. Western blot and immunocytochemistry did not reveal WASP expression in four WAS patients, whereas two WAS patients (with a moderate clinical course) expressed trace amounts of mutant WASP. In contrast, the XLT patient expressed normal amounts of WASP. Furthermore, cell lines from WAS and XLT patients also markedly differed in F-actin polymerization and alpha-actinin distribution. In particular, severe defects of cytoplasmic F-actin expression and of F-actin-positive microvillus formation, and impaired capping of alpha-actinin, were observed in all patients who lacked WASP. As a whole, the degree of impairment of WASP protein expression in WAS/XLT seems to correlate with anomalies of cytoskeletal organization, strongly supporting a role for WASP in the regulation of F-actin polymerization.

Structural and functional basis for JAK3-deficient severe combined immunodeficiency.

Mutations of the Janus family kinase JAK3 have been found to be responsible for autosomal recessive severe combined immunodeficiency (SCID) in humans. We report here the analysis of four new unrelated patients affected by JAK3-deficient SCID. The genetic defects were heterogeneous and included a large intragenic deletion as well as different point mutations, leading to missense substitutions, early stop codons, or splicing defects. We performed a series of studies of the biochemical events induced by cytokines on lymphoblastoid B-cell lines obtained from these patients. Abnormalities in tyrosine phosphorylation of JAK3 in response to interleukin-2 (IL-2) and IL-4 were present in all patients. Accordingly, IL-2-mediated phosphorylation of STAT5 was also absent or barely detectable. On the contrary, in all cases, we could show reduced but clear phosphorylation of STAT6 upon IL-4 stimulation. In one patient carrying a single amino acid change (Glu481Gly) in the JH3 domain of JAK3, we observed partially conserved IL-2 responses resulting in reduced but detectable levels of JAK3 and STAT5 phosphorylation. Interestingly, the patient bearing this mutation developed a substantial number of circulating CD4(+)/CD45RO+ activated T lymphocytes that were functionally impaired. In two cases, patients' cells expressed JAK3 proteins with mutations in the JH2 pseudo-kinase domain. A single cysteine to arginine substitution (Cys759Arg) in this region resulted in high basal levels of constitutive JAK3 tyrosine phosphorylation unresponsive to either downregulation by serum starvation or cytokine-mediated upregulation. The characterization of the genetic defects and biochemical abnormalities in these JAK3-deficient patients will help define the role of JAK3 in the ontogeny of a competent immune system and may lead to a better understanding of the JAK3 functional domains.

In-utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with X-linked severe combined immunodeficiency (SCIDXI).

BACKGROUND: X-linked severe combined immunodeficiency (SCIDXI) is an inherited immune defect which leads to death in infancy from severe infections. The defect is caused by mutations of the IL-2RG gene that encodes for the common gamma chain shared by several cytokine receptors. The disease is characterised by lack of T and NK cells with normal numbers of B cells. SCIDXI can be cured by bone marrow transplantation (BMT) or prevented by abortion after prenatal diagnosis. METHODS: A male fetus was diagnosed as having SCIDXI by molecular, immunophenotypic, and functional analyses. The fetus was injected intraperitoneally under ultrasound guidance with CD34 haematopoietic progenitor cells purified from paternal bone marrow and T-cell depleted by E rosetting. Chimerism analysis was by HLA-DQ alpha typing and gamma-chain staining on cord blood. FINDINGS: A healthy 3.6 kg boy was delivered by caesarean section at 38 weeks of gestation with no clinical or laboratory signs of graft-versus-host disease. Engraftment of donor-derived CD2 cells was found at birth. At 3.5 months of age the infant is well and his T-cell counts and function are normal. INTERPRETATION: In-utero transplantation of haematopoietic progenitor cells allowed immune reconstitution of a fetus with SCIDXI and may be an alternative to elective abortion. Our report should encourage applications of this method to other inherited disorders curable by BMT.