Direct comparison of current cell-based and cell-free approaches towards the repair of craniofacial bone defects - A preclinical study.

For craniofacial bone defect repair, several alternatives to bone graft (BG) exist, including the combination of biphasic calcium phosphate (BCP) biomaterials with total bone marrow (TBM) and bone marrow-derived mesenchymal stromal cells (MSCs), or the use of growth factors like recombinant human bone morphogenic protein-2 (RhBMP-2) and various scaffolds. Therefore, clinicians might be unsure as to which approach will offer their patients the most benefit. Here, we aimed to compare different clinically relevant bone tissue engineering methods in an "all-in-one" study in rat calvarial defects. TBM, and MSCs committed or not, and cultured in two- or three-dimensions were mixed with BCP and implanted in bilateral parietal bone defects in rats. RhBMP-2 and BG were used as positive controls. After 7 weeks, significant de novo bone formation was observed in rhBMP-2 and BG groups, and in a lesser amount, when BCP biomaterials were mixed with TBM or committed MSCs cultured in three-dimensions. Due to the efficacy and safety of the TBM/BCP combination approach, we recommend this one-step procedure for further clinical investigation. STATEMENT OF SIGNIFICANCE: For craniofacial repair, total bone marrow (BM) and BM mesenchymal stem cell (MSC)-based regenerative medicine have shown to be promising in alternative to bone grafting (BG). Therefore, clinicians might be unsure as to which approach will offer the most benefit. Here, BM and MSCs committed or not were mixed with calcium phosphate ceramics (CaP) and implanted in bone defects in rats. RhBMP-2 and BG were used as positive controls. After 7 weeks, significant bone formation was observed in rhBMP-2 and BG groups, and when CaP were mixed with BM or committed MSCs. Since the BM-based procedure does not require bone harvest or cell culture, but provides de novo bone formation, we recommend consideration of this strategy for craniofacial applications.

Hydrogel/calcium phosphate composites require specific properties for three-dimensional culture of human bone mesenchymal cells.

To provide multipotent cells with a three-dimensional environment closer to bone matrix, an engineered construct mimicking bone components has been designed and evaluated. A biocompatible hydrogel (silated hydroxypropylmethyl cellulose) was used as an extra-cellular matrix while biphasic calcium phosphate ceramic particles were used to replace mineralized matrix. Finally, human bone mesenchymal cells were cultured in three dimensions in the resulting constructs to study their cell viability, proliferation, interactions within the composites, and maintenance of their osteogenic potential. This approach resulted in homogeneous structures in which cells were viable and retained their osteoblastic differentiation potential. However, the cells did not proliferate nor colonize the constructs, possibly because of a lack of suitable interactions with their micro-environment.

3D environment on human mesenchymal stem cells differentiation for bone tissue engineering.

In this work a novel method was developed to create a three dimensional environment at a cellular level for bone tissue engineering. Biphasic calcium phosphate (BCP) particles of 140-200 microm were used in association with human mesenchymal stem cells (hMSCs). The cells seeded on these particles adhered and proliferated more rapidly in the first day of culture compared to culture on plastic. Analyses of hMSCs cultured without osteogenic factors on BCP particles revealed an abundant extracellular matrix production forming 3-dimensional (3D) hMSCs/BCP particles constructs after few days. Bone morphogenetic 2 (BMP-2), bone sialoprotein (BSP) and ALP gene expression using real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed that expression profiles were modified by the culture substrate while the addition of osteogenic medium enhanced bone markers expression. These results indicate that BCP particles alone are able to induce an osteoblastic differentiation of hMSCs that might be of interest for bone tissue engineering.

Critical factors in the design of growth factor releasing scaffolds for cartilage tissue engineering.

BACKGROUND: Trauma or degenerative diseases of the joints are common clinical problems resulting in high morbidity. Although various orthopedic treatments have been developed and evaluated, the low repair capacities of articular cartilage renders functional results unsatisfactory in the long term. Over the last decade, a different approach (tissue engineering) has emerged that aims not only to repair impaired cartilage, but also to fully regenerate it, by combining cells, biomaterials mimicking extracellular matrix (scaffolds) and regulatory signals. The latter is of high importance as growth factors have the potency to induce, support or enhance the growth and differentiation of various cell types towards the chondrogenic lineage. Therefore, the controlled release of different growth factors from scaffolds appears to have great potential to orchestrate tissue repair effectively. OBJECTIVE: This review aims to highlight considerations and limitations of the design, materials and processing methods available to create scaffolds, in relation to the suitability to incorporate and release growth factors in a safe and defined manner. Furthermore, the current state of the art of signalling molecules release from scaffolds and the impact on cartilage regeneration in vitro and in vivo is reported and critically discussed. METHODS: The strict aspects of biomaterials, scaffolds and growth factor release from scaffolds for cartilage tissue engineering applications are considered. CONCLUSION: Engineering defined scaffolds that deliver growth factors in a controlled way is a task seldom attained. If growth factor delivery appears to be beneficial overall, the optimal delivery conditions for cartilage reconstruction should be more thoroughly investigated.

Pharmaceutical aspects of drug eluting stents.

This review focuses on the pharmaceutical aspects of the development of drug eluting stents. It discusses the different processes that can be used to obtain a controlled release of a drug from the stent as well as the coatings therefore applied. Results obtained for stents already available on the market or in a far stage of development are discussed. In a final part possible future research areas as well as expected new evolutions in the design of drug eluting stents are presented.

Tailored release of TGF-beta1 from porous scaffolds for cartilage tissue engineering.

In view of cartilage tissue engineering, the possibility to prepare porous scaffolds releasing transforming growth factor-beta(1) (TGF-beta(1)) in a well controlled fashion was investigated by means of an emulsion-coating method. Poly(ether-ester) multiblock copolymers were used to prepare emulsions containing TGF-beta(1) which were subsequently applied onto prefabricated scaffolds. This approach resulted in defined porous structures (66%) with interconnected porosity, suitable to allow tissue ingrowth. The scaffolds were effectively associated with TGF-beta(1) and allowed to tailor precisely the release of the growth factor from 12 days to more than 50 days by varying the copolymer composition of the coating. An incomplete release was measured by ELISA, possibly linked to the rapid concentration decrease of the protein in solution. The released growth factor retained its biological activity as was assessed by a cell proliferation assay and by the ability of the released protein to induce chondrogenic differentiation of bone marrow-derived mesenchymal stem cells. However, exact bioactivity quantification was rendered difficult by the protein concentration decrease during storage. Therefore, this study confirms the interest of poly(ether-ester) multiblock copolymers for controlled release of growth factors, and indicates that emulsion-coated scaffolds are promising candidates for cartilage tissue engineering applications requiring precise TGF-beta(1) release rates.

Kinetics and energetics of ligand binding determined by microcalorimetry: insights into active site mobility in a psychrophilic alpha-amylase.

A new microcalorimetric method for recording the kinetic parameters k(cat), K(m) and K(i) of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic alpha-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic alpha-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic alpha-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site.

Dual release of proteins from porous polymeric scaffolds.

To create porous scaffolds releasing in a controlled and independent fashion two different proteins, a novel approach based on protein-loaded polymeric coatings was evaluated. In this process, two water-in-oil emulsions are forced successively through a prefabricated scaffold to create coatings, containing each a different protein and having different release characteristics. In a first step, a simplified three-layered system was designed with model proteins (myoglobin and lysozyme). Poly(ether-ester) multiblock copolymers were chosen as polymer matrix, to allow the diffusion of proteins through the coatings. The model system showed the independent release of the two proteins. The myoglobin release was tailored from a burst to a linear release still on-going after 60 days, while the lysozyme release rate was kept constant. Macro-porous scaffolds, with a porosity of 59 vol.%, showed the same ability to control the release rate of the model proteins independently. The relation between the coatings properties and their release characteristics were investigated with the use of a mathematical diffusion model based on Fick's second law. It confirmed that the multiple coated scaffolds are biphasic system, where each coating controls the release of the protein that it contains. This approach could be of value for tissue engineering applications.

Biodegradable poly(ether-ester) multiblock copolymers for controlled release applications: An in vivo evaluation.

Multiblock poly(ether-ester)s based on poly(ethylene glycol), butylene terephthalate, and butylene succinate segments were evaluated for their in vivo degradation and biocompatibility in order to establish a correlation with previously reported in vitro results. Porous polymer sheets were implanted subcutaneously for 32 weeks in rats. The degradation was monitored visually (histology), by molecular weight (GPC), and by copolymer composition (NMR). Substitution of the aromatic terephthalate units by aliphatic succinate units was shown to accelerate the degradation rate of the copolymers. Direct correlation of the in vivo and in vitro degradation of the porous implants showed a slightly faster initial molecular weight decrease in vivo. Besides hydrolysis, oxidation occurs in vivo due to the presence of radicals produced by inflammatory cells. In addition, the higher molecular weight plateau of the residue found in vivo indicated a higher solubility of the oligomers in the extracellular fluid compared to a phosphate buffer. Minor changes in the poly(ether-ester) compositions were noted due to degradation. Microscopically, fragmentation of the porous implants was observed in time. At later stages of degradation, macrophages were observed phagocytozing small polymer particles. Both in vitro cytotoxicity studies and histology on in vivo samples proved the biocompatibility of the poly(ether-ester)s.

A novel method to obtain protein release from porous polymer scaffolds: emulsion coating.

To obtain the controlled release of proteins from macro-porous polymeric scaffolds, a novel emulsion-coating method has been developed. In this process, a water-in-oil emulsion, from an aqueous protein solution and a polymer solution, is forced through a prefabricated scaffold by applying a vacuum. After solvent evaporation, a polymer film, containing the protein, is then deposited on the porous scaffold surface. This paper reports the effect of processing parameters on the emulsion coating characteristics, scaffold structure, and protein release and stability. Poly(ether-ester) multiblock copolymers were chosen as the polymer matrix for both scaffolds and coating. Macro-porous scaffolds, with a porosity of 77 vol% and pores of approximately 500 microm were prepared by compression moulding/salt leaching. A micro-porous, homogeneous protein-loaded coating could be obtained on the scaffold surface. Due to the coating, the scaffold porosity was decreased, whereas the pore interconnection was increased. A model protein (lysozyme) could effectively be released in a controlled fashion from the scaffolds. Complete lysozyme release could be achieved within 3 days up to more than 2 months by adjusting the coated emulsion parameters. In addition, the coating process did not reduce the enzymatic activity. This new method appears to be promising for tissue engineering applications.

Release of small water-soluble drugs from multiblock copolymer microspheres: a feasibility study.

Poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) multiblock copolymer was investigated as a possible matrix for controlled delivery of small water-soluble drugs. Two molecules were selected as sustained release candidates from microspheres: leuprorelin acetate (peptide of Mw = 1270 D) and vitamin B(12) (Mw = 1355 D). First, vitamin B(12)-loaded microspheres were prepared using a double emulsion method and preparation parameters were varied (surfactant in the first emulsion and copolymer composition). The resulting microsphere structure, entrapment efficiency and release rate were evaluated. Vitamin B(12)-loaded microsphere parameters could easily be tailored to achieve specific requirements. The addition of surfactant in the first preparation process led to a significant increase of the microsphere entrapment efficiency, whereas the decrease of the PEGT copolymer content allowed the release rates from microspheres to be precisely decreased. However, leuprorelin acetate-loaded microspheres did not show the same characteristics when prepared with the same parameters, possibly because of a high water solubility discrepancy between the vitamin B(12) and the peptide. This study shows the suitability of PEGT/PBT microspheres as a controlled release system for vitamin B(12), but not for leuprorelin acetate. It also underlines the necessity of tailored development for each individual drug and emphasizes the risk of using model molecules.

[Weber-Christian syndrome and chronic pancreatitis (author's transl)]. / Syndrome de Weber-Christian et pancréatite chronique.

The authors describe a case of cytosteatonecrosis associated with chronic pancreatitis. Clinical, biological (amylasemia and amylasuria) and radiological (arteriography) signs of pancreatic disease are noticed as well as important radiological lesions of the limbs. The cutaneous lesions are hypodermic nodules located on the lower limbs with swelling of the right big toe and a cutaneous abrasion.

[Ultrastructural similarities between the planus-like reaction of the graft versus host disease and the idiopathic lichen planus (author's transl)]. / Ultrastructure de la phase lichénienne de la réaction chronique du greffon contre l'hôte. Similitudes avec le lichen plan idiopathique.

An ultrastructural study was performed on skin biopsies of eight patients with lichenoid eruption of chronic graft-versus-host disease (GVHD). The lichenoid eruption is similar to idiopathic lichen planus with the injury of the basal cells, the distribution of the numerous colloid bodies, and the presence of regenerative cells. Lymphocytes satellites of injured keratinocytes were observed in those lichenoid eruptions, as well as in the acute phase of GVHD. The numerous mast cells in the lichenoid eruption of GVHD may be related to the connective tissue changes of the patients progressing to a late sclerotic phase of chronic GVHD.

[Cutaneous manifestations of graft versus host disease (author's transl)]. / Les signes cutanés de la maladie du greffon contre l'hôte.

Skin is a target organ for Graft v Host Reaction (GvHR). Cutaneous changes sometimes are the first symptoms for GvHR. They offer the opportunity for its early recognition. They include: -- A primarily acute phase, as red maculopapular rash, on face, palms and soles, which can spread over large areas, resembling scarlatiniform rash. Some courses have resulted in severe epidermal necrolysis similar to Lyell syndrome. -- A chronic phase, described with a variable time of onset after the acute episode. Two stages have been recognised: -- the early chronic phase has a clinical and histological picture similar to Lichen Planus, a frequent dermatosis which is thought to be the result of a chronic immune injury against epidermis. As early phase is fading, an atrophic aspect of the skin may develop, whose severe type is described as Poïkiloderma. -- The late chronic phase, in which densification of dermal collagen is become the essential features. In some patients, sclerotic changes develop, which are resembling to scleroderma ones. The cutaneous manifestations of GvH Disease can be responsible for considerable morbidity, mortality and sequelles. They may serve as a biological model for several skin diseases.

The lichen planus like and sclerotic phases of the graft versus host disease in man: an ultrastructural study of six cases.

Skin biopsies from 6 patients with chronic graft-versus-host disease (GVHD) were studied ultrastructurally. The 6 patients experienced an early lichenoid phase 65-135 days after the graft and 3 of them progressed to a late sclerotic phase 200-340 days after the grafting Damage to the basal membrane and to the keratinocytes of the basal layer and low spinous layers, and presence of epidermal regenerative cells were features common to the lichenoid phase of chronic GVHD and idiopathic lichen planus. The late sclerotic phase of GVHD with persistence of basal cell injury, normal periodicity and structure of the collagen fibres and numerous active fibroblasts in the upper third of the dermis were findings that distinguished GVHD from scleroderma. Satellite cell necrosis, i.e. lymphocyte satellites of necrotic keratinocyte, was observed in the two phases of chronic GVHD. Thus at the ultrastructural level the early phase of chronic GVHD mimics lichen planus, but the late sclerotic phase is distinct from scleroderma.

Bone marrow transplantation in Fanconi anaemia.

Five patients with Fanconi anaemia have been treated by bone marrow transplantation from HLA identical donors. Only one patient survived for more than 3 years. She is now perfectly healthy with complete haematological reconstitution with chimaerism and disparition of chromosomal abnormalities. In contrast, four patients died of acute severe GVHD soon after grafting. In addition, all had signs of severe cyclophosphamide toxicity. This evolution could be explained by a special sensitivity of FA cells to alkylating agents and may indicate the need to modify the conditioning regimen in FA patients.

[Sezary syndrome with serum monoclonal immunoglobulin. Absence of detectable shared idiotype between the monoclonal immunoglobulin and membrane structures of the Sezary cells (author's transl)]. / Syndrome de Sézary avec immunoglobuline monoclonale sérique. Absence de communauté idiotypique décelable entre l'immunoglobuline monoclonale et les structures de membrane des cellules de Sézary.

In a case of Sezary syndrome with monoclonal serum immunoglobulin (IgM kappa) haematological investigations and a study of lymphocyte membrane markers failed to demonstrate any proliferation of B-lymphocytes. In order to test the hypothesis that the T-cells of Sezary syndrome and the B-cells producing the monoclonal immunoglobulin derived from the same stem cells and belonged to the same clone, an antiserum specific for the idiotype determinants of the IgM was used to detect by membrane immunofluorescence the possible presence in Sezary cells of surface receptors containing these determinants. The results were negative. The significance of the association--probably relatively too frequent to be fortuitous--between T-lymphocyte proliferation and monoclonal serum immunoglobulin therefore remains uncertain.