X-Ray Phase Contrast Tomography Reveals Early Vascular Alterations and Neuronal Loss in a Multiple Sclerosis Model.

The degenerative effects of multiple sclerosis at the level of the vascular and neuronal networks in the central nervous system are currently the object of intensive investigation. Preclinical studies have demonstrated the efficacy of mesenchymal stem cell (MSC) therapy in experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis, but the neuropathology of specific lesions in EAE and the effects of MSC treatment are under debate. Because conventional imaging techniques entail protocols that alter the tissues, limiting the reliability of the results, we have used non-invasive X-ray phase-contrast tomography to obtain an unprecedented direct 3D characterization of EAE lesions at micro-to-nano scales, with simultaneous imaging of the vascular and neuronal networks. We reveal EAE-mediated alterations down to the capillary network. Our findings shed light on how the disease and MSC treatment affect the tissues, and promote X-ray phase-contrast tomography as a powerful tool for studying neurovascular diseases and monitoring advanced therapies.

Quantitative 3D investigation of Neuronal network in mouse spinal cord model.

The investigation of the neuronal network in mouse spinal cord models represents the basis for the research on neurodegenerative diseases. In this framework, the quantitative analysis of the single elements in different districts is a crucial task. However, conventional 3D imaging techniques do not have enough spatial resolution and contrast to allow for a quantitative investigation of the neuronal network. Exploiting the high coherence and the high flux of synchrotron sources, X-ray Phase-Contrast multiscale-Tomography allows for the 3D investigation of the neuronal microanatomy without any aggressive sample preparation or sectioning. We investigated healthy-mouse neuronal architecture by imaging the 3D distribution of the neuronal-network with a spatial resolution of 640 nm. The high quality of the obtained images enables a quantitative study of the neuronal structure on a subject-by-subject basis. We developed and applied a spatial statistical analysis on the motor neurons to obtain quantitative information on their 3D arrangement in the healthy-mice spinal cord. Then, we compared the obtained results with a mouse model of multiple sclerosis. Our approach paves the way to the creation of a "database" for the characterization of the neuronal network main features for a comparative investigation of neurodegenerative diseases and therapies.

Imaging collagen packing dynamics during mineralization of engineered bone tissue.

The structure and organization of the Type I collagen microfibrils during mineral nanoparticle formation appear as the key factor for a deeper understanding of the biomineralization mechanism and for governing the bone tissue physical properties. In this work we investigated the dynamics of collagen packing during ex-vivo mineralization of ceramic porous hydroxyapatite implant scaffolds using synchrotron high resolution X-ray phase contrast micro-tomography (XPCµT) and synchrotron scanning micro X-ray diffraction (SµXRD). While XPCµT provides the direct 3D image of the collagen fibers network organization with micrometer spatial resolution, SµXRD allows to probe the structural statistical fluctuations of the collagen fibrils at nanoscale. In particular we imaged the lateral spacing and orientation of collagen fibrils during the anisotropic growth of mineral nanocrystals. Beyond throwing light on the bone regeneration multiscale process, this approach can provide important information in the characterization of tissue in health, aging and degeneration conditions. STATEMENT OF SIGNIFICANCE: BONE grafts are the most common transplants after the blood transfusions. This makes the bone-tissue regeneration research of pressing scientific and social impact. Bone is a complex hierarchical structure, where the interplay of organic and inorganic mineral phases at different length scale (from micron to atomic scale) affect its functionality and health. Thus, the understanding of bone tissue regeneration requires to image its spatial-temporal evolution (i) with high spatial resolution and (ii) at different length scale. We exploited high spatial resolution X-ray Phase Contrast micro Tomography and Scanning micro X-ray Diffraction in order to get new insight on the engineered tissue formation mechanisms. This approach could open novel routes for the early detection of different degenerative conditions of tissue.

Early stage mineralization in tissue engineering mapped by high resolution X-ray microdiffraction.

The specific routes of biomineralization in nature are here explored using a tissue engineering approach in which bone is formed in porous ceramic constructs seeded with bone marrow stromal cells and implanted in vivo. Unlike previous studies this model system reproduces mammalian bone formation, here investigated at high temporal resolution. Different mineralization stages were monitored at different distances from the scaffold interface so that their spatial analysis corresponded to temporal monitoring of the bone growth and mineralization processes. The micrometer spatial resolution achieved by our diffraction technique ensured highly accurate reconstruction of the different temporal mineralization steps and provided some hints to the challenging issue of the mineral deposit first formed at the organic-mineral interface. Our results indicated that in the first stage of biomineralization organic tissue provides bioavailable calcium and phosphate ions, ensuring a constant reservoir of amorphous calcium phosphate (ACP) during hydroxyapatite (HA) nanocrystal formation. In this regard we suggest a new role of ACP in HA formation, with a continuous organic-mineral transition assisted by a dynamic pool of ACP. After HA nanocrystals formed, the scaffold and collagen act as templates for nanocrystal arrangement on the microscopic and nanometric scales, respectively.

Biodegradation of porous calcium phosphate scaffolds in an ectopic bone formation model studied by X-ray computed microtomograph.

Three types of ceramic scaffolds with different composition and structure [namely synthetic 100% hydroxyapatite (HA; Engipore), synthetic calcium phosphate multiphase biomaterial containing 67% silicon stabilized tricalcium phosphate (Si-TCP; Skelite) and natural bone mineral derived scaffolds (Bio-oss)] were seeded with mesenchymal stem cells (MSC) and ectopically implanted for 8 and 16 weeks in immunodeficient mice. X-ray synchrotron radiation microtomography was used to derive 3D structural information on the same scaffolds both before and after implantation. Meaningful images and morphometric parameters such as scaffold and bone volume fraction, mean thickness and thickness distribution of the different phases as a function of the implantation time, were obtained. The used imaging algorithms allowed a direct comparison and registration of the 3D structure before and after implantation of the same sub-volume of a given scaffold. In this way it was possible to directly monitor the tissue engineered bone growth and the complete or partial degradation of the scaffold. Further, the detailed kinetics studies on Skelite scaffolds implanted for different length of times from 3 days to 24 weeks, revealed in the X-ray absorption histograms two separate peaks associated to HA and TCP. It was therefore possible to observe that the progressive degradation of the Skelite scaffolds was mainly due to the resorption of TCP. The different saturation times in the tissue engineered bone growth and in the TCP resorption confirmed that the bone growth was not limited the scaffold regions that were resorbed but continued in the inward direction with respect to the pore surface.

Platelet lysate favours in vitro expansion of human bone marrow stromal cells for bone and cartilage engineering.

The heterogeneous population of non-haematopoietic cells residing in the bone marrow (bone marrow stromal cells, BMSCs) and the different fractions and components obtained from platelet-rich plasma provide an invaluable source of autologous cells and growth factors for bone and other connective tissue reconstruction. In this study, we investigated the effect of an allogenic platelet lysate on human BMSCs proliferation and differentiation. Cell proliferation and number of performed cell doublings were enhanced in cultures supplemented with the platelet-derived growth factors (platelet lysate, PL), either with or without the concomitant addition of fetal bovine serum (FBS), compared to cultures performed in the presence of FBS and FGF2. Both in vitro and in vivo osteogenic differentiation were unaltered in cells maintained in medium supplemented with PL and not FBS (Only PL) and in cells maintained in medium containing FBS and FGF2. Interestingly, the in vitro cartilage formation was more effective in the pellet of BMSCs expanded in the Only PL medium. In particular, a chondrogenic differentiation was observed in pellets of some in vitro-expanded BMSCs in the Only PL medium, whereas pellets from parallel cell cultures in medium containing FBS did not respond to the chondrogenic induction. We conclude that the platelet lysate from human source is an effective and even more beneficial substitute for fetal bovine serum to support the in vitro expansion of human BMSCs for subsequent tissue-engineering applications.

Regeneration of large bone defects in sheep using bone marrow stromal cells.

Bone repair was addressed in a critical-sized defect model in sheep, combining a ceramic biomaterial and mesenchymal progenitor cells. The defects in the tibial mid-diaphysis were treated with autologous bone or with a silicon-stabilized tricalcium phosphate biomaterial, implemented or not by the addition of expanded bone marrow stromal cells. An internal locking compression plate and an external fixator were applied for stabilization. Radiographies were taken during the 8 months follow-up: the pixel grey levels of the lesion areas were determined to evaluate the repair process radiologically. Microradiography, histology and vascular density tests were performed. The autologous bone-treated group performed best, as assessed radiologically, within 20-24 weeks after surgery. Very limited healing was detected in the other experimental group: a partial bone deposition occurred at the periphery of the bony stumps only in the cell-seeded scaffolds. Interestingly, this effect ended within 20-24 weeks, as for the autologous bone, suggesting similar kinetics of the repair processes involved. Moreover, bone deposition was located where a significant reduction of the ceramic scaffold was detected. Faxitron microradiography and histology data confirmed these results. Vascular density analysis evidenced that cell-seeded scaffolds supported an increased vascular ingrowth. Thus, the interactions with the proper microenvironment and the oxygen and nutrient supply in the inner part of the constructs seem fundamental to initiate scaffold substitution and to improve cell performance in tissue-engineered approaches to bone repair.

Kinetics of in vivo bone deposition by bone marrow stromal cells within a resorbable porous calcium phosphate scaffold: an X-ray computed microtomography study.

Resorbable ceramic scaffolds based on Silicon stabilized tricalcium phosphate (Si-TCP) were seeded with bone marrow stromal cells (BMSC) and ectopically implanted for 2, 4, and 6 months in immunodeficient mice. Qualitative and quantitative evaluation of the scaffold material was performed by X-ray synchrotron radiation computed microtomography (microCT) with a spatial resolution lower than 5 microm. Unique to these experiments was that microCT data were first collected on the scaffolds before implantation and then on the same scaffolds after they were seeded with BMSC, implanted in the mice and rescued after different times. Volume fraction, mean thickness and thickness distribution were evaluated for both new bone and scaffold phases as a function of the implantation time. New bone thickness increased from week 8 to week 16. Data for the implanted scaffolds were compared with those derived from the analysis of the same scaffolds prior to implantation and with data derived from 100% hydroxyapatite (HA) scaffold treated and analyzed in the same way. At variance with findings with the 100% HA scaffolds a significant variation in the density of the different Si-TCP scaffold regions in the pre- and post-implantation samples was observed. In particular a post-implantation decrease in the density of the scaffolds, together with major changes in the scaffold phase composition, was noticeable in areas adjacent to newly formed bone. Histology confirmed a better integration between new bone and scaffold in the Si-TCP composites in comparison to 100% HA composites where new bone and scaffold phases remained well distinct.

Bulk and interface investigations of scaffolds and tissue-engineered bones by X-ray microtomography and X-ray microdiffraction.

This review is presented of recent investigations concerning the structure of ceramic scaffolds and tissue-engineered bones and focused on two techniques based on X-ray radiation, namely microtomography (microCT) and microdiffraction. Bulk 3D information, with micro-resolution, is mainly obtained by microCT, whereas microdiffraction provides useful information on interfaces to the atomic scale, i.e. of the order of the nanometer. Since most of the reported results were obtained using synchrotron radiation, a brief description of the European Synchrotron Radiation Facility (ESRF) is presented, followed by a description of the two techniques. Then examples of microstructural investigations of scaffolds are reported together with studies on bone architecture. Finally, studies on ex vivo tissue-engineered bone and on bone microstructure in vivo are presented.

Engineering of bone using bone marrow stromal cells and a silicon-stabilized tricalcium phosphate bioceramic: evidence for a coupling between bone formation and scaffold resorption.

Resorbable porous ceramic constructs, based on silicon-stabilized tricalcium phosphate, were implanted in critical-size defects of sheep tibias, either alone or after seeding with bone marrow stromal cells (BMSC). Only BMSC-loaded ceramics displayed a progressive scaffold resorption, coincident with new bone deposition. To investigate the coupled mechanisms of bone formation and scaffold resorption, X-ray computed microtomography (muCT) with synchrotron radiation was performed on BMSC-seeded ceramic cubes. These were analyzed before and after implantation in immunodeficient mice for 2 or 6 months. With increasing implantation time, scaffold thickness significantly decreased while bone thickness increased. The muCT data evidenced that all scaffolds showed a uniform density distribution before implantation. Areas of different segregated densities were instead observed, in the same scaffolds, once seeded with cells and implanted in vivo. A detailed muX-ray diffraction analysis revealed that only in the contact areas between deposited bone and scaffold, the TCP component of the biomaterial decreased much faster than the HA component. This event did not occur at areas away from the bone surface, highlighting coupling and cell-dependency of the resorption and matrix deposition mechanisms. Moreover, in scaffolds implanted without cells, both the ceramic density and the TCP:HA ratio remained unchanged with respect to the pre-implantation analysis.

Engineered bone from bone marrow stromal cells: a structural study by an advanced x-ray microdiffraction technique.

The mechanism of mineralized matrix deposition was studied in a tissue engineering approach in which bone tissue is formed when porous ceramic constructs are loaded with bone marrow stromal cells and implanted in vivo. We investigated the local interaction between the mineral crystals of the engineered bone and the biomaterial by means of microdiffraction, using a set-up based on an x-ray waveguide. We demonstrated that the newly formed bone is well organized inside the scaffold pore, following the growth model of natural bone. Combining wide angle (WAXS) and small angle (SAXS) x-ray scattering with high spatial resolution, we were able to determine the orientation of the crystallographic c-axis inside the bone crystals, and the orientation of the mineral crystals and collagen micro-fibrils with respect to the scaffold. In this work we analysed six samples and for each of them two pores were studied in detail. Similar results were obtained in all cases but we report here only the most significant sample.

Kinetics of in vivo bone deposition by bone marrow stromal cells into porous calcium phosphate scaffolds: an X-ray computed microtomography study.

In a typical bone tissue engineering application, osteogenic cells are harvested and seeded on a three-dimensional (3D) synthetic scaffold that acts as guide and stimulus for tissue growth, creating a tissue engineering construct or living biocomposite. Despite the large number of performed experiments in different laboratories, information on the kinetics of bone growth into the scaffolds is still scarce. Highly porous hydroxyapatite scaffolds were investigated before the implantation and after they were seeded with in vitro expanded bone marrow stromal cells (BMSC) and implanted for 8, 16, or 24 weeks in immunodeficient mice. Synchrotron x-ray computed microtomography (microCT) was used for qualitative and quantitative 3D characterization of the scaffold material and 3D evaluation of tissue engineered bone growth kinetics after in vivo implantation. Experiments were performed taking advantage of a dedicated set up at the European Synchrotron Radiation Facility (ESRF, Grenoble, France), which allowed quantitative imaging at a spatial resolution of about 5 microm. A peculiarity of these experiments was the fact that at first the data were obtained on the different pure scaffolds, then the same scaffolds were seeded by BMSC, implanted, and brought again to ESRF for investigating the formation of new bone. The volume fraction, average thickness, and distribution of the newly formed bone were evaluated as a function of the implantation time. New bone thickness increased from week 8 to week 16, but deposition of new bone was arrested from week 16 to week 24. Instead, mineralization of the newly deposited bone matrix continued up to week 24.

Tissue engineering of bone: search for a better scaffold.

BACKGROUND: Large bone defects still represent a major problem in orthopedics. Traditional bone-repair treatments can be divided into two groups: the bone transport (Ilizarov technology) and the graft transplant (autologous or allogeneic bone grafts). Thus far, none of these strategies have proven to be always resolving. As an alternative, a tissue engineering approach has been proposed where osteogenic cells, bioceramic scaffolds, growth factors and physical forces concur to the bone defect repair. Different sources of osteoprogenitor cells have been suggested, bone marrow stromal cells (BMSC) being in most cases the first choice. METHODS AND RESULTS: In association with mineral tridimensional scaffolds, BMSC form a primary bone tissue which is highly vascularized and colonized by host hemopoietic marrow. The chemical composition of the scaffold is crucial for the osteoconductive properties and the resorbability of the material. In addition, scaffolds should have an internal structure permissive for vascular invasion. Porous bioceramics [hydroxyapatite (HA) and tricalcium phosphate] are osteoconductive and are particularly advantageous for bone tissue engineering application as they induce neither an immune nor an inflammatory response in the implanted host. Earlier, we first reported a cell-based tissue engineering procedure to treat three patients with long bone segmental defects. Cells were loaded on a 100% HA porous ceramic. These scaffolds proved to have good osteoconductive properties resulting in a good functional recovery, but they have not been resorbed after more than 5 years from the implant. In addition, due to the high density of the mineral and the relatively low porosity (50-60%), it was very difficult to monitor the patient recovery during the post-surgery time using X-rays. CONCLUSIONS: We report here some pre-clinical testing of new scaffolds. To compare these second generation ceramic scaffolds more suitable for a tissue engineering approach we had to first establish animal models and analysis procedures including the use of X-ray-computed microtomography associated with X-rays synchroton radiation.

Bone and cartilage formation by skeletal muscle derived cells.

In adult individuals when most tissues have progressively lost the ability to regenerate, bone maintains the potential for a continuous self remodeling. The bone marrow has been so far the main recognized source of osteoprogenitor cells that contribute to the turnover of the skeletal scaffold. The possibility though exists that a pool of osteoprogenitor cells resides within other adult tissues and in particular, as reported previously, in other connective tissues such as fat and skeletal muscle. In an attempt to identify an alternative source of osteoprogenitor cells other than bone marrow we looked into the skeletal muscle. A plastic adhering cell population, from now on referred to as skeletal muscle derived cells (SMDCs), was obtained from biopsies of human skeletal muscle. SMDCs were clonogenic and displayed a fibroblast-like morphology. The isolated cell population had a mesenchymal origin as indicated by abundant expression of type I collagen, fibronectin, and vimentin and appeared heterogeneous. SMDCs were positive for alpha smooth actin, and to a lesser extent for desmin and alpha sarcomeric myosin, two specific markers of the myogenic phenotype. Surprisingly though SMDCs expressed early markers of an osteogenic commitment as indicated by positive staining for alkaline phosphatase, osteopontin, and osteonectin. Under the appropriate stimuli, these cells deposited in vitro a mineralized bone matrix and a proteoglycan rich matrix. In addition, SMDCs cultured in the presence of low serum and insulin differentiated towards adipocytes developing abundant lipid droplets in the cytoplasm. Furthermore SMDCs formed three-dimensional bone tissue in vivo when implanted in an immunodeficient mouse, and a mature cartilage rudiment when maintained as a pellet culture. In summary, we report the isolation and characterization of a cell population from the human skeletal muscle not only able to express in vitro specific markers of distinct mesenchymal lineages (adipogenic, chondrogenic, and osteogenic), but most importantly, able to complete the differentiation pathway leading to the formation of bone and cartilage. In this respect SMDCs resemble bone marrow stromal cells (BMSCs).

Synchrotron radiation microtomography of bone engineered from bone marrow stromal cells.

Osteoprogenitor cells expanded in vitro and associated with porous ceramic scaffolds have been proposed as bone substitutes. Animal models have been developed to test the efficacy of various cell populations and scaffolds in promoting bone repair. Qualitative analysis of the new bone formed within the ceramic scaffold is relatively easy by conventional histology. On the other hand, quantitative data are difficult to obtain. X-ray computed microtomography was used as a possible experimental technique to obtain quantitative data on the three-dimensional structure of newly formed bone and of remaining scaffold in implants after 8 weeks in vivo. Measurements were performed at the European Synchrotron Radiation Facility on beamline ID19 with a spatial resolution of about 5 microm. This study clearly indicates the possibility of nondestructive quantitative analysis of bone-engineered constructs. The technique appears suitable to compare different scaffolds (and possibly different cell populations) with regard to bone formation efficiency and reabsorbability of biomaterials in the immunodeficient mouse model.

[Hepatic hydatidosis in Puglia: analysis of the hospital discharge cards in the years 1996-2000]. / L'echinococcosi epatica in Puglia: analisi delle schede di dimissione ospedaliera (S.D.O.) negli anni 1996-2000.

The authors valueted the distribution of Hepatic Hydatidosis in the Region Puglia (Italy) by hospital dimission cards (schede di dimissione ospedaliera). At present, the SDO represent the most important information tool to monitor hospital activity. The authors examined 468 cases of hepatic Hydatidosis admitted in regional hospital during 1996-2000. 54% were men and 46% women, 75% of whom aged between 40-60 anni. The median hospitalization time was of 12 days and 80% were surgical departments. All the cases examined were uniformedly distributed in surgical hospitals. No hospital acts as a pole of attraction. The periodal prevalence was 6.4/100.000. 40% of cases had surgical DRG with quadriennal sanitary cost of 4 miliardi liras. The Surgical procedure was 53% hepatic lesion demolition, 14% partial epatectomy, 7.2% lobectomy, 5.2% marsupializzazione, 2.6% hepatectomy, and 19% others procedures. At present, due to physiopathological considerations, radical surgery is preferred to conservative surgery. Hepatic hydatidosis is considered a public health problem whose epidemiologic monitoring and precautionary measures could be oriented to control the related sanitary costs.

Effect of different growth factors on the chondrogenic potential of human bone marrow stromal cells.

OBJECTIVE: The aim of this study was to investigate the effects of different growth factors on the chondrogenic potential of human bone marrow stromal cells (BMSC). DESIGN: Different growth factors which have been shown to sustain the osteogenic potential of BMSC during their 'in vitro' expansion were assayed for the maintenance of the chondrogenic potential. We compared the ability of BMSC to reconstitute cartilage in vitro with their ability to form bone on hydroxyapatite microporous particles in an ectopic bone formation assay. RESULTS: Among the factors assayed, fibroblast growth factor 2 (FGF2) was the most effective in promoting growth of BMSC 'in vitro'. For all growth factors tested, we have found a complete overlap of the enhancement of chondrogenic and osteogenic potential. Any factor, either promoting or depressing bone formation, exerted the same effect on the chondrogenic potential of human BMSC. In particular, FGF2, either alone or in combination with other factors, strongly supported the formation of bone as well as of cartilage. CONCLUSIONS: We conclude that FGF2 maintains human BMSC in an immature state allowing their 'in vitro' expansion. Expanded cells retain the chondro- osteogenic potential. Interestingly, the chondrogenic potential of BMSC 'in vitro' is directly related to their ability to form bone 'in vivo'. BMSC expanded 'ex vivo' are presently being proposed for cell therapy of bone defects. 'In vitro' chondrogenesis may be regarded as a rapid prediction assay to assess cell ability to form bone after 'in vivo' transplant.

Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy.

Bone marrow stromal cells (BMSC) are an attractive target for novel strategies in the gene/cell therapy of hematologic and skeletal pathologies, involving BMSC in vitro expansion/transfection and reinfusion. We investigated the effects of in vitro expansion on BMSC pluripotentiality, proliferative ability, and bone-forming efficiency in vivo. BMSC from three marrow donors were cultured to determine their growth kinetics. At each passage, their differentiation potential was verified by culture in inductive media and staining with alizarin red, alcian blue, or Sudan black, and by immunostaining for osteocalcin or collagen II. First passage cells were compared to fresh marrow for their bone-forming efficiency in vivo. Stromal cell clones were isolated from five donors and characterized for their multidifferentiation ability. The lifespan and differentiation kinetics of five of these clones were determined. After the first passage, BMSC had a markedly diminish proliferation rate and gradually lost their multiple differentiation potential. Their bone-forming efficiency in vivo was reduced by about 36 times at first confluence as compared to fresh bone marrow. Experiments on the clones yielded comparable results. Culture expansion causes BMSC to gradually lose their early progenitor properties. Both the duration and the conditions of culture could be crucial to successful clinical use of these cells and must be considered when designing novel therapeutic strategies involving stromal mesenchymal progenitor manipulation and reinfusion.

Retinoic acid induces cell proliferation and modulates gelatinases activity in human osteoclast-like cell lines.

The effect of Retinoic Acid (RA) on human osteoclast-like cell lines, obtained from Giant Cell tumors (GCT) of bone, has been investigated evaluating its action on bone resorption, cell proliferation, microtubular organization and gelatinases expression and activity. Increasing concentrations of RA significantly dose-dependently decreased GCTs bone resorption, while 10(-7) M RA promoted an increase of cell proliferation. By immunofluorescence we demonstrated that GCTs express A and B gelatinases and, by zymography, that their activity was enhanced in medium collected from GCTs cultured in the presence of 10(-7) M RA. These data indicate that RA increases cell proliferation and modulates metalloproteinases (MMPs) activity, crucial events during the migration of osteoclast precursors toward bone surfaces.