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Carvalho M.,University of Coimbra | Costa L.M.,University of Trás os Montes e Alto Douro | Pereira J.E.,University of Trás os Montes e Alto Douro | Shirosaki Y.,Kyushu Institute of Technology | And 8 more authors.
Neurological Research | Year: 2015

Objectives: Post-operative scarring process on lumbar surgery is object of several studies mainly because of the epidural fibrosis formation. Hybrid chitosan have shown promising effect on fibrosis prevention. The aim of this study was to determine the influence of chitosan-silane membrane on the lumbar surgery scarring process. These membranes have improved mechanical strength which makes them suitable to maintain a predefined shape.Methods: A two level lumbar laminectomy was performed in 14 New Zealand male rabbits. Laminectomy sites were randomly selected for biomaterial or control. Chitosan membranes were prepared and care was taken in order to make it adapted to the bone defect dimensions covering the totality of the defect including the bone margins. Histological analysis was performed by haematoxylin/eosin and by Masson’s trichrome staining four weeks after laminectomy.Results: Microscope observations revealed the presence of a well-organized regenerating tissue, integrated in the surrounding vertebral bone tissue with a regular and all-site interface on the chitosan sites, in clear contrast with the presence of a disorganized regenerating tissue with aspects consistent with the persistence of a chronic inflammatory condition, on control sites.Discussion: The results of this study clearly demonstrated that hybrid chitosan had an organizing effect on post-operative scarring process. The presence of the hybrid chitosan membrane resulted on a wellorganized tissue integrated in the surrounding vertebral bone tissue with signs of regenerative bone tissue in continuity with native bone. This can be a major feature on the dynamics of epidural fibrosis formation. © W. S. Maney & Son Ltd 2015.

Atayde L.M.,Abel Salazar Biomedical Sciences Institute | Atayde L.M.,Institute Ciencias e Tecnologias Agrarias e Agro Alimentares ICETA | Cortez P.P.,Abel Salazar Biomedical Sciences Institute | Cortez P.P.,Institute Ciencias e Tecnologias Agrarias e Agro Alimentares ICETA | And 8 more authors.
Journal of Materials Science: Materials in Medicine | Year: 2014

Presently, several bone graft substitutes are being developed or already available for clinical use. However, the limited number of clinical and in vivo trials for direct comparison between these products may complicate this choice. One of the main reasons for this scarcity it is the use of models that do not readily allow the direct comparison of multiple bone graft substitutes, due especially to the small number of implantation sites. Although sheep cancellous bone models are now well established for these purposes, the limited availability of cancellous bone makes it difficult to find multiple comparable sites within a same animal. These limitations can be overcome by the monocortical model here proposed as it consists in 5-6 holes (5 mm Ø), in the femoral diaphysis, with similar bone structure, overlying soft tissue and loading pattern for all defects. Associated to this model, it is also described a fast histomorphometric analysis method using a computer image segmentation test (Threshold method) to assess bone regeneration parameters. The information compiled through the experimental use of 45 sheep in several studies allowed determining that this ovine model has the potential to demonstrate differences in bone-forming performance between various scaffolds. Additionally, the described histomorphometric method is fast, accurate and reproducible. © 2014 Springer Science+Business Media.

Gartner A.,Abel Salazar Biomedical Sciences Institute | Gartner A.,Institute Ciencias e Tecnologias Agrarias e Agro Alimentares ICETA | Pereira T.,Abel Salazar Biomedical Sciences Institute | Pereira T.,Institute Ciencias e Tecnologias Agrarias e Agro Alimentares ICETA | And 19 more authors.
Journal of Stem Cells and Regenerative Medicine | Year: 2014

Peripheral nerves have the intrinsic capacity of self-regeneration after traumatic injury but the extent of the regeneration is often very poor. Increasing evidence demonstrates that mesenchymal stem/stromal cells (MSCs) may play an important role in tissue regeneration through the secretion of soluble trophic factors that enhance and assist in repair by paracrine activation of surrounding cells. In the present study, the therapeutic value of a population of umbilical cord tissue-derived MSCs, obtained by a proprietary method (UCX®), was evaluated on end-to-end rat sciatic nerve repair. Furthermore, in order to promote both, end-to-end nerve fiber contacts and MSC cell-cell interaction, as well as reduce the flush away effect of the cells after administration, a commercially available haemostatic sealant, Floseal®, was used as vehicle. Both, functional and morphologic recoveries were evaluated along the healing period using extensor postural thrust (EPT), withdrawal reflex latency (WRL), ankle kinematics analysis, and either histological analysis or stereology, in the hyper-acute, acute and chronic phases of healing. The histological analysis of the hyper-acute and acute phase studies revealed that in the group treated with UCX® alone the Wallerian degeneration was improved for the subsequent process of regeneration, the fiber organization was higher, and the extent of fibrosis was lower. The chronic phase experimental groups revealed that treatment with UCX® induced an increased number of regenerated fibers and thickening of the myelin sheet. Kinematics analysis showed that the ankle joint angle determined for untreated animals was significantly different from any of the treated groups at the instant of initial contact (IC). At opposite toe off (OT) and heel rise (HR), differences were found between untreated animals and the groups treated with either UCX® alone or UCX® administered with Floseal®. Overall, the UCX® application presented positive effects in functional and morphologic recovery, in both the acute and chronic phases of the regeneration process. Kinematics analysis has revealed positive synergistic effects brought by Floseal® as vehicle for MSCs. © Journal of Stem Cells and Regenerative Medicine. All rights reserved.

Alexandre N.,University of Évora | Ribeiro J.,Institute Ciencias e Tecnologias Agrarias e Agro Alimentares ICETA | Ribeiro J.,Abel Salazar Biomedical Sciences Institute | Gartner A.,Institute Ciencias e Tecnologias Agrarias e Agro Alimentares ICETA | And 17 more authors.
Journal of Biomedical Materials Research - Part A | Year: 2014

Polyvinyl alcohol hydrogel (PVA) is a synthetic polymer with an increasing application in the biomedical field that can potentially be used for vascular grafting. However, the tissue and blood-material interactions of such gels and membranes are unknown in detail. The objectives of this study were to: (a) assess the biocompatibility and (b) hemocompatibility of PVA-based membranes in order to get some insight into its potential use as a vascular graft. PVA was evaluated isolated or in copolymerization with dextran (DX), a biopolymer with known effects in blood coagulation homeostasis. The effects of the mesenchymal stem cells (MSCs) isolated from the umbilical cord Wharton's jelly in the improvement of PVA biocompatibility and in the vascular regeneration were also assessed. The biocompatibility of PVA was evaluated by the implantation of membranes in subcutaneous tissue using an animal model (sheep). Histological samples were assessed and the biological response parameters such as polymorphonuclear neutrophilic leucocytes and macrophage scoring evaluated in the implant/tissue interface by International Standards Office (ISO) Standard 10993-6 (annex E). According to the scoring system based on those parameters, a total value was obtained for each animal and for each experimental group. The in vitro hemocompatibility studies included the classic hemolysis assay and both human and sheep bloods were used. Relatively to biocompatibility results, PVA was slightly irritant to the surrounding tissues; PVA-DX or PVA plus MSCs groups presented the lowest score according to ISO Standard 10993-6. Also, PVA was considered a nonhemolytic biomaterial, presenting the lowest values for hemolysis when associated to DX. © 2014 Wiley Periodicals, Inc.

PubMed | Institute Ciencias e Tecnologias Agrarias e Agro Alimentares ICETA, Polytechnic Institute of Leiria, University of Trás os Montes e Alto Douro, University of Turin and 3 more.
Type: | Journal: BioMed research international | Year: 2014

In peripheral nerves MSCs can modulate Wallerian degeneration and the overall regenerative response by acting through paracrine mechanisms directly on regenerating axons or upon the nerve-supporting Schwann cells. In the present study, the effect of human MSCs from Whartons jelly (HMSCs), differentiated into neuroglial-like cells associated to poly (DL-lactide--caprolactone) membrane, on nerve regeneration, was evaluated in the neurotmesis injury rat sciatic nerve model. Results in vitro showed successful differentiation of HMSCs into neuroglial-like cells, characterized by expression of specific neuroglial markers confirmed by immunocytochemistry and by RT-PCR and qPCR targeting specific genes expressed. In vivo testing evaluated during the healing period of 20 weeks, showed no evident positive effect of HMSCs or neuroglial-like cell enrichment at the sciatic nerve repair site on most of the functional and nerve morphometric predictors of nerve regeneration although the nociception function was almost normal. EPT on the other hand, recovered significantly better after HMSCs enriched membrane employment, to values of residual functional impairment compared to other treated groups. When the neurotmesis injury can be surgically reconstructed with an end-to-end suture or by grafting, the addition of a PLC membrane associated with HMSCs seems to bring significant advantage, especially concerning the motor function recovery.

PubMed | University of Porto, Abel Salazar Biomedical Sciences Institute, Institute Ciencias e Tecnologias Agrarias e Agro Alimentares ICETA, University of Lisbon and Polytechnic Institute of Leiria
Type: | Journal: Stem cells international | Year: 2014

Skeletal muscle has good regenerative capacity, but the extent of muscle injury and the developed fibrosis might prevent complete regeneration. The in vivo application of human mesenchymal stem cells (HMSCs) of the umbilical cord and the conditioned media (CM) where the HMSCs were cultured and expanded, associated with different vehicles to induce muscle regeneration, was evaluated in a rat myectomy model. Two commercially available vehicles and a spherical hydrogel developed by our research group were used. The treated groups obtained interesting results in terms of muscle regeneration, both in the histological and in the functional assessments. A less evident scar tissue, demonstrated by collagen type I quantification, was present in the muscles treated with HMSCs or their CM. In terms of the histological evaluation performed by ISO 10993-6 scoring, it was observed that HMSCs apparently have a long-term negative effect, since the groups treated with CM presented better scores. CM could be considered an alternative to the in vivo transplantation of these cells, as it can benefit from the local tissue response to secreted molecules with similar results in terms of muscular regeneration. Searching for an optimal vehicle might be the key point in the future of skeletal muscle tissue engineering.

Shirosaki Y.,Kyushu Institute of Technology | Hayakawa S.,Okayama University | Osaka A.,Okayama University | Lopes M.A.,University of Porto | And 4 more authors.
BioMed Research International | Year: 2014

The treatment of peripheral nerve injuries remains one of the greatest challenges of neurosurgery, as functional recover is rarely satisfactory in these patients. Recently, biodegradable nerve guides have shown great potential for enhancing nerve regeneration. A major advantage of these nerve guides is that no foreign material remains after the device has fulfilled its task, which spares a second surgical intervention. Recently, we studied peripheral nerve regeneration using chitosan-γ-glycidoxypropyltrimethoxysilane (chitosan-GPTMS) porous hybrid membranes. In our studies, these porous membranes significantly improved nerve fiber regeneration and functional recovery in rat models of axonotmetic and neurotmetic sciatic nerve injuries. In particular, the number of regenerated myelinated nerve fibers and myelin thickness were significantly higher in rat treated with chitosan porous hybrid membranes, whether or not they were used in combination with mesenchymal stem cells isolated from the Wharton's jelly of the umbilical cord. In this review, we describe our findings on the use of chitosan-GPTMS hybrids for nerve regeneration. © 2014 Yuki Shirosaki et al.

Atayde L.M.,Abel Salazar Biomedical Sciences Institute | Atayde L.M.,Institute Ciencias e Tecnologias Agrarias e Agro Alimentares ICETA | Cortez P.P.,Abel Salazar Biomedical Sciences Institute | Cortez P.P.,Institute Ciencias e Tecnologias Agrarias e Agro Alimentares ICETA | And 5 more authors.
Journal of Biomedical Materials Research - Part B Applied Biomaterials | Year: 2015

In the last decades, the well-known disadvantages of autografts and allografts have driven to the development of synthetic bone grafts for bone regeneration. BonelikeVR , a glass-reinforced hydroxyapatite (HA) composite was developed and registered for bone grafting. This biomaterial is composed by a modified HA matrix, with α- and β-tricalcium phosphate secondary phases. Aiming to improve the biological characteristics of Bonelike® , new spherical pelleted granules, of different shape and size, were developed with controlled micro and macrostructure. In the present study, it was compared the physicochemical properties and in vivo performance of different Bonelike® granule presentations-Bonelike® polygonal (500-1000 μm size) and Bonelike spherical (250-500 μm; 500-1000 μm size). For the in vivo study, Bonelike® was implanted on sheep femurs, with various implantation times (30 days, 60 days, 120 days, and 180 days). X-ray diffraction analysis revealed that the phase composition of different granules presentations was similar. Bonelike® spherical 500-1000 mm was the most porous material (global porosity and intraporosity) and Bonelike® polygonal 500-1000 mm the less porous. Considering the in vivo study, both polygonal and spherical granules presented osteoconductive proprieties. The spherical granules showed several advantages, including easier medical application through syringe and improved osteointegration, osteoconduction, and degradation, by the presence of larger pores, controlled micro- and macrosctructure and suitable particle format that adapts to bone growth. Bonelike® spherical 500-1000 mm showed improved new bone invasion throughout the material's structure and Bonelike® spherical 250-500 mm appeared to induce faster bone regeneration, presenting less unfilled areas and less lacunae in the histological analysis. © 2014 Wiley Periodicals Inc.

Goncalves C.,University of Porto | Ribeiro J.,Abel Salazar Biomedical Sciences Institute | Ribeiro J.,Institute Ciencias e Tecnologias Agrarias E Agro Alimentares ICETA | Pereira T.,Abel Salazar Biomedical Sciences Institute | And 7 more authors.
Journal of Biomedical Materials Research - Part A | Year: 2016

Peripheral nerve regeneration is a serious clinical problem. Presently, there are several nerve tube-guides available in the market, however with some limitations. The goal of this work was the development of a biomaterial with high electrical conductivity to produce tube-guides for nerve regeneration after neurotmesis injuries whenrver an end-to-end suture without tension is not possible. A matrix of poly(vinyl alcohol) (PVA) was used loaded with the following electrical conductive materials: COOH-functionalized multiwall carbon nanotubes (MWCNTs), poly(pyrrole) (PPy), magnesium chloride (MgCl2), and silver nitrate (AgNO3). The tube-guide production was carried out by a freezing/thawing process (physical crosslinking) with a final annealing treatment. After producing the tube-guide for nerve regeneration, the physicochemical characterization was performed. The most interesting results were achieved by loading PVA with 0.05% of PPy or COOH- functionalized CNTs. These tubes combined the electrical conductivity of carbon nanotubes (CNTs) and PPy with the biocompatibility of PVA matrix, with potential clinical application for nerve regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1981–1987, 2016. © 2016 Wiley Periodicals, Inc.

Ribeiro J.,Abel Salazar Biomedical Sciences Institute | Ribeiro J.,Institute Ciencias e Tecnologias Agrarias e Agro Alimentares ICETA | Pereira T.,Abel Salazar Biomedical Sciences Institute | Pereira T.,Institute Ciencias e Tecnologias Agrarias e Agro Alimentares ICETA | And 17 more authors.
International Journal of Medical Sciences | Year: 2014

The healing process of the skin is a dynamic procedure mediated through a complex feedback of growth factors secreted by a variety of cells types. Despite the most recent advances in wound healing management and surgical procedures, these techniques still fail up to 50%, so cellular therapies involving mesenchymal stem cells (MSCs) are nowadays a promising treatment of skin ulcers which are a cause of high morbidity. The MSCs modulate the inflammatory local response and induce cell replacing, by a paracrine mode of action, being an important cell therapy for the impaired wound healing. The local application of human MSCs (hMSCs) isolated from the umbilical cord Wharton's jelly together with a poly(vinyl alcohol) hydrogel (PVA) membrane, was tested to promote wound healing in two dogs that were referred for clinical examination at UPVET Hospital, showing non-healing large skin lesions by the standard treatments. The wounds were infiltrated with 1000 cells/μl hMSCs in a total volume of 100 μl per cm2 of lesion area. A PVA membrane was applied to completely cover the wound to prevent its dehydration. Both animals after the treatment demonstrated a significant progress in skin regeneration with decreased extent of ulcerated areas confirmed by histological analysis. The use of Wharton's jelly MSCs associated with a PVA membrane showed promising clinical results for future application in the treatment of chronic wounds in companion animals and humans. © Ivyspring International Publisher.

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