RESUMO
Designing a new scaffold with an optimal ability of osteogenesis differentiation is a significant step bone tissue engineering along with the growing demands for bone craft in recent decades. Herein, we used Polyurethane (PU), a novel biocompatible and flexible polymer, and Hydroxyapatite (HA), the major component of human hard tissues matrix for developing new scaffolds and analyzing the in vitro osteogenic differentiation potential of human adipose-derived mesenchymal stem cells (Ad-MSCs) in basal and induction media. Gene expression analysis was performed to evaluate the expression level of four osteogenic differentiation genes. MTT assays were also done to assess the attachment and proliferation of the cells after 7 and 21 days of seeding to scaffolds. The expression level of RUNX2 was increased in seeded cells on PU/HA scaffolds compared with the PU. Cellular adhesion and proliferation of the Ad-MSCs were higher in PU/HA than PU scaffolds according to the histology analysis. The PU and PU/HA scaffolds supported the attachment, proliferation, and differentiation of Ad-MSCs, and they are suitable candidates for producing constructs in bone regeneration. However, further in-vitro and in-vivo studies on these scaffolds are needed to introduce an appropriate candidate for clinical bone regeneration.
Assuntos
Durapatita , Poliuretanos , Humanos , Poliuretanos/farmacologia , Osteogênese , Alicerces Teciduais , Engenharia Tecidual/métodos , Diferenciação Celular , Proliferação de CélulasRESUMO
PURPOSE: To report clinical and radiographic outcomes, rate of complications and influence on spinal alignment on long-term follow-up (FU) of patients who underwent lumbar total disc arthroplasty (TDR), bringing some evidence to determine the profile of the most well-suited patients for TDR. METHODS: A retrospective review of patients underwent TDR for low back pain from degenerative disc disease (DDD) resistant to conservative treatment was performed. Demographic features, surgical data, clinical and radiographic outcomes, complications and spinopelvic parameters were evaluated. RESULTS: Thirty patients (32 TDR) were included with a mean FU of 164 ± 36.5 months. The clinical outcomes measured by visual analogue scale and Oswestry Disability Index showed a significant improvement between preoperative and 1-year FU (p < 0.01). No significant temporal variance has been identified between 1-year and long-term follow-up (p > 0.05). The surgical revision rate was 10%. The overall rate of complications was 20%. At final follow-up, the mobility of the prosthesis was preserved in 68.75% of the cases, and 73.3% of the patients were globally well aligned. CONCLUSION: The optimal surgical indication is crucial to achieve excellent clinical and radiological outcomes. According to the literature and to our experience, we underline the importance of a coronal deformity < 15° Cobb angle and a Roussouly type 1 or 2 as the profile of the most well-suited patient for TDR. Our long-term results confirm the existing evidence about efficacy and safety of TDR as a reliable option, in optimal surgery indication, to treat DDD. These slides can be retrieved under Electronic Supplementary Material.
Assuntos
Degeneração do Disco Intervertebral , Disco Intervertebral , Dor Lombar , Vértebras Lombares , Substituição Total de Disco , Adolescente , Adulto , Feminino , Seguimentos , Humanos , Disco Intervertebral/cirurgia , Degeneração do Disco Intervertebral/complicações , Degeneração do Disco Intervertebral/diagnóstico por imagem , Degeneração do Disco Intervertebral/cirurgia , Dor Lombar/diagnóstico , Dor Lombar/etiologia , Dor Lombar/cirurgia , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/cirurgia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do Tratamento , Escala Visual Analógica , Adulto JovemRESUMO
To understand the regenerative effect of platelet-released molecules in bone repair one should investigate the cascade of events involving the resident osteoblast population during the reconstructive process. Here the in vitro response of human osteoblasts to a platelet lysate (PL) stimulus is reported. Quiescent or very slow dividing osteoblasts showed a burst of proliferation after PL stimulation and returned to a none or very slow dividing condition when the PL was removed. PL stimulated osteoblasts maintained a differentiation capability in vitro and in vivo when tested in absence of PL. Since angiogenesis plays a crucial role in the bone healing process, we investigated in PL stimulated osteoblasts the activation of hypoxia-inducible factor 1-alpha (HIF-1α) and signal transducer and activator of transcription 3 (STAT3) pathways, involved in both angiogenesis and bone regeneration. We observed phosphorylation of STAT3 and a strong induction, nuclear translocation and DNA binding of HIF-1α. In agreement with the induction of HIF-1α an enhanced secretion of vascular endothelial growth factor (VEGF) occurred. The double effect of the PL on quiescent osteoblasts, i.e., resumption of proliferation and activation of pathways promoting both angiogenesis and bone formation, provides a rationale to the application of PL as therapeutic agent in post-traumatic bone repair.
Assuntos
Plaquetas/metabolismo , Regeneração Óssea , Osso e Ossos/irrigação sanguínea , Osso e Ossos/lesões , Neovascularização Fisiológica , Osteoblastos/citologia , Adulto , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Osteoblastos/metabolismo , Osteogênese , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND AIMS: The amniotic fluid is a new source of multipotent stem cells with therapeutic potential for human diseases. In agreement with the regulatory requirement to reduce and possibly to avoid animal-derived reagents in the culture of cells intended for cell therapy, bovine serum, the most common supplement in the culture medium, was replaced by human platelet-derived growth factors. METHODS: We tested a new culture medium to expand monolayers of human amniotic fluid stem cells (hAFSC) for clinical use. The AFSC were isolated by c-Kit selection and expanded in media supplemented with either bovine serum or a human platelet lysate (Lyset). RESULTS: We compared proliferation kinetics, colony-forming unit percentage, multilineage differentiation, immunophenotypic characterization and inhibition of peripheral blood mononuclear cell proliferation of the two AFSC cell cultures and we found no significant differences. Moreover, the karyotype analysis of the cells expanded in the presence of the platelet lysate did not present cytogenetic abnormalities and in vitro and in vivo studies revealed no cell tumorigenicity. CONCLUSIONS: Platelet derivatives represent a rich source of growth factors that can play a safety role in the homeostasis, proliferation and remodeling of tissue healing. We propose human platelet extracts as a preferential alternative to animal serum for the expansion of stem cells for clinical applications.
Assuntos
Líquido Amniótico/citologia , Técnicas de Cultura de Células/métodos , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco/citologia , Animais , Bovinos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Leucócitos Mononucleares/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/fisiologia , Células-Tronco/efeitos dos fármacosRESUMO
BACKGROUND AIMS: Platelet derivatives have been proposed as alternatives to animal sera given that for cell therapy applications, the use of fetal bovine/calf serum (FBS/FCS) is subjected to severe limitations for safety and ethical concerns. We developed a cell culture medium additive obtained by the combination of two blood-derived standardized components. METHODS: A platelet lysate (PL) and a platelet-poor plasma (PPP) were produced in a lyophilized form. Each component was characterized for its growth factor content (platelet-derived growth factor-BB/vascular endothelial growth factor). PL and PPP were used as single components or in combination in different ratio at cumulative 5% final concentration in the culture medium. RESULTS: The single components were less effective than the component combination. In primary cell cultures (bone marrow stromal cells, adipose derived adult stem cells, osteoblasts, chondrocytes, umbilical cord-derived mesenchymal stromal cells, lymphocytes), the PL/PPP supplement promoted an increased cell proliferation in respect to the standard FCS culture in a dose-dependent manner, maintaining the cell functionality, clonogenicity, phenotype and differentiative properties throughout the culture. At a different component ratio, the supplement was also used to support proliferation of a cell line (U-937). CONCLUSIONS: The PL/PPP supplement is an efficient cell culture medium additive that can replace FCS to promote cell proliferation. It can outdo FCS, especially when adopted in primary cultures from tissue biopsies. Moreover, the dual component nature of the supplement allows the researcher to determine the more appropriate ratio of the two components for the nutritional and functional requirements of the cell type of interest.
Assuntos
Plaquetas/metabolismo , Diferenciação Celular , Meios de Cultura Livres de Soro/metabolismo , Células-Tronco Mesenquimais/citologia , Adulto , Células-Tronco Adultas/metabolismo , Animais , Becaplermina , Bovinos , Técnicas de Cultura de Células , Extratos Celulares/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Condrócitos/metabolismo , Meios de Cultura Livres de Soro/química , Humanos , Osteoblastos/metabolismo , Plasma/citologia , Proteínas Proto-Oncogênicas c-sis/metabolismo , Linfócitos T/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Serum of animal origin and in particular fetal bovine serum is the most commonly utilized cell culture medium additive for in vitro cell growth and differentiation. However, several major concerns have been raised by the scientific community regarding the use of animal sera for human cell-based culture applications. Among the possible alternatives to the animal serum, platelet-derived compounds have been proposed since more than 10 years. Nevertheless, the high degree of variability between the different platelet preparations, and the lack of standardized manufacturing and quality control procedures, made difficult to reach a consensus on the applicability of this novel cell culture medium supplement. In this study, we describe the preparation of a standardized platelet-rich plasma (PRP) derivative obtained starting from human-certified buffy coat samples with a defined platelet concentration and following protocols including also freeze-drying, gamma irradiation and biological activity testing prior the product release as cell culture medium additive. Biological activity testing of the different preparations was done by determining the capability of the different PRP preparations to sustain human bone marrow mesenchymal stem cell (MSC) clone formation and proliferation. Taking advantage of a developed MSC in vitro clonogenicity test, we also determined biological activity and stability of the freeze-dried gamma-sterilized PRP preparations after their storage for different times and at different temperatures. The PRP effects on cell proliferation were determined both on primary cell cultures established from different tissues and on a cell line. Results were compared with those obtained in "traditional" parallel control cultures performed in the presence of bovine serum [10% fetal calf serum (FCS)]. Compared to FCS, the PRP addition to the culture medium increased the MSC colony number and average size. In primary cell cultures and in cell line cultures, the PRP promoted cell proliferation also in conditions where the FCS had not a proliferation stimulating effect due to either the nature of the cells and the tissue of origin (such as human articular chondrocytes from elderly patients) or to the critical low density cell seeding (such as for HeLa cells). In summary, the standardized PRP formulation would provide an "off-the-shelf" product to be used for the selection and expansion of several cell types also in critical cell culture conditions.
Assuntos
Plaquetas , Meios de Cultura , Liofilização/métodos , Animais , Bovinos , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Células Cultivadas , Meios de Cultura/efeitos da radiação , Células HeLa , HumanosRESUMO
Pluripotent stem cells are defined as cells that can generate cells of lineages from all three germ layers, ectoderm, mesoderm, and endoderm. On the contrary, unipotent and multipotent stem cells develop into one or more cell types respectively, but their differentiation is limited to the cells present in the tissue of origin or, at most, from the same germ layer. Multipotent and unipotent stem cells have been isolated from a variety of adult tissues, Instead, the presence in adult tissues of pluripotent stem cells is a very debated issue. In the early embryos, all cells are pluripotent. In mammalians, after birth, pluripotent cells are maintained in the bone-marrow and possibly in gonads. In fact, pluripotent cells were isolated from marrow aspirates and cord blood and from cultured bone-marrow stromal cells (MSCs). Only in few cases, pluripotent cells were isolated from other tissues. In addition to have the potential to differentiate toward lineages derived from all three germ layers, the isolated pluripotent cells shared other properties, including the expression of cell surface stage specific embryonic antigen (SSEA) and of transcription factors active in the early embryos, but they were variously described and named. However, it is likely that they are part of the same cell population and that observed diversities were the results of different isolation and expansion strategies. Adult pluripotent stem cells are quiescent and self-renew at very low rate. They are maintained in that state under the influence of the "niche" inside which they are located. Any tissue damage causes the release in the blood of inflammatory cytokines and molecules that activate the stem cells and their mobilization and homing in the injured tissue. The inflammatory response could also determine the dedifferentiation of mature cells and their reversion to a progenitor stage and at the same time stimulate the progenitors to proliferate and differentiate to replace the damaged cells. In this review we rate articles reporting isolation and characterization of tissue resident pluripotent cells. In the attempt to reconcile observations made by different authors, we propose a unifying picture that could represent a starting point for future experiments.
RESUMO
Injuries of the meniscus remain a burden for the development of premature cartilage degeneration and osteoarthritis. This review surveys all treatment options and focuses on the recent development of tissue engineering. Tissue engineering of the meniscus means a successful combination of cells, scaffolds and specific stimuli. Each element of the combination can be subject to variation. Studies investigating the optimum meniscus implant and previous steps in producing these implants are presented in this article. A comprehensive search of the English and German literature was performed in PubMed to retrieve appropriate manuscripts for review. Based on the literatures, autografts and allografts can delay the progress of osteoarthritis for a restricted time period, but several concerns persist. The biomechanical properties of the native meniscus are not copied entirely by the current existing autografts. Congruence, fixation, biocompatibility and potential infection will always remain as limitations for the users of allografts. Long-term results are still not available for meniscus prosthesis and even though it permits fast recovery, several aspects are questionable: bioincompatibility and a lack of cellular adhesion are likely to compromise their long-term fate. Currently, there is no ideal implant generated by means of tissue engineering. However, meniscus tissue engineering is a fast developing field, which promises to develop an implant that mimics histological and biomechanical properties of the native meniscus. At present several cell sources and scaffolds have been used successfully to grow 3-dimensional constructs. In future, optimal implants have to be developed using growth factors, modified scaffolds and stimuli that support cellular proliferation and differentiation to regenerate the native meniscus more closely.
Assuntos
Meniscos Tibiais/cirurgia , Osteoartrite do Joelho/terapia , Lesões do Menisco Tibial , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Osteoartrite do Joelho/etiologia , Osteoartrite do Joelho/prevenção & controle , Plasma Rico em Plaquetas , Próteses e Implantes , Regeneração , Engenharia Tecidual/tendências , Alicerces Teciduais , Transplante Autólogo , Transplante Homólogo , TransplantesRESUMO
Maintenance of tissue homeostasis and tissue regeneration after an insult are essential functions of adult stem cells (SCs). In adult tissues, SCs proliferate at a very slow rate within "stem cell niches", but, during tissue development and regeneration, before giving rise to differentiated cells, they give rise to multipotent and highly proliferative cells, known as transit-amplifying cells (TACs). Although differences exist in diverse tissues, TACs are not only a transitory phase from SCs to post-mitotic cells, but they also actively control proliferation and number of their ancestor SCs and proliferation and differentiation of their progeny toward tissue specific functional cells. Autocrine signals and negative and positive feedback and feedforward paracrine signals play a major role in these controls. In the present review we will consider the generation and the role played by TACs during development and regeneration of lining epithelia characterized by a high turnover including epidermis and hair follicles, ocular epithelial surfaces, and intestinal mucosa. A comparison between these different tissues will be made. There are some genes and molecular pathways whose expression and activation are common to most TACs regardless their tissue of origin. These include, among others, Wnt, Notch, Hedgehog and BMP pathways. However, the response to these molecular signals can vary in TACs of different tissues. Secondly, we will consider cultured cells derived from tissues of mesodermal origin and widely adopted for cell therapy treatments. These include mesenchymal stem cells and dedifferentiated chondrocytes. The possible correlation between cell dedifferentiation and reversion to a transit amplifying cell stage will be discussed.
RESUMO
BACKGROUND: The current challenge for immunotherapies is to generate effective antitumor immunity. Since tumor immune escape mechanisms do not impact pre-existing and consolidated immune responses, we tested the hypothesis of redirecting a pregenerated immunity to cancer: to recall a non-tumor antigen response against the tumor, silk fibroin nanoparticles (SFNs) have been selected as 'Trojan-horse' carriers, promoting the antigen uptake by the tumor cells. METHODS: SFNs have been loaded with either ovalbumin (OVA) or CpG oligonucleotide (CpG) as antigen or adjuvant, respectively. In vitro uptake of SFNs by tumor (B16/F10 melanoma and MB49 bladder cancer) or dendritic cells, as well as the presence of OVA-specific T cells in splenic and tumor-infiltrating lymphocytes, were assessed by cytometric analyses. Proof-of-concept of in vivo efficacy was achieved in an OVA-hyperimmune B16/F10 murine melanoma model: SFNs-OVA or SFNs-CpG were injected, separately or in association, into the subcutaneous peritumoral area. Cancer dimensions/survival time were monitored, while, at the molecular level, system biology approaches based on graph theory and experimental proteomic data were performed. RESULTS: SFNs were efficiently in vitro uptaken by cancer and dendritic cells. In vivo peritumor administration of SFNs-OVA redirected OVA-specific cytotoxic T cells intratumorally. Proteomics and systems biology showed that peritumoral treatment with either SFNs-OVA or SFNs-CpG dramatically modified tumor microenvironment with respect to the control (CTR), mainly involving functional modules and hubs related to angiogenesis, inflammatory mediators, immune function, T complex and serpins expression, redox homeostasis, and energetic metabolism. Both SFNs-OVA and SFNs-CpG significantly delayed melanoma growth/survival time, and their effect was additive. CONCLUSIONS: Both SFNs-OVA and SFNs-CpG induce effective anticancer response through complementary mechanisms and show the efficacy of an innovative active immunotherapy approach based on the redirection of pre-existing immunity against cancer cells. This approach could be universally applied for solid cancer treatments if translated into the clinic using re-call antigens of childhood vaccination.
Assuntos
Fibroínas , Melanoma Experimental , Camundongos , Animais , Proteômica , Linfócitos T Citotóxicos , Adjuvantes Imunológicos , Ovalbumina , Microambiente TumoralRESUMO
Cutaneous chronic wounds are a major global health burden in continuous growth, because of population aging and the higher incidence of chronic diseases, such as diabetes. Different treatments have been proposed: biological, surgical, and physical. However, most of these treatments are palliative and none of them can be considered fully satisfactory. During a spontaneous wound healing, endogenous regeneration mechanisms and resident cell activity are triggered by the released platelet content. Activated stem and progenitor cells are key factors for ulcer healing, and they can be either recruited to the wound site from the tissue itself (resident cells) or from elsewhere. Transplant of skin substitutes, and of stem cells derived from tissues such as bone marrow or adipose tissue, together with platelet-rich plasma (PRP) treatments have been proposed as therapeutic options, and they represent the today most promising tools to promote ulcer healing in diabetes. Although stem cells can directly participate to skin repair, they primarily contribute to the tissue remodeling by releasing biomolecules and microvesicles able to stimulate the endogenous regeneration mechanisms. Stem cells and PRP can be obtained from patients as autologous preparations. However, in the diabetic condition, poor cell number, reduced cell activity or impaired PRP efficacy may limit their use. Administration of allogeneic preparations from healthy and/or younger donors is regarded with increasing interest to overcome such limitation. This review summarizes the results obtained when these innovative treatments were adopted in preclinical animal models of diabetes and in diabetic patients, with a focus on allogeneic preparations.
RESUMO
Mesenchymal stem cell (MSC) culturing for cell therapies needs a step forward to be routinely used in clinical settings. Main concerns regard the use of animal origin reagents, in particular supplementing the culture medium with FBS. Lately, Human Platelet Lysate (HPL) has been proposed as animal-free alternative, described as an excellent supplement for culturing MSCs. The aim of this systematic review was to analyze the current literature on the effect of HPL and FBS on ASCs and BMSCs. The primary outcome was the proliferation rate of cells cultured with FBS and HPL. Differences in terms of doubling time (DT) and population doubling (PD) were evaluated by meta-analysis, subgrouping data according to the cell type. A total of 35 articles were included. BMSCs and ASCs were used in 65.7% (23) and 28.6% (10) studies, respectively. Only two studies included both cell types. Overall, 22 studies were eligible for the meta-analysis. Among them, 9 articles described ASCs and 13 BMSCs. The results showed that BMSCs and ASCs cultured with 10% HPL and 5% HPL have lower DT and higher PD compared to cells cultured with 10% FBS. A possible correlation between the DT decrease and the application of at least 3 freeze/thaw cycles to induce platelet lysis was found. Additionally, HPL increased VEGF secretion and maintained the immuno-modulatory abilities for both cell types. The clarification reported here of the higher efficiency of HPL compared to FBS can help the transition of the scientific community towards clinical-related procedures. 1. The meta-analysis shows that HPL induces a population doubling increase and a doubling time decrease of both ASCs and BMSCs compared to FBS. 2. When at least 3 freeze/thaw cycles are applied to induce platelet lysis, the doubling time of HPL-cultured cells is lower than FBS-cultured cells (Created with BioRender.com).
Assuntos
Plaquetas , Técnicas de Cultura de Células , Células-Tronco Mesenquimais , Soroalbumina Bovina , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Humanos , Células-Tronco Mesenquimais/citologiaRESUMO
Standard treatments of chronic skin ulcers based on the direct application of dressings still present several limits with regard to a complete tissue regeneration. Innovative strategies in tissue engineering offer materials that can tune cell behavior and promote growth tissue favoring cell recruitment in the early stages of wound healing. A combination of Alginate (Alg), Sericin (SS) with Platelet Lysate (PL), as a freeze-dried sponge, is proposed to generate a bioactive wound dressing to care skin lesions. Biomembranes at different composition were tested for the release of platelet growth factors, cytotoxicity, protective effects against oxidative stress and cell proliferation induction. The highest level of the growth factors release occurred within 48 h, an optimized time to burst a healing process in vivo; the presence of SS differently modulated the release of the factors by interaction with the proteins composing the biomembranes. Any cytotoxicity was registered, whereas a capability to protect cells against oxidative stress and induce proliferation was observed when PL was included in the biomembrane. In a mouse skin lesion model, the biomembranes with PL promoted the healing process, inducing an accelerated and more pronounced burst of inflammation, formation of granulation tissue and new collagen deposition, leading to a more rapid skin regeneration.
RESUMO
Chronic wounds account for 3% of total healthcare expenditure of developed countries; thus, innovative therapies, including Mesenchymal Stem Cells (MSCs) end their exosomes are increasingly considered, even if the activity depends on the whole secretome, made of both soluble proteins and extracellular vesicles. In this work, we prove for the first time the in vivo activity of the whole secretome formulated in a sponge-like alginate wound dressing to obtain the controlled release of bioactive substances. The product has been prepared in a public GMP-compliant facility by a scalable process; based on the murine model, treated wounds healed faster than controls without complications or infections. The treatment induced a higher acute inflammatory process in a short time and sustained the proliferative phase by accelerating fibroblast migration, granulation tissue formation, neovascularization and collagen deposition. The efficacy was substantially supported by the agreement between histological and proteomic findings. In addition to functional modules related to proteolysis, complement and coagulation cascades, protein folding and ECM remodeling, in treated skin, emerged the role of specific wound healing related proteins, including Tenascin (Tnc), Decorin (Dcn) and Epidermal growth factor receptor (EGFR). Of note, Decorin and Tenascin were also components of secretome, and network analysis suggests a potential role in regulating EGFR. Although further experiments will be necessary to characterize better the molecular keys induced by treatment, overall, our results confirm the whole secretome efficacy as novel "cell-free therapy". Also, sponge-like topical dressing containing the whole secretome, GMP- compliant and "ready-off-the-shelf", may represent a relevant point to facilitate its translation into the clinic.
Assuntos
Alginatos/administração & dosagem , Bandagens , Modelos Animais de Doenças , Esponja de Gelatina Absorvível/administração & dosagem , Proteômica/métodos , Cicatrização/efeitos dos fármacos , Alginatos/farmacocinética , Animais , Esponja de Gelatina Absorvível/farmacocinética , Masculino , Camundongos , Cicatrização/fisiologiaRESUMO
X-ray Synchrotron radiation-based techniques, in particular Micro-tomography and Micro-diffraction, were exploited to investigate the structure of bone deposited in vivo within a porous ceramic scaffold. Bone formation was studied by implanting Mesenchymal Stem Cell (MSC) seeded ceramic scaffolds in a mouse model. Osteoblasts derived from the seeded MSC and from differentiation of cells migrated within the scaffold together with the blood vessels, deposited within the scaffold pores an organic collagenous matrix on which a precursor mineral amorphous liquid-phase, containing Ca++ and PO4-- crystallized filling the gaps between the collagen molecules. Histology offered a valid instrument to investigate the engineered tissue structure, but, unfortunately, limited itself to a macroscopic analysis. The evolution of the X-ray Synchrotron radiation-based techniques and the combination of micro X-ray diffraction with X-ray phase-contrast imaging enabled to study the dynamic of the structural and morphological changes occurring during the new bone deposition, biomineralization and vascularization. In fact, the unique features of Synchrotron radiation, is providing the high spatial resolution probe which is necessary for the study of complex materials presenting heterogeneity from micron-scale to meso- and nano-scale. Indeed, this is the occurrence in the heterogeneous and hierarchical bone tissue where an organic matter, such as the collagenous matrix, interacts with mineral nano-crystals to generate a hybrid multiscale biomaterial with unique physical properties. In this framework, the use of advanced synchrotron radiation techniques allowed to understand and to clarify fundamental aspects of the bone formation process within the bioceramic, i.e. biomineralization and vascularization, including to obtain deeper knowledge on bone deposition, mineralization and reabsorption in different health, aging and pathological conditions. In this review we present an overview of the X-ray Synchrotron radiation techniques and we provide a general outlook of their applications on bone Tissue Engineering, with a focus on our group work. STATEMENT OF SIGNIFICANCE: Synchrotron Radiation techniques for Tissue Engineering In this review we report recent applications of X-ray Synchrotron radiation-based techniques, in particular Microtomography and Microdiffraction, to investigations on the structure of ceramic scaffolds and bone tissue regeneration. Tissue engineering has made significant advances in bone regeneration by proposing the use of mesenchymal stem cells in combination with various types of scaffolds. The efficacy of the biomaterials used to date is not considered optimal in terms of resorbability and bone formation, resulting in a poor vascularization at the implant site. The review largely based on our publications in the last ten years could help the study of the regenerative model proposed. We also believe that the new imaging technologies we describe could be a starting point for the development of additional new techniques with the final aim of transferring them to the clinical practice.
Assuntos
Osso e Ossos/metabolismo , Diferenciação Celular , Cerâmica/química , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Síncrotrons , Engenharia Tecidual , Alicerces Teciduais/química , Difração de Raios X , Animais , Calcificação Fisiológica , Camundongos , PorosidadeRESUMO
: Injured blood vessel repair and blood circulation re-establishment are crucial events for tissue repair. We investigated in primary cultures of human umbilical vein endothelial cells (HUVEC), the effects of platelet lysate (PL), a cocktail of factors released by activated platelets following blood vessel disruption and involved in the wound-healing process triggering. PL exerted a protective effect on HUVEC in an inflammatory milieu by inhibiting IL-1α-activated NF-κB pathway and by inducing the secretion of PGE2, a pro-resolving molecule in the wound microenvironment. Moreover, PL enhanced HUVEC proliferation, without affecting their capability of forming tube-like structures on matrigel, and activated resting quiescent cells to re-enter cell cycle. In agreement with these findings, proliferation-related pathways Akt and ERK1/2 were activated. The expression of the cell-cycle activator Cyclin D1 was also enhanced, as well as the expression of the High Mobility Group Box-1 (HMGB1), a protein of the alarmin group involved in tissue homeostasis, repair, and remodeling. These in vitro data suggest a possible in vivo contribution of PL to new vessel formation after a wound by activation of cells resident in vessel walls. Our biochemical study provides a rationale for the clinical use of PL in the treatment of wound healing-related pathologies.
Assuntos
Fatores de Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Plaquetas/citologia , Diferenciação Celular , Células Cultivadas , Ciclina D1/metabolismo , Proteína HMGB1/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
Autologous platelet-rich plasma (PRP) is used clinically to induce repair of different tissues through the release of bioactive molecules. In some patients, the production of efficient autologous PRP is unfeasible due to their compromised health. Allogeneic PRP mismatched for AB0 and Rh antigens was developed. The effect of allogeneic PRP on immune response should be defined to use it in clinical practice avoiding side effects. Thus, whether PRP affects the differentiation of peripheral blood monocytes to dendritic cells upon stimulation with granulocyte monocyte colony stimulating factor and interleukin-4 was investigated. Indeed, these cells are the main players of immune response and tissue repair. PRP inhibited the differentiation of monocytes to CD1a+ dendritic cells and favoured the expansion of phagocytic CD163+ CD206+ fibrocyte-like cells. These cells produced interleukin-10 and prostaglandin-E2 , but not interferon-γ, upon stimulation with lipopolysaccharides. Moreover, they promoted the expansion of regulatory CD4+ CD25+ FoxP3+ T cells upon allostimulation or antigen specific priming. Finally, the conditioned medium harvested from monocytes differentiated with PRP triggered a strong chemotactic effect on mesenchymal cells in both scratch and transwell migration assays. These results strongly suggest that allogeneic PRP can foster the differentiation of monocytes to a regulatory anti-inflammatory population, possibly favouring wound healing. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Microambiente Celular , Células Dendríticas/citologia , Monócitos/citologia , Plasma Rico em Plaquetas/metabolismo , Cicatrização/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Microambiente Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Dinoprostona/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Humanos , Interferon gama/biossíntese , Interleucina-10/biossíntese , Teste de Cultura Mista de Linfócitos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Fagócitos/citologia , Fagócitos/efeitos dos fármacos , Fenótipo , Toxina Tetânica/farmacologia , Transplante Homólogo , Leveduras/efeitos dos fármacos , Leveduras/metabolismoRESUMO
Skin chronic wounds are non-healing ulcerative defects, which arise in association with a morbidity state, such as diabetes and vascular insufficiency or as the consequence of systemic factors including advanced age. Platelet Rich Plasma, a platelet-rich blood fraction, can significantly improve the healing of human skin chronic ulcers. Given that the subcutaneous adipose tissue is located beneath the skin and plays a role in the skin homeostasis, in this study, we investigated the in vitro response of human subcutaneous adipose tissue cells to platelet content in a model mimicking in vitro the in situ milieu of a deep skin injury. Considering that, at the wound site, plasma turn to serum, platelets are activated and inflammation occurs, human adipose-derived stromal cells (hASC) were cultured with Human Serum (HS) supplemented or not with Platelet Lysate (PL) and/or IL-1α. We observed that HS sustained hASC proliferation more efficiently than FBS and induced a spontaneous adipogenic differentiation in the cells. PL added to HS enhanced hASC proliferation, regardless the presence of IL-1α. In the presence of PL, hASC progressively lessened the adipogenic phenotype, possibly because the proliferation of less committed cells was induced. However, these cells resumed adipogenesis in permissive conditions. Accordingly, PL induced in quiescent cells activation of the proliferation-related pathways ERK, Akt, and STAT-3 and expression of Cyclin D1. Moreover, PL induced an early and transient increase of the pro-inflammatory response triggered by IL-1α, by inducing COX-2 expression and secretion of a large amount of PGE2, IL-6, and IL-8. Media conditioned by PL-stimulated hASC exerted a chemotactic activity on human keratinocytes and favored the healing of an in vitro scratch wound. In order to bridge the gap between in vitro results and possible in vivo events, the stimuli were also tested in ex vivo cultures of in toto human adipose tissue biopsies (hAT). PL induced cell proliferation in hAT and outgrowth of committed progenitor cells able to differentiate in permissive conditions. In conclusion, we report that the adipose tissue responds to the wound microenvironment by activating the proliferation of adipose tissue progenitor cells and promoting the release of factors favoring wound healing.
RESUMO
Human platelet lysate (hPL) is a pool of growth factors and cytokines able to induce regeneration of different tissues. Despite its good potentiality as therapeutic tool for regenerative medicine applications, hPL has been only moderately exploited in this field. A more widespread adoption has been limited because of its rapid degradation at room temperature that decreases its functionality. Another limiting factor for its extensive use is the difficulty of handling the hPL gels. In this work, silk fibroin-based patches were developed to address several points: improving the handling of hPL, enabling their delivery in a controlled manner and facilitating their storage by creating a device ready to use with expanded shelf life. Patches of fibroin loaded with hPL were synthesized by electrospinning to take advantage of the fibrous morphology. The release kinetics of the material was characterized and tuned through the control of fibroin crystallinity. Cell viability assays, performed with primary human dermal fibroblasts, demonstrated that fibroin is able to preserve the hPL biological activity and prolong its shelf-life. The strategy of storing and preserving small active molecules within a naturally-derived, protein-based fibrous scaffold was successfully implemented, leading to the design of a biocompatible device, which can potentially simplify the storage and the application of the hPL on a human patient, undergoing medical procedures such as surgery and wound care. STATEMENT OF SIGNIFICANCE: Human platelets lysate (hPL) is a mixture of growth factors and cytokines able to induce the regeneration of damaged tissues. This study aims at enclosing hPL in a silk fibroin electrospun matrix to expand its utilization. Silk fibroin showed the ability to preserve the hPL activity at temperature up to 60⯰C and the manipulation of fibroin's crystallinity provided a tool to modulate the hPL release kinetic. This entails the possibility to fabricate the hPL silk fibroin patches in advance and store them, resulting in an easy and fast accessibility and an expanded use of hPL for wound healing.