RESUMEN
Titanium plates are the current gold standard for fracture fixation of the mandible. Magnesium alloys such as WE43 are suitable biodegradable alternatives due to their high biocompatibility and elasticity modulus close to those of cortical bone. By surface modification, the reagibility of magnesium and thus hydrogen gas accumulation per time are further reduced, bringing plate fixation with magnesium closer to clinical application. This study aimed to compare bone healing in a monocortical mandibular fracture model in sheep with a human-standard size, magnesium-based, plasma electrolytic-oxidation (PEO) surface modified miniplate fixation system following 4 and 12 weeks. Bone healing was analyzed using micro-computed tomography and histological analysis with Movat's pentachrome and Giemsa staining. For evaluation of the tissue's osteogenic activity, polychrome fluorescent labeling was performed, and vascularization was analyzed using immunohistochemical staining for alpha-smooth muscle actin. Bone density and bone mineralization did not differ significantly between titanium and magnesium (BV/TV: T1: 8.74 ± 2.30%, M1: 6.83 ± 2.89%, p = 0.589 and T2: 71.99 ± 3.13%, M2: 68.58 ± 3.74%, p = 0.394; MinB: T1: 26.16 ± 9.21%, M1: 22.15 ± 7.99%, p = 0.818 and T2: 77.56 ± 3.61%, M2: 79.06 ± 4.46%, p = 0.699). After 12 weeks, minor differences were observed regarding bone microstructure, osteogenic activity, and vascularization. There was significance with regard to bone microstructure (TrTh: T2: 0.08 ± 0.01 mm, M2: 0.06 ± 0.01 mm; p = 0.041). Nevertheless, these differences did not interfere with bone healing. In this study, adequate bone healing was observed in both groups. Only after 12 weeks were some differences detected with larger trabecular spacing and more vessel density in magnesium vs titanium plates. However, a longer observational time with full resorption of the implants should be targeted in future investigations.
Asunto(s)
Placas Óseas , Magnesio , Mandíbula , Titanio , Animales , Magnesio/farmacología , Titanio/química , Titanio/farmacología , Ovinos , Mandíbula/cirugía , Mandíbula/diagnóstico por imagen , Curación de Fractura/efectos de los fármacos , Propiedades de Superficie , Osteogénesis/efectos de los fármacos , Fracturas Mandibulares/cirugía , Fracturas Mandibulares/diagnóstico por imagen , Microtomografía por Rayos X , Aleaciones/químicaRESUMEN
Magnesium as a biodegradable material offers promising results in recent studies of different maxillo-facial fracture models. To overcome adverse effects caused by the fast corrosion of pure magnesium in fluid surroundings, various alloys, and surface modifications are tested in animal models. In specified cases, magnesium screws already appeared for clinical use in maxillofacial surgery. The present study aims to compare the bone healing outcome in a non-load-bearing fracture scenario of the forehead in sheep when fixed with standard-sized WE43 magnesium fixation plates and screws with plasma electrolytic oxidation (PEO) surface modification in contrast to titanium osteosynthesis. Surgery was performed on 24 merino mix sheep. The plates and screws were explanted en-bloc with the surrounding tissue after four and twelve weeks. The outcome of bone healing was investigated with micro-computed tomography, histological, immunohistological, and fluorescence analysis. There was no significant difference between groups concerning the bone volume, bone volume/ total volume, and newly formed bone in volumetric and histological analysis at both times of investigation. The fluorescence analysis revealed a significantly lower signal in the magnesium group after one week, although there was no difference in the number of osteoclasts per mm2. The magnesium group had significantly fewer vessels per mm2 in the healing tissue. In conclusion, the non-inferiority of WE43-based magnesium implants with PEO surface modification was verified concerning fracture healing under non-load-bearing conditions in a defect model. STATEMENT OF SIGNIFICANCE: Titanium implants, the current gold standard of fracture fixation, can lead to adverse effects linked to the implant material and often require surgical removal. Therefore, degradable metals like the magnesium alloy WE43 with plasma electrolytic oxidation (PEO) surface modification gained interest. Yet, miniplates of this alloy with PEO surface modification have not been examined in a fracture defect model of the facial skeleton in a large animal model. This study shows, for the first time, the non-inferiority of magnesium miniplates compared to titanium miniplates. In radiological and histological analysis, bone healing was undisturbed. Magnesium miniplates can reduce the number of interventions for implant removal, thus reducing the risk for the patient and minimizing the costs.
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The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged organs, including limbs, brain, heart, tail, and others. Studies on axolotl shed light on cellular and molecular pathways ruling progenitor activation and tissue restoration after injury. This knowledge can be applied to facilitate the healing of regeneration-incompetent injuries, such as bone non-union. In the current protocol, the femur osteotomy stabilization using an internal plate fixation system is described. The procedure was adapted for use in aquatic animals (axolotl, Ambystoma mexicanum). ≥20 cm snout-to-tail tip axolotls with fully ossified, mouse-size comparable femurs were used, and special attention was paid to the plate positioning and fixation, as well as to the postoperative care. This surgical technique allows for standardized and stabilized bone fixation and could be useful for direct comparison to axolotl limb regeneration and analogous studies of bone healing across amphibians and mammals.
Asunto(s)
Ambystoma mexicanum , Placas Óseas , Fémur , Osteotomía , Animales , Ambystoma mexicanum/cirugía , Osteotomía/métodos , Fémur/cirugíaRESUMEN
Breast cancer often metastasizes to bone, causing osteolytic lesions. Structural and biophysical changes are rarely studied yet are hypothesized to influence metastasis. We developed a mouse model of early bone metastasis and multimodal imaging to quantify cancer cell homing, bone (re)modeling, and onset of metastasis. Using tissue clearing and three-dimensional (3D) light sheet fluorescence microscopy, we located enhanced green fluorescent protein-positive cancer cells and small clusters in intact bones and quantified their size and spatial distribution. We detected early bone lesions using in vivo microcomputed tomography (microCT)-based time-lapse morphometry and revealed altered bone (re)modeling in the absence of detectable lesions. With a new microCT image analysis tool, we tracked the growth of early lesions over time. We showed that cancer cells home in all bone compartments, while osteolytic lesions are only detected in the metaphysis, a region of high (re)modeling. Our study suggests that higher rates of (re)modeling act as a driver of lesion formation during early metastasis.
Asunto(s)
Neoplasias Óseas , Osteólisis , Animales , Ratones , Microtomografía por Rayos X/métodos , Neoplasias Óseas/complicaciones , Neoplasias Óseas/secundario , Huesos/diagnóstico por imagen , Osteólisis/diagnóstico por imagen , Osteólisis/etiología , Osteólisis/patología , Modelos Animales de Enfermedad , Línea Celular TumoralRESUMEN
Bone fracture healing is regulated by mechanobiological cues. Both, extracellular matrix (ECM) deposition and microvascular assembly determine the dynamics of the regenerative processes. Mechanical instability as by inter-fragmentary shear or compression is known to influence early ECM formation and wound healing. However, it remains unclear how these external cues shape subsequent ECM and microvascular network assembly. As transcriptional coactivators, the mechanotransducers yes-associated protein 1 (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) translate physical cues into downstream signaling events, yet their role in sprouting angiogenesis into the hematoma after injury is unknown. Using bone healing as model system for scar-free regeneration, the role of endothelial YAP/TAZ in combination with tuning the extrinsic mechanical stability via fracture fixation is investigated. Extrinsically imposed shear across the gap delayed hematoma remodeling and shaped the morphology of early collagen fiber orientations and microvascular networks, suggesting that enhanced shear increased the nutrient exchange in the hematoma. In contrast, endothelial YAP/TAZ deletion has little impact on the overall vascularization of the fracture gap, yet slightly increases the collagen fiber deposition under semi-rigid fixation. Together, these data provide novel insights into the respective roles of endothelial YAP/TAZ and extrinsic mechanical cues in orchestrating the process of bone regeneration.
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Hematoma , Mecanotransducción Celular , Colágeno/metabolismo , Mecanotransducción Celular/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAP/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Curación de Fractura/fisiología , Humanos , Hematoma/metabolismo , Hematoma/patología , Huesos/metabolismo , Huesos/patologíaRESUMEN
Multipotent stromal cells are considered attractive sources for cell therapy and tissue engineering. Despite numerous experimental and clinical studies, broad application of stromal cell therapeutics is not yet emerging. A major challenge is the functional diversity of available cell sources. Here, we investigated the regenerative potential of clinically relevant human stromal cells from bone marrow (BMSCs), white adipose tissue, and umbilical cord compared with mature chondrocytes and skin fibroblasts in vitro and in vivo. Although all stromal cell types could express transcription factors related to endochondral ossification, only BMSCs formed cartilage discs in vitro that fully regenerated critical-size femoral defects after transplantation into mice. We identified cell type-specific epigenetic landscapes as the underlying molecular mechanism controlling transcriptional stromal differentiation networks. Binding sites of commonly expressed transcription factors in the enhancer and promoter regions of ossification-related genes, including Runt and bZIP families, were accessible only in BMSCs but not in extraskeletal stromal cells. This suggests an epigenetically predetermined differentiation potential depending on cell origin that allows common transcription factors to trigger distinct organ-specific transcriptional programs, facilitating forward selection of regeneration-competent cell sources. Last, we demonstrate that viable human BMSCs initiated defect healing through the secretion of osteopontin and contributed to transient mineralized bone hard callus formation after transplantation into immunodeficient mice, which was eventually replaced by murine recipient bone during final tissue remodeling.
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Cartílago , Células del Estroma , Humanos , Ratones , Animales , Células del Estroma/metabolismo , Cartílago/metabolismo , Condrocitos , Osteogénesis , Ingeniería de Tejidos , Diferenciación Celular , Factores de Transcripción/metabolismo , Células de la Médula Ósea , Regeneración ÓseaRESUMEN
Bone regeneration after fracture is a complex process with high and dynamic energy demands. The impact of metabolism on bone healing progression and outcome, however, is so far understudied. Our comprehensive molecular profiling reveals that central metabolic pathways, such as glycolysis and the citric acid cycle, are differentially activated between rats with successful or compromised bone regeneration (young versus aged female Sprague-Dawley rats) early in the inflammatory phase of bone healing. We also found that the citric acid cycle intermediate succinate mediates individual cellular responses and plays a central role in successful bone healing. Succinate induces IL-1ß in macrophages, enhances vessel formation, increases mesenchymal stromal cell migration, and potentiates osteogenic differentiation and matrix formation in vitro. Taken together, metabolites-here particularly succinate-are shown to play central roles as signaling molecules during the onset of healing and in steering bone tissue regeneration.
Asunto(s)
Regeneración Ósea , Osteogénesis , Ratas , Femenino , Animales , Osteogénesis/genética , Ratas Sprague-Dawley , Regeneración Ósea/genética , Huesos , SuccinatosRESUMEN
The bone structure is very dynamic and continuously adapts its geometry to external stimuli by modeling and remodeling the mineralized tissue. In vivo microCT-based time-lapse morphometry is a powerful tool to study the temporal and spatial dynamics of bone (re)modeling. Here an advancement in the methodology to detect and quantify site-specific differences in bone (re)modeling of 12-week-old BALB/c nude mice is presented. We describe our method of quantifying new bone surface interface readouts and how these are influenced by bone curvature. This method is then used to compare bone surface (re)modeling in mice across different anatomical regions to demonstrate variations in the rate of change and spatial gradients thereof. Significant differences in bone (re)modeling baseline parameters between the metaphyseal and epiphyseal, as well as cortical and trabecular bone of the distal femur and proximal tibia are shown. These results are validated using conventional static in vivo microCT analysis. Finally, the insights from these new baseline values of physiological bone (re)modeling were used to evaluate pathological bone (re)modeling in a pilot breast cancer bone metastasis model. The method shows the potential to be suitable to detect early pathological events and track their spatio-temporal development in both cortical and trabecular bone. This advancement in (re)modeling surface analysis and defined baseline parameters according to distinct anatomical regions will be valuable to others investigating various disease models with site-distinct local alterations in bone (re)modeling.
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Huesos , Tibia , Animales , Densidad Ósea/fisiología , Huesos/diagnóstico por imagen , Ratones , Ratones Desnudos , Tibia/diagnóstico por imagen , Tibia/fisiología , Imagen de Lapso de Tiempo , Microtomografía por Rayos X/métodosRESUMEN
Bone morphogenetic protein-2 (BMP-2) is a known key mediator of physiological bone regeneration and is clinically approved for selected musculoskeletal interventions. Yet, broad usage of this growth factor is impeded due to side effects that are majorly evoked by high dosages and burst release kinetics. In this study, mesoporous bioactive glass microspheres (MBGs), produced by an aerosol-assisted spray-drying scalable process, were loaded with BMP-2 resulting in prolonged, low-dose BMP-2 release without affecting the material characteristics. In vitro, MBGs were found to be cytocompatible and to induce a pro-osteogenic response in primary human mesenchymal stromal cells (MSCs). In a pre-clinical rodent model, BMP-2 loaded MBGs significantly enhanced bone formation and influenced the microarchitecture of newly formed bone. The MBG carriers alone performed equal to the untreated (empty) control in most parameters tested, while additionally exerting mild pro-angiogenic effects. Using MBGs as a biocompatible, pro-regenerative carrier for local and sustained low dose BMP-2 release could limit side effects, thus enabling a safer usage of BMP-2 as a potent pro-osteogenic growth factor.
RESUMEN
Hydrogels with patterned biophysical and biochemical properties have found increasing attention in the biomaterials community. In this work, we explore alginate-based materials with two orthogonal crosslinking mechanisms: the spontaneous Diels-Alder reaction and the ultraviolet light-initiated thiol-ene reaction. Combining these mechanisms in one material and spatially restricting the location of the latter using photomasks, enables the formation of dual-crosslinked hydrogels with patterns in stiffness, biomolecule presentation and degradation, granting local control over cell behavior. Patterns in stiffness are characterized morphologically by confocal microscopy and mechanically by uniaxial compression and microindentation measurement. Mouse embryonic fibroblasts seeded on stiffness-patterned substrates attach preferably and attain a spread morphology on stiff compared to soft regions. Human mesenchymal stem cells demonstrate preferential adipogenic differentiation on soft surfaces and osteogenic differentiation on stiff surfaces. Patterns in biomolecule presentation reveal favored attachment of mouse pre-osteoblasts on stripe regions, where thiolated cell-adhesive biomolecules have been coupled. Patterns in degradation are visualized by microindentation measurement following collagenase exposure. Patterned tissue infiltration into degradable regions on the surface is discernible in n=5/12 samples, when these materials are implanted subcutaneously into the backs of mice. Taken together, these results demonstrate that our hydrogel system with patterns in biophysical and biochemical properties enables the study of how environmental cues affect multiple cell behaviors in vitro and could be applied to guide endogenous tissue growth in diverse healing scenarios in vivo. STATEMENT OF SIGNIFICANCE: Hydrogels with patterns in biophysical and biochemical properties have been explored in the biomaterials community in order to spatially control or guide cell behavior. In our alginate-based system, we demonstrate the effect of local substrate stiffness and biomolecule presentation on the in vitro cell attachment, morphology, migration and differentiation behavior of two different mouse cell lines and human primary cells. Additionally, the effect of degradation patterns on the in vivo tissue infiltration is analyzed following subcutaneous implantation into a mouse model. The achievement of patterned tissue infiltration following the hydrogel template represents an important step towards guiding endogenous healing responses, thus inviting application in various tissue engineering contexts.
Asunto(s)
Alginatos , Osteogénesis , Animales , Fibroblastos , Hidrogeles , Ratones , Ingeniería de TejidosRESUMEN
Local pH is stated to acidify after bone fracture. However, the time course and degree of acidification remain unknown. Whether the acidification pattern within a fracture hematoma is applicable to adjacent muscle hematoma or is exclusive to this regenerative tissue has not been studied to date. Thus, in this study, we aimed to unravel the extent and pattern of acidification in vivo during the early phase post musculoskeletal injury. Local pH changes after fracture and muscle trauma were measured simultaneously in two pre-clinical animal models (sheep/rats) immediately after and up to 48 h post injury. The rat fracture hematoma was further analyzed histologically and metabolomically. In vivo pH measurements in bone and muscle hematoma revealed a local acidification in both animal models, yielding mean pH values in rats of 6.69 and 6.89, with pronounced intra- and inter-individual differences. The metabolomic analysis of the hematomas indicated a link between reduction in tricarboxylic acid cycle activity and pH, thus, metabolic activity within the injured tissues could be causative for the different pH values. The significant acidification within the early musculoskeletal hematoma could enable the employment of the pH for novel, sought-after treatments that allow for spatially and temporally controlled drug release.
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Fracturas Óseas/metabolismo , Metabolómica/métodos , Músculo Esquelético/lesiones , Animales , Ciclo del Ácido Cítrico , Femenino , Fracturas Óseas/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Concentración de Iones de Hidrógeno , Músculo Esquelético/química , Ratas , OvinosRESUMEN
Biomaterials with tunable biophysical properties hold great potential for tissue engineering. The adaptive immune system plays an important role in bone regeneration. Our goal is to investigate the regeneration potential of cell-laden alginate hydrogels depending on the immune status of the animal model. Specifically, the regeneration potential of rat mesenchymal stromal cell (MSC)-laden, void-forming alginate hydrogels, with a stiffness optimized for osteogenic differentiation, is studied in 5-mm critical-sized femoral defects, in both T cell-deficient athymic Rowett Nude (RNU) rats and immunocompetent Sprague Dawley rats. Bone volume fraction, bone mineral density, and tissue mineral density are higher for athymic RNU nude rats 6 weeks postsurgery. In addition, these animals show a significantly higher number of total cells and cells with non-lymphocyte morphology at the defect site, while the number of cells with lymphocyte-like morphology is lower. Hydrogel degradation is slower and the remaining alginate fragments are surrounded by a thicker fibrous capsule. Ossification islands originating from alginate residues suggest that encapsulated MSCs differentiate into the osteogenic lineage and initiate the mineralization process. However, this effect is insufficient to fully bridge the bone defect in both animal models. Alginate hydrogels can be used to deliver MSCs and thereby recruit endogenous cells through paracrine signaling, but additional osteogenic stimuli are needed to regenerate critical-sized segmental femoral defects.
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Alginatos , Regeneración Ósea , Hidrogeles , Inmunocompetencia , Inmunidad Adaptativa , Animales , Densidad Ósea , Diferenciación Celular , Modelos Animales de Enfermedad , Fémur , Ácidos Hexurónicos , Hidrogeles/farmacología , Células Madre Mesenquimatosas , Osteogénesis , Ratas , Ratas Sprague-DawleyRESUMEN
Fracture repair is initiated by a multitude of immune cells and induction of an inflammatory cascade. Alterations in the early healing response due to an aged adaptive immune system leads to impaired bone repair, delayed healing or even formation of non-union. However, immuno-senescence is not limited to the adaptive immunity, but is also described for macrophages, main effector cells from the innate immune system. Beside regulation of pro- and anti-inflammatory signaling, macrophages contribute to angiogenesis and granulation tissue maturation. Thus, it seems likely that an altered macrophage function due to aging may affect bone repair at various stages and contribute to age related deficiencies in bone regeneration. To prove this hypothesis, we analyzed the expression of macrophage markers and angiogenic factors in the early bone hematoma derived from young and aged osteotomized Spraque Dawley rats. We detected an overall reduced expression of the monocyte/pan-macrophage markers CD14 and CD68 in aged rats. Furthermore, the analysis revealed an impaired expression of anti-inflammatory M2 macrophage markers in hematoma from aged animals that was connected to a diminished revascularization of the bone callus. To verify that the age related disturbed bone regeneration was due to a compromised macrophage function, CD14+ macrophage precursors were transplanted locally into the osteotomy gap of aged rats. Transplantation rescued bone regeneration partially after 6 weeks, demonstrated by a significantly induced deposition of new bone tissue, reduced fibrosis and significantly improved callus vascularization.
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Envejecimiento/inmunología , Regeneración Ósea/inmunología , Fracturas Óseas/inmunología , Macrófagos/inmunología , Cicatrización de Heridas/inmunología , Factores de Edad , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/inmunología , Antígenos de Diferenciación Mielomonocítica/metabolismo , Biomarcadores/metabolismo , Regeneración Ósea/genética , Regeneración Ósea/fisiología , Huesos/irrigación sanguínea , Huesos/inmunología , Huesos/lesiones , Femenino , Expresión Génica/genética , Expresión Génica/inmunología , Receptores de Lipopolisacáridos/genética , Receptores de Lipopolisacáridos/inmunología , Receptores de Lipopolisacáridos/metabolismo , Macrófagos/clasificación , Macrófagos/metabolismo , Osteotomía , Ratas Sprague-Dawley , Cicatrización de Heridas/genéticaRESUMEN
Enzymatically-degradable materials recapitulate the dynamic and reciprocal interactions between cells and their native microenvironment by allowing cells to actively shape the degradation process. In order to engineer a synthetic 3D environment enabling cells to orchestrate the degradation of the surrounding material, norbornene-modified alginate was crosslinked with two different peptide crosslinkers susceptible to cleavage by matrix metalloproteinases using UV-initiated thiol-ene chemistry. Resulting hydrogels were characterized for their initial mechanical and rheological properties, and their degradation behavior was measured by tracking changes in wet weight upon enzyme incubation. This process was found to be a function of the crosslinker type and enzyme concentration, indicating that degradation kinetics could be controlled and tuned. When mouse embryonic fibroblasts were encapsulated in 3D, cell number remained constant and viability was high in all materials, while cell spreading and extensive filopodia formation was observed only in the degradable gels, not in non-degradable controls. After implanting hydrogels into the backs of C57/Bl6 mice for 8 weeks, histological stainings of recovered gel remnants and surrounding tissue revealed higher tissue and cell infiltration into degradable materials compared to non-degradable controls. This alginate-based material platform with cell-empowered enzymatic degradation could prove useful in diverse tissue engineering contexts, such as regeneration and drug delivery.
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Alginatos/farmacología , Movimiento Celular , Hidrogeles/farmacología , Metaloproteinasas de la Matriz/metabolismo , Especificidad de Órganos , Animales , Recuento de Células , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Reactivos de Enlaces Cruzados/química , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Ratones Endogámicos C57BL , Especificidad de Órganos/efectos de los fármacos , Péptidos/química , ReologíaRESUMEN
Bone formation as well as bone healing capacity is known to be impaired in the elderly. Although bone formation is outpaced by bone resorption in aged individuals, we hereby present a novel path that considerably impacts bone formation and architecture: Bone formation is substantially reduced in aged individual owing to the experience of the adaptive immunity. Thus, immune-aging in addition to chronological aging is a potential risk factor, with an experienced immune system being recognized as more pro-inflammatory. The role of the aging immune system on bone homeostasis and on the bone healing cascade has so far not been considered. Within this study mice at different age and immunological experience were analyzed toward bone properties. Healing was assessed by introducing an osteotomy, immune cells were adoptively transferred to disclose the difference in biological vs. chronological aging. In vitro studies were employed to test the interaction of immune cell products (cytokines) on cells of the musculoskeletal system. In metaphyseal bone, immune-aging affects bone homeostasis by impacting bone formation capacity and thereby influencing mass and microstructure of bone trabeculae leading to an overall reduced mechanical competence as found in bone torsional testing. Furthermore, bone formation is also impacted during bone regeneration in terms of a diminished healing capacity observed in young animals who have an experienced human immune system. We show the impact of an experienced immune system compared to a naïve immune system, demonstrating the substantial differences in the healing capacity and bone homeostasis due to the immune composition. We further showed that in vivo mechanical stimulation changed the immune system phenotype in young mice toward a more naïve composition. While this rescue was found to be significant in young individuals, aged mice only showed a trend toward the reconstitution of a more naïve immune phenotype. Considering the immune system's experience level in an individual, will likely allow one to differentiate (stratify) and treat (immune-modulate) patients more effectively. This work illustrates the relevance of including immune diagnostics when discussing immunomodulatory therapeutic strategies for the progressively aging population of the industrial countries.
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Inmunidad Adaptativa , Regeneración Ósea , Remodelación Ósea/inmunología , Huesos/inmunología , Huesos/metabolismo , Homeostasis , Osteogénesis , Animales , Biomarcadores , Huesos/diagnóstico por imagen , Huesos/patología , Diferenciación Celular , Citocinas/metabolismo , Femenino , Humanos , Fenómenos Mecánicos , Ratones , Transducción de Señal , Cicatrización de Heridas , Microtomografía por Rayos X/métodosRESUMEN
Degradable biomaterials aim to recapitulate the dynamic microenvironment that cells are naturally exposed to. By oxidizing the alginate polymer backbone, thereby rendering it susceptible to hydrolysis, and crosslinking it via norbornene-tetrazine click chemistry, we can control rheological, mechanical, and degradation properties of resulting hydrogels. Chemical modifications were confirmed by nuclear magnetic resonance (NMR) and the resulting mechanical properties measured by rheology and unconfined compression testing, demonstrating that these are both a function of norbornene coupling and oxidation state. The degradation behavior was verified by tracking mechanical and swelling behavior over time, showing that degradation could be decoupled from initial mechanical properties. The cell compatibility was assessed in 2D and 3D using a mouse pre-osteoblast cell line and testing morphology, proliferation, and viability. Cells attached, spread and proliferated in 2D and retained a round morphology and stable number in 3D, while maintaining high viability in both contexts over 7 days. Finally, oxidized and unoxidized control materials were implanted subcutaneously into the backs of C57/Bl6 mice, and recovered after 8 weeks. Histological staining revealed morphological differences and fibrous tissue infiltration only in oxidized materials. These materials with tunable and decoupled mechanical and degradation behavior could be useful in many tissue engineering applications.
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Alginatos/química , Química Clic/métodos , Hidrogeles/química , Ingeniería de Tejidos/métodos , Animales , Línea Celular , Espectroscopía de Resonancia Magnética , Ratones , Estructura MolecularRESUMEN
In-situ tissue regeneration aims to utilize the body's endogenous healing capacity through the recruitment of host stem or progenitor cells to an injury site. Stromal cell-derived factor-1α (SDF-1α) is widely discussed as a potent chemoattractant. Here we use a cell-free biomaterial-based approach to (i) deliver SDF-1α for the recruitment of endogenous bone marrow-derived stromal cells (BMSC) into a critical-sized segmental femoral defect in rats and to (ii) induce hydrogel stiffness-mediated osteogenic differentiation in-vivo. Ionically crosslinked alginate hydrogels with a stiffness optimized for osteogenic differentiation were used. Fast-degrading porogens were incorporated to impart a macroporous architecture that facilitates host cell invasion. Endogenous cell recruitment to the defect site was successfully triggered through the controlled release of SDF-1α. A trend for increased bone volume fraction (BV/TV) and a significantly higher bone mineral density (BMD) were observed for gels loaded with SDF-1α, compared to empty gels at two weeks. A trend was also observed, albeit not statistically significant, towards matrix stiffness influencing BV/TV and BMD at two weeks. However, over a six week time-frame, these effects were insufficient for bone bridging of a segmental femoral defect. While mechanical cues combined with ex-vivo cell encapsulation have been shown to have an effect in the regeneration of less demanding in-vivo models, such as cranial defects of nude rats, they are not sufficient for a SDF-1α mediated in-situ regeneration approach in segmental femoral defects of immunocompetent rats, suggesting that additional osteogenic cues may also be required. STATEMENT OF SIGNIFICANCE: Stromal cell-derived factor-1α (SDF-1α) is a chemoattractant used to recruit host cells for tissue regeneration. The concept that matrix stiffness can direct mesenchymal stromal cell (MSC) differentiation into various lineages was described a decade ago using in-vitro experiments. Recently, alginate hydrogels with an optimized stiffness and ex-vivo encapsulated MSCs were shown to have an effect in the regeneration of skull defects of nude rats. Here, we apply this material system, loaded with SDF-1α and without encapsulated MSCs, to (i) recruit endogenous cells and (ii) induce stiffness-mediated osteogenic differentiation in-vivo, using as model system a load-bearing femoral defect in immunocompetent rats. While a cell-free approach is of great interest from a translational perspective, the current limitations are described.
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Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Quimiocina CXCL12 , Fémur , Hidrogeles , Osteogénesis/efectos de los fármacos , Animales , Densidad Ósea/efectos de los fármacos , Células de la Médula Ósea/patología , Quimiocina CXCL12/química , Quimiocina CXCL12/farmacocinética , Quimiocina CXCL12/farmacología , Implantes de Medicamentos/química , Implantes de Medicamentos/farmacocinética , Implantes de Medicamentos/farmacología , Femenino , Fémur/lesiones , Fémur/metabolismo , Fémur/patología , Hidrogeles/química , Hidrogeles/farmacocinética , Hidrogeles/farmacología , Ratas , Ratas Sprague-Dawley , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
Controlled revascularization and inflammation are key elements regulating endogenous regeneration after (bone) tissue trauma. Peripheral blood-derived cell subsets, such as regulatory T-helper cells and circulating (endothelial) progenitor cells, respectively, can support endogenous tissue healing, whereas effector T cells that are associated with an aged immune system can hinder bone regeneration. CD31 is expressed by diverse leukocytes and is well recognized as a marker of circulating endothelial (precursor) cells; however, CD31 is absent from the surface of differentiated effector T cells. Thus, we hypothesized that by separating the inhibitory fractions from the supportive fractions of circulating cells within the peripheral blood (PB) using the CD31 marker, bone regeneration in biologically compromised conditions, such as those observed in aged patients, could be improved. In support of our hypothesis, we detected an inverse correlation between CD31+ cells and effector T cells in the hematomas of human fracture patients, dependent on the age of the patient. Furthermore, we demonstrated the regenerative capacity of human PB-CD31+ cells in vitro. These findings were translated to a clinically relevant rat model of impaired bone healing. The transplantation of rat PB-CD31+ cells advanced bone tissue restoration in vivo and was associated with an early anti-inflammatory response, the stimulation of (re)vascularization, and reduced fibrosis. Interestingly, the depletion or enrichment of the highly abundant CD31+/14+ monocytes from the mixed CD31+ cell population diminished tissue regeneration at different levels, suggesting combined effects within the PB-CD31+ subsets. In summary, an intraoperative enrichment of PB-CD31+ cells might be a novel option to facilitate endogenous regeneration under biologically impaired situations by supporting immunomodulation and vascularization. © 2016 American Society for Bone and Mineral Research.
Asunto(s)
Regeneración Ósea/inmunología , Células Progenitoras Endoteliales/inmunología , Células Progenitoras Endoteliales/trasplante , Inmunomodulación , Neovascularización Fisiológica/inmunología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Trasplante de Células Madre , Adolescente , Adulto , Anciano , Animales , Células Progenitoras Endoteliales/citología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ratas , Ratas Sprague-DawleyRESUMEN
Fracture healing is a complex regeneration process which produces new bone tissue without scar formation. However, fracture healing disorders occur in approximately 10% of human patients and cause severe pain and reduced quality of life. Recently, the development of more standardized, sophisticated and commercially available osteosynthesis techniques reflecting clinical approaches has increased the use of small rodents such as rats and mice in bone healing research dramatically. Nevertheless, there is no standard for pain assessment, especially in these species, and consequently limited information regarding the welfare aspects of osteotomy models. Moreover, the selection of analgesics is restricted for osteotomy models since non-steroidal anti-inflammatory drugs (NSAIDs) are known to affect the initial, inflammatory phase of bone healing. Therefore, opioids such as buprenorphine and tramadol are often used. However, dosage data in the literature are varied. Within this review, we clarify the background of osteotomy models, explain the current status and challenges of animal welfare assessment, and provide an example score sheet including model specific parameters. Furthermore, we summarize current refinement options and present a brief outlook on further 3R research.
Asunto(s)
Bienestar del Animal , Osteotomía/efectos adversos , Dimensión del Dolor/métodos , Dolor Postoperatorio/prevención & control , Proyectos de Investigación , Animales , Modelos Animales de Enfermedad , Ratones , RatasRESUMEN
Structured hydrogels showing form stability and elastic properties individually tailorable on different length scales are accessible in a one-step process. They support cell adhesion and differentiation and display growing pore size during degradation. In vivo experiments demonstrate their efficacy in biomaterial-induced bone regeneration, not requiring addition of cells or growth factors.