RESUMEN
We used the tarsus of an adult Xenopus laevis frog as an in vivo load-bearing model to study the regeneration of critical-size defects (CSD) in long bones. We found the CSD for this bone to be about 35% of the tarsus length. To promote regeneration, we implanted biocompatible 1,6 hexanediol diacrylate scaffolds soaked with bone morphogenetic proteins-4 and vascular endothelial growth factors. In contrast to studies that use scaffolds as templates for bone formation, we used scaffolds as a growth factor delivery vehicle to promote cartilage-to-bone regeneration. Defects in control frogs were filled with scaffolds lacking growth factors. The limbs were harvested at a series of time points ranging from 3 weeks to 6 months after implantation and evaluated using micro-computed tomography and histology. In frogs treated with growth factor-loaded scaffolds, we observed a cartilage-to-bone regeneration in the skeletal defect. Five out of eight defects were completely filled with cartilage by 6 weeks. Blood vessels had invaded the cartilage, and bone was beginning to form in ossifying centers. By 3 months, these processes were well advanced, and extensive ossification was observed in 6-month samples. In contrast, the defects in control frogs showed only formation of fibrous scar tissue. This study demonstrates the utility of a Xenopus model system for tissue engineering research and that the normal in vivo mechanism of endochondral bone development and fracture repair can be mimicked in the repair of CSD with scaffolds used as growth factor delivery mechanisms.
Asunto(s)
Proteína Morfogenética Ósea 4/farmacología , Regeneración Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Huesos/patología , Modelos Animales , Factor A de Crecimiento Endotelial Vascular/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Huesos/cirugía , Humanos , Implantes Experimentales , Microscopía Electrónica de Rastreo , Porosidad/efectos de los fármacos , Andamios del Tejido/química , Xenopus laevisRESUMEN
BACKGROUND: Following amputation, urodele salamander limbs reprogram somatic cells to form a blastema that self-organizes into the missing limb parts to restore the structure and function of the limb. To help understand the molecular basis of blastema formation, we used quantitative label-free liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)-based methods to analyze changes in the proteome that occurred 1, 4 and 7 days post amputation (dpa) through the mid-tibia/fibula of axolotl hind limbs. RESULTS: We identified 309 unique proteins with significant fold change relative to controls (0 dpa), representing 10 biological process categories: (1) signaling, (2) Ca2+ binding and translocation, (3) transcription, (4) translation, (5) cytoskeleton, (6) extracellular matrix (ECM), (7) metabolism, (8) cell protection, (9) degradation, and (10) cell cycle. In all, 43 proteins exhibited exceptionally high fold changes. Of these, the ecotropic viral integrative factor 5 (EVI5), a cell cycle-related oncoprotein that prevents cells from entering the mitotic phase of the cell cycle prematurely, was of special interest because its fold change was exceptionally high throughout blastema formation. CONCLUSION: Our data were consistent with previous studies indicating the importance of inositol triphosphate and Ca2+ signaling in initiating the ECM and cytoskeletal remodeling characteristic of histolysis and cell dedifferentiation. In addition, the data suggested that blastema formation requires several mechanisms to avoid apoptosis, including reduced metabolism, differential regulation of proapoptotic and antiapoptotic proteins, and initiation of an unfolded protein response (UPR). Since there is virtually no mitosis during blastema formation, we propose that high levels of EVI5 function to arrest dedifferentiated cells somewhere in the G1/S/G2 phases of the cell cycle until they have accumulated under the wound epidermis and enter mitosis in response to neural and epidermal factors. Our findings indicate the general value of quantitative proteomic analysis in understanding the regeneration of complex structures.
Asunto(s)
Ambystoma/fisiología , Extremidades/fisiología , Proteómica , Regeneración/fisiología , Amputación Quirúrgica , Animales , Señalización del Calcio/genética , Cromatografía Líquida de Alta Presión , Matriz Extracelular/metabolismo , Extremidades/cirugía , Inositol 1,4,5-Trifosfato/metabolismo , Mapeo Peptídico , Espectrometría de Masas en Tándem , Cicatrización de HeridasRESUMEN
In this study, we present strategies for experimental design that minimize variation in Xenopus hindlimb regeneration results. We have standardized our laboratory culture conditions for older stage Xenopus tadpoles. We have established a normal tadpole growth curve for our laboratory and characterized normal tadpole behaviors in an effort to eliminate abnormal tadpoles from our experiments. We have used large sample sizes and statistical analysis to establish normal regeneration performances for seven amputation planes in stages 55-57 tadpole hindlimbs. We have demonstrated that regeneration performance of abnormal tadpoles is significantly different than that of normal tadpoles. We have examined the kinetics of ossification in developing Xenopus hindlimbs, and have found that increasing ossification rates and rates of regeneration decline are inversely correlated.
Asunto(s)
Miembro Posterior/fisiología , Regeneración/fisiología , Proyectos de Investigación , Animales , Regeneración Ósea/fisiología , Larva/crecimiento & desarrollo , Larva/fisiología , Reproducibilidad de los Resultados , XenopusRESUMEN
The existing table of stages of the normal development of the axolotl (Ambystoma mexicanum) ends just after hatching. At this time, the forelimbs are small buds. In this study, we extend the staging series through completion of development of the forelimbs and hindlimbs.
Asunto(s)
Ambystoma mexicanum/embriología , Animales , Huesos/embriología , Cartílago/embriología , Extremidades/embriología , Esbozos de los Miembros/embriologíaRESUMEN
Urodele amphibians have been widely used for studies of limb regeneration. In this article, we review studies on blastema cell proliferation and propose a model of blastemal self-organization and patterning. The model is based on local cell interactions that intercalate positional identities within circumferential and proximodistal boundaries that outline the regenerate. The positional identities created by the intercalation process appear to be reflected in the molecular composition of the cell surface. Transcription factors and signaling molecules involved in patterning are discussed within the context of the boundary/intercalation model.
Asunto(s)
Extremidades/fisiología , Regeneración/fisiología , Urodelos/fisiología , Animales , Epidermis/fisiología , Fenómenos Fisiológicos del Sistema Nervioso , Transducción de Señal/fisiología , Factores de Transcripción/fisiologíaRESUMEN
Urodele amphibians, newts and salamanders, can regenerate lesioned spinal cord at any stage of the life cycle and are the only tetrapod vertebrates that regenerate spinal cord completely as adults. The ependymal cells play a key role in this process in both gap replacement and caudal regeneration. The ependymal response helps to produce a different response to neural injury compared with mammalian neural injury. The regenerating urodele cord produces new neurons as well as supporting axonal regrowth. It is not yet clear to what extent urodele spinal cord regeneration recapitulates embryonic anteroposterior and dorsoventral patterning gene expression to achieve functional reconstruction. The source of axial patterning signals in regeneration would be substantially different from those in developing tissue, perhaps with signals propagated from the stump tissue. Examination of the effects of fibroblast growth factor and epidermal growth factor on ependymal cells in vivo and in vitro suggest a connection with neural stem cell behavior as described in developing and mature mammalian central nervous system. This review coordinates the urodele regeneration literature with axial patterning, stem cell, and neural injury literature from other systems to describe our current understanding and assess the gaps in our knowledge about urodele spinal cord regeneration.