RESUMEN
BACKGROUND: Anterior cruciate ligament (ACL) reconstruction is the current standard of care for ACL tears. However, the results are not consistently successful; autografts or allografts have certain disadvantages; and synthetic grafts have had poor clinical results. PURPOSE: To determine if recellularization of decellularized tendons combined with mechanical stimulation in a bioreactor could replicate the mechanical properties of the native ACL and be successfully used for ACL reconstruction in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: Porcine tibialis tendons were decellularized and then recellularized with human adult bone marrow-derived stem cells. Tendons were cultured in a tissue bioreactor that provided biaxial cyclic loading for up to 7 days. To reproduce mechanical stresses similar to those experienced by the ACL within the knee joint, the tendons were subjected to simultaneous tension and torsion in the bioreactor. Expression of tendon-specific genes and newly synthesized collagen and glycosaminoglycan were used to quantify the efficacy of recellularization and dynamic bioreactor culture. The ultimate tensile load to failure and stiffness of recellularized constructs were measured after dynamic stimulation. Finally, the tissue-engineered tendons were used to reconstruct the ACL in 24 pigs, and ultimate tensile load to failure and stiffness were assessed after 3 months. RESULTS: Dynamic bioreactor culture significantly increased the expression of tendon-specific genes, the quantity of newly synthesized collagen and glycosaminoglycan, and the ultimate tensile load and stiffness of recellularized tendons. After in vivo reconstruction, the ultimate tensile load and stiffness of the tissue-engineered tendons increased significantly up to 3 months after surgery and were within 80% of the ultimate tensile load of the natural ACL. CONCLUSION: This translational study indicates that recellularization and dynamic mechanical stimuli can significantly enhance matrix synthesis and ultimate tensile load of decellularized porcine tibialis tendons. This approach to tissue engineering can be very useful for ACL reconstruction and may overcome some of the disadvantages of autografts and allografts. CLINICAL RELEVANCE: Dynamic bioreactor cultivation of tissue-engineered tendons may overcome the limitations of autografts and allografts.
Asunto(s)
Lesiones del Ligamento Cruzado Anterior/cirugía , Reconstrucción del Ligamento Cruzado Anterior , Ligamento Cruzado Anterior/cirugía , Tendones/trasplante , Ingeniería de Tejidos , Animales , Fenómenos Biomecánicos , Proteínas de Caenorhabditis elegans/química , Colágeno/química , Galactosiltransferasas/química , Humanos , Articulación de la Rodilla/cirugía , Masculino , Células Madre Mesenquimatosas/citología , Estrés Mecánico , Porcinos , Porcinos Enanos , Resistencia a la TracciónRESUMEN
Biologic augmentation for rotator cuff repair is a challenging treatment in patients with chronic large, massive, and irreparable rotator cuff injuries. Particularly, the use of an extracellular matrix (ECM) patch such as dermal tissue offered improved biomechanical properties in previous studies. Cytokines induce cell chemotaxis, proliferation, matrix synthesis, and cell differentiation. Moreover, osteoinductive growth factors such as bone morphogenetic protein-2 (BMP-2) affect the formation of new bone and fibrocartilage in lesions. However, the effects of using a dermal patch in combination with BMP-2 have not been evaluated to date, although many researchers have recognized the importance thereof. In this study, rhBMP-2-coated dermal patch (1 cm × 2 cm) isolated from human cadaveric donor was inserted in a rabbit model of chronic rotator cuff injury for in vivo evaluation. Bone mineral density and biomechanical strength were tested and histological and histomorphometric analyses were performed. The results showed that insertion of an rhBMP-2-coated acellular dermal patch not only significantly ameliorated new bone formation, it also improved biomechanical properties such as ultimate tensile strength. Thus, the use of this combination may improve the chronic rotator cuff injury-healing rate and clinical outcomes after rotator cuff repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1840-1846, 2017.
Asunto(s)
Apósitos Biológicos , Proteína Morfogenética Ósea 2 , Materiales Biocompatibles Revestidos , Lesiones del Manguito de los Rotadores/terapia , Piel , Animales , Proteína Morfogenética Ósea 2/química , Proteína Morfogenética Ósea 2/farmacocinética , Proteína Morfogenética Ósea 2/farmacología , Enfermedad Crónica , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacocinética , Materiales Biocompatibles Revestidos/farmacología , Masculino , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologíaRESUMEN
This study examines the hypothesis that injectable collagen gel can be an effective carrier for recombinant human bone morphogenetic protein-2 (rhBMP-2)'s localization to the healing tendon-bone interface. In 36 mature New Zealand White rabbits, the upper long digital extensor tendon was cut and inserted into the proximal tibial bone tunnel. Then a rhBMP-2-containing collagen gel was injected into the tendon-bone tunnel interface, using a syringe. Histological and biomechanical assessments of the tendon-bone interface were conducted at 3 and 6 weeks after implantation. In vitro testing showed that the semi-viscous collagen gel at room temperature was transformed into a firm gel state at 37°C. The rhBMP-2 release profile showed that rhBMP-2 was released from the collagen gel for more than 28 days. In vivo testing showed that fibrocartilage and new bone are formed at the interface at 6 weeks after injection of rhBMP-2. On radiography, spotty calcification appeared and enthesis-like tissue was produced successfully in the tendon at 6 weeks after injection of rhBMP-2. Use of the viscous collagen gel and rhBMP-2 mixture increased the fusion rate between the bone tunnel and tissue graft. This study demonstrates that viscous collagen gel can be an effective carrier for rhBMP-2 delivery into surgical sites, and that the injectable rhBMP-2-containing collagen gel may be applied for the enhancement of tendon-bone interface healing in the future. Copyright © 2015 John Wiley & Sons, Ltd.
Asunto(s)
Proteína Morfogenética Ósea 2/farmacología , Colágeno/farmacología , Traumatismos de los Tendones/tratamiento farmacológico , Tendones/metabolismo , Tibia/metabolismo , Animales , Proteína Morfogenética Ósea 2/química , Colágeno/química , Implantes de Medicamentos , Humanos , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Traumatismos de los Tendones/metabolismo , Traumatismos de los Tendones/patología , Tendones/patología , Tibia/lesionesRESUMEN
Sequentially chemical-treated bovine bone was not only evaluated by mechanical and chemical analyses but also implanted into the gluteal muscles of rats for 12 weeks to investigate potential local pathological effects and systemic toxicities. The test (chemical treated bone) and control (heat treated bone) materials were compared using scanning electron microscope (SEM), x-ray diffraction pattern, inductively coupled plasma analysis, and bending strength test. In the SEM images, the micro-porous structure of heat-treated bone was changed to sintered ceramic-like structure. The structure of bone mineral from test and control materials was analyzed as100% hydroxyapatite. The ratio of calcium (Ca) to potassium (P), the main inorganic elements, was same even though the Ca and P percentages of the control material was relatively higher than the test material. No death or critical symptoms arose from implantation of the test (chemical treated bone) and control (physiological saline) materials during 12 weeks. The implanted sites were macroscopically examined, with all the groups showing non-irritant results. Our results indicate that chemical processed bovine bone has a better mechanical property than the heat treated bone and the implantation of this material does not produce systemic or pathological toxicity.
Asunto(s)
Trasplante Óseo/métodos , Huesos/efectos de los fármacos , Músculo Esquelético/cirugía , Animales , Fenómenos Biomecánicos , Trasplante Óseo/efectos adversos , Huesos/química , Huesos/diagnóstico por imagen , Huesos/ultraestructura , Nalgas , Calcio/análisis , Bovinos , Durapatita/análisis , Femenino , Xenoinjertos , Calor , Masculino , Microscopía Electrónica de Rastreo , Porosidad , Potasio/análisis , Radiografía , Ratas , Ratas Sprague-Dawley , Medición de Riesgo , Espectrofotometría Atómica , Estrés Mecánico , Factores de Tiempo , Pruebas de Toxicidad Subcrónica , Trasplante Heterólogo , Difracción de Rayos XRESUMEN
Decellularized tissues have been successfully used in tissue engineering and regenerative medicine for the purpose of removing antigens present in the cellular components. However, this decellularization technique uses ionic solutions or chemical treatments such as enzyme treatments that might damage the biophysical properties or reduce the physical strength of tissue. This study aimed to improve the strength of decellularized tissues. We designed a tissue bioreactor that can repeatedly deliver physical stimulation, such as tensile and torsional deformation, to the upper and lower parts of a tissue. To decellularized porcine Tibialis tendons, we used an enzymatic solution to remove the primary cells, and then applied ultrasonic cleansing using a combination of ionic solution and distilled water to destroy residual cells by differing from the osmotic pressure between the inside and outside of the cell membrane. The total DNA content of decellularized tissue was decreased by 77% compared with that of the original tissue and the ultimate tensile strength of the decellularized tissue was 20% lower than that of the normal tissue. Decellularized tissues were then cultivated in the tissue bioreactor with repeated physical stimulation of 110% tension, 90° torsion, and frequency of once per a second, and the ultimate tensile strength was found to be greater than that of the normal ligament at 7 day culture. This study showed that decellularization using enzyme and mechanical treatment is safe and use of a tissue bioreactor can increase the physical strength of tendons, making this a potential mechanism to reconstruct human ligaments.
Asunto(s)
Reactores Biológicos , Tendones/citología , Tendones/fisiología , Técnicas de Cultivo de Tejidos/instrumentación , Animales , Fenómenos Biomecánicos , Electroforesis en Gel de Poliacrilamida , Humanos , Coloración y Etiquetado , Sus scrofa , Tendones/ultraestructura , Resistencia a la Tracción/fisiologíaRESUMEN
BACKGROUND: Xenografts, unlike other grafting products, cannot be commercialized unless they conform to stringent safety regulations. Particularly with bovine-derived materials, it is essential to remove viruses and inactivate infectious factors because of the possibility that raw materials are imbrued with infectious viruses. The removal of the characteristics of infectious viruses from the bovine bone grafting materials need to be proved and inactivation process should satisfy the management provision of the Food and Drug Administration (FDA). To date, while most virus inactivation studies were performed in human allograft tissues, there have been almost no studies on bovine bone. METHODS: To evaluate the efficacy of virus inactivation after treatment of bovine bone with 70% ethanol, 4% sodium hydroxide, and gamma irradiation, we selected a variety of experimental model viruses that are known to be associated with bone pathogenesis, including bovine parvovirus (BPV), bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), and bovine parainfluenza-3 virus (BPIV-3). The cumulative virus log clearance factor or cumulative virus log reduction factor for the manufacturing process was obtained by calculating the sum of the individual virus log clearance factors or log reduction factors determined for individual process steps with different physicochemical methods. RESULTS: The cumulative log clearance factors achieved by three different virus inactivation processes were as follows: BPV ≥ 17.73, BHV ≥ 20.53, BVDV ≥ 19.00, and BPIV-3 ≥ 16.27. On the other hand, the cumulative log reduction factors achieved were as follows: BPV ≥ 16.95, BHV ≥ 20.22, BVDV ≥ 19.27, and BPIV-3 ≥ 15.58. CONCLUSIONS: Treatment with 70% ethanol, 4% sodium hydroxide, or gamma irradiation was found to be very effective in virus inactivation, since all viruses were at undetectable levels during each process. We have no doubt that application of this established process to bovine bone graft manufacture will be effective and essential.