RESUMEN
Total hip replacement is the most effective treatment for late stage osteoarthritis. However, adverse local tissue reactions (ALTRs) have been observed in patients with modular total hip implants. Although the detailed mechanisms of ALTRs are still unknown, fretting corrosion and the associated metal ion release from the CoCrMo femoral head at the modular junction has been reported to be a major factor. The purpose of this study is to increase the fretting corrosion resistance of the CoCrMo alloy and the associated metal ion release by applying hard coatings to the surface. Cathodic arc evaporation technique (arc-PVD) was used to deposit TiSiN and ZrN hard coatings on CoCrMo substrates. The morphology, chemical composition, crystal structures and residual stress of the coatings were characterized by scanning electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffractometry. Hardness, elastic modulus, and adhesion of the coatings were measured by nano-indentation, nano-scratch test, and the Rockwell C test. Fretting corrosion resistance tests of coated and uncoated CoCrMo discs against Ti6Al4V spheres were conducted on a four-station fretting testing machine in simulated body fluid at 1Hz for 1 million cycles. Post-fretting samples were analyzed for morphological changes, volume loss and metal ion release. Our analyses showed better surface finish and lower residual stress for ZrN coating, but higher hardness and better scratch resistance for TiSiN coating. Fretting results demonstrated substantial improvement in fretting corrosion resistance of CoCrMo with both coatings. ZrN and TiSiN decreased fretting volume loss by more than 10 times and 1000 times, respectively. Both coatings showed close to 90% decrease of Co ion release during fretting corrosion tests. Our results suggest that hard coating deposition on CoCrMo alloy can significantly improve its fretting corrosion resistance and could thus potentially alleviate ALTRs in metal hip implants.
Asunto(s)
Aleaciones , Prótesis de Cadera , Corrosión , Humanos , Ensayo de Materiales , Propiedades de SuperficieRESUMEN
The nucleus pulposus (NP) is composed of NP and notochord cell. It is a paucicellular tissue and if it is to be used as a source of cells for tissue engineering the cell number will have to be expanded by cell passaging. The hypothesis of this study is that passaged NP and notochordal cells grown in three-dimensional (3D) culture in the presence of transforming growth factor ß (TGFß) will show enhanced NP tissue formation compared with cells grown in the absence of this growth factor. Bovine NP cells isolated by sequential enzymatic digestion from caudal intervertebral discs were either placed directly in 3D culture (P0) or serially passaged up to passage 3 (P3) prior to placement in 3D culture. Serial cell passage in monolayer culture led to de-differentiation, increased senescence and oxidative stress and decreases in the gene expression of NP and notochordal associated markers and increases in de-differentiation markers. The NP tissue regeneration capacity of cells in 3D culture decreases with passaging as indicated by diminished tissue thickness and total collagen content when compared with tissues formed by P0 cells. Immunohistochemical studies showed that type II collagen accumulation appeared to decrease. TGFß1 or TGFß3 treatment enhanced the ability of cells at each passage to form tissue, in part by decreasing cell death. However, neither TGFß1 nor TGFß3 were able to restore the notochordal phenotype. Although TGFß1/3 recovered NP tissue formation by passaged cells, to generate NP in vitro that resembles the native tissue will require identification of conditions facilitating retention of notochordal cell differentiation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:438-449, 2020.
Asunto(s)
Notocorda/citología , Núcleo Pulposo/citología , Ingeniería de Tejidos/métodos , Factor de Crecimiento Transformador beta1 , Factor de Crecimiento Transformador beta3 , Animales , Bovinos , Senescencia Celular , Estrés Oxidativo , Cultivo Primario de CélulasRESUMEN
Calcium phosphate-based biomaterials are extensively used for bone replacement and regeneration in orthopedic, dental, and maxillofacial surgical applications. The injury induced by surgical implantation of bone replacement graft materials initiates a cascade of host responses, starting with blood-biomaterial contact, protein adsorption on the material surface, blood coagulation, and leukocyte responses. During the initial acute inflammatory response, polymorphonuclear neutrophils (PMNs) and monocytes, abundant circulating leukocytes of the myeloid lineage, are recruited to the site of inflammation. In addition to responding to pathogenic challenges, these cells respond to particulate substances within the body including crystals of monosodium urate (MSU). Host responses toward grafts impact short- and long-term success in tissue engineering and regenerative applications. Although multinucleated osteoclasts, formed by monocyte/macrophage fusion, are generally thought to be responsible for resorption of implant biomaterials, the ability of different biomaterials to trigger PMNs, which are invariably present at the early stages after implant surgery, and are abundant in the oral cavity, has never been tested. In this article, we present analysis of the response of human blood-derived PMNs and monocytes toward brushite, monetite, and calcium polyphosphate (CPP) biomaterial substrates and compare this to the response to MSU crystals, the latter serving as a positive control. Employing multicolor flow cytometry to look at PMN and monocyte cell surface markers of activation to gauge the response to different biomaterials, we found that both types of myeloid cells are highly activated after exposure to brushite, monetite, and MSU but not CPP. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:253-262, 2020.
Asunto(s)
Materiales Biocompatibles/farmacología , Sustitutos de Huesos/farmacología , Fosfatos de Calcio/farmacología , Leucocitos/metabolismo , Ensayo de Materiales , Polifosfatos/farmacología , Humanos , Osteoclastos/metabolismoRESUMEN
Integration of in vitro-formed cartilage on a suitable substrate to form tissue-engineered implants for osteochondral defect repair is a considerable challenge. In healthy cartilage, a zone of calcified cartilage (ZCC) acts as an intermediary for mechanical force transfer from soft to hard tissue, as well as an effective interlocking structure to better resist interfacial shear forces. We have developed biphasic constructs that consist of scaffold-free cartilage tissue grown in vitro on, and interdigitated with, porous calcium polyphosphate (CPP) substrates. However, as CPP degrades, it releases inorganic polyphosphates (polyP) that can inhibit local mineralization, thereby preventing the formation of a ZCC at the interface. Thus, we hypothesize that coating CPP substrate with a layer of hydroxyapatite (HA) might prevent or limit this polyP release. To investigate this we tested both inorganic or organic sol-gel processing methods, asa barrier coating on CPP substrate to inhibit polyP release. Both types of coating supported the formation of ZCC in direct contact with the substrate, however the ZCC appeared more continuous in the tissue formed on the organic HA sol gel coated CPP. Tissues formed on coated substrates accumulated comparable quantities of extracellular matrix and mineral, but tissues formed on organic sol-gel (OSG)-coated substrates accumulated less polyP than tissues formed on inorganic sol-gel (ISG)-coated substrates. Constructs formed with OSG-coated CPP substrates had greater interfacial shear strength than those formed with ISG-coated and non-coated substrates. These results suggest that the OSG coating method can modify the location and distribution of ZCC and can be used to improve the mechanical integrity of tissue-engineered constructs formed on porous CPP substrates. STATEMENT OF SIGNIFICANCE: Articular cartilage interfaces with bone through a zone of calcified cartilage. This study describes a method to generate an "osteochondral-like" implant that mimics this organization using isolated deep zone cartilage cells and a sol-gel hydroxyapatite coated bone substitute material composed of calcium polyphosphate (CPP). Developing a layer of calcified cartilage at the interface should contribute to enhancing the success of this "osteochondral-like" construct following implantation to repair cartilage defects.
Asunto(s)
Cartílago , Durapatita , Ensayo de Materiales , Membranas Artificiales , Polifosfatos , Ingeniería de Tejidos/métodos , Animales , Cartílago/lesiones , Cartílago/metabolismo , Cartílago/patología , Bovinos , Durapatita/química , Durapatita/farmacología , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Transición de Fase , Polifosfatos/química , Polifosfatos/farmacología , PorosidadRESUMEN
Disc degeneration is associated with low back pain for which currently there is no optimal therapy so there is a great need to identify new treatment approaches. Inorganic polyphosphates (polyP) are linear polymers of orthophosphate units varying in chain length and present in many cell types. As polyP has anabolic effects on chondrocytes, we hypothesized that polyP treatment would enhance matrix accumulation by nucleus pulposus (NP) cells. NP cells isolated from bovine caudal discs were grown in 3D culture under normoxic or in select experiments under hypoxic conditions, in the presence or absence of various concentrations and sizes of polyP. Gene expression was determined using RT-PCR. Matrix accumulation was quantified by measuring proteoglycan and collagen contents. DAPI fluorescence shift was used to stain for polyP in tissue. DAPI staining showed polyP present predominantly in the pericellular region of in vitro formed tissue. PolyP treatment enhanced matrix accumulation in a concentration and chain length dependant manner. NP cells exposed to polyP-22 (22 phosphate units length) showed an increase in gene expression of aggrecan, Collagen II, Sox 9, and MMP-13 which was maintained for the 14 days of culture. This suggests that polyP may enhance NP tissue formation in vitro by upregulating the expression of matrix genes. As polyP enhances proteoglycan accumulation even under hypoxic conditions, this raises the possibility that polyP may be a novel treatment to induce NP regeneration. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:41-50, 2017.
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Anabolizantes/uso terapéutico , Degeneración del Disco Intervertebral/tratamiento farmacológico , Núcleo Pulposo/efectos de los fármacos , Polifosfatos/uso terapéutico , Anabolizantes/farmacología , Animales , Bovinos , ADN/metabolismo , Evaluación Preclínica de Medicamentos , Matriz Extracelular/metabolismo , Expresión Génica/efectos de los fármacos , Hipoxia/metabolismo , Núcleo Pulposo/metabolismo , Polifosfatos/farmacología , Cultivo Primario de CélulasRESUMEN
This article addresses the effects of glycerol (GLY) concentrations on the mechanical properties of calcium polyphosphate (CPP) bone substitute structures manufactured using binder jetting additive manufacturing. To achieve this goal, nine types of water-based binder solutions were prepared with 10, 12.5, and 15 wt % GLY liquid-binding agent, mixed, respectively, with 0, 0.75, and 1.5 wt % ethylene glycol diacetate (EGD) flow enhancer. The print quality of each of the solutions was established quantitatively using an image processing algorithm. The print quality analysis narrowed down the solutions to three batches containing 1.5 wt % EGD and variable amount of GLY. These solutions were used to manufacture porous CPP bone substitute samples, which were characterized physically to determine shrinkage, porosity, microstructure, and compression strength. The 12.5 wt % GLY, 1.5 wt % EGD solution resulted in the highest mechanical strength after sintering (34.6 ± 5.8 MPa), illustrating similar mechanical properties when compared to previous studies (33.9 ± 6.3 MPa) of additively manufactured CPP bone substitutes using a commercially available binder. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 828-835, 2017.
Asunto(s)
Sustitutos de Huesos/química , Fuerza Compresiva , Glicerol/química , Polifosfatos/química , Impresión Tridimensional , Glicoles de Etileno/química , PorosidadRESUMEN
This study investigates the characteristics of porous calcium polyphosphate particulates (CPPp) formed using two different processing treatments as bone void fillers in non- or minimally load-bearing sites. The two calcium polyphosphate particulate variants (grades) were formed using different annealing conditions during particulate preparation to yield either more slowly degrading calcium polyphosphate particulates (SD-CPPp) or faster degrading particulates (FD-CPPp) as suggested by a previous degradation study conducted in vitro (Hu et al., Submitted for publication 2016). The two CPPp grades were compared as bone void fillers in vivo by implanting particulates in defects created in rabbit femoral condyle sites (critical size defects). The SD-CPPp and FD-CPPp were implanted for 4- and 16-week periods. The in vivo study indicated a significant difference in amount of new bone formed in the prepared sites with SD-CPPp resulting in more new bone formation compared with FD-CPPp. The lower bone formation characteristic of the FD-CPPp was attributed to its faster degradation rate and resulting higher local concentration of released polyphosphate degradation products. The study results indicate the importance of processing conditions on preparing calcium polyphosphate particulates for potential use as bone void fillers in nonload-bearing sites. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 874-884, 2017.
Asunto(s)
Sustitutos de Huesos , Fémur , Osteogénesis/efectos de los fármacos , Polifosfatos , Animales , Sustitutos de Huesos/química , Sustitutos de Huesos/farmacología , Fémur/lesiones , Fémur/metabolismo , Fémur/patología , Polifosfatos/química , Polifosfatos/farmacología , ConejosRESUMEN
A 2-Step sinter/anneal treatment has been reported previously for forming porous CPP as biodegradable bone substitutes [9]. During the 2-Step annealing treatment, the heat treatment used strongly affected the rate of CPP degradation in vitro. In the present study, x-ray diffraction and (31)P solid state nuclear magnetic resonance were used to determine the phases that formed using different heat treating processes. The effect of in vitro degradation (in PBS at 37 °C, pH 7.1 or 4.5) was also studied. During CPP preparation, ß-CPP and γ-CPP were identified in powders formed from a calcium monobasic monohydrate precursor after an initial calcining treatment (10 h at 500 °C). Melting of this CPP powder (at 1100 °C), quenching and grinding formed amorphous CPP powders. Annealing powders at 585 °C (Step-1) resulted in rapid sintering to form amorphous porous CPP. Continued annealing to 650 °C resulted in crystallization to form a multi-phase structure of ß-CPP primarily plus lesser amounts of α-CPP, calcium ultra-phosphates and retained amorphous CPP. Annealing above 720 °C and up to 950 °C transformed this to ß-CPP phase. In vitro degradation of the 585 °C (Step-1 only) and 650 °C Step-2 annealed multi-phase samples occurred significantly faster than the ß-CPP samples formed by Step-2 annealing at or above 720 °C. This faster degradation was attributable to preferential degradation of thermodynamically less stable phases that formed in samples annealed at 650 °C (i.e. α-phase, ultra-phosphate and amorphous CPP). Degradation in lower pH solutions significantly increased degradation rates of the 585 and 650 °C annealed samples but had no significant effect on the ß-CPP samples.
Asunto(s)
Sustitutos de Huesos/química , Fosfatos de Calcio/química , Materiales Biocompatibles/química , Cementos para Huesos/química , Huesos/fisiología , Humanos , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Microscopía Electrónica de Rastreo , Isótopos de Fósforo/química , Polifosfatos/química , Porosidad , Polvos , Temperatura , Termodinámica , Difracción de Rayos XRESUMEN
Porous calcium polyphosphate (CPP) structures proposed as bone-substitute implants and made by sintering CPP powders to form bending test samples of approximately 35 vol % porosity were machined from preformed blocks made either by additive manufacturing (AM) or conventional gravity sintering (CS) methods and the structure and mechanical characteristics of samples so made were compared. AM-made samples displayed higher bending strengths (≈1.2-1.4 times greater than CS-made samples), whereas elastic constant (i.e., effective elastic modulus of the porous structures) that is determined by material elastic modulus and structural geometry of the samples was ≈1.9-2.3 times greater for AM-made samples. X-ray diffraction analysis showed that samples made by either method displayed the same crystal structure forming ß-CPP after sinter annealing. The material elastic modulus, E, determined using nanoindentation tests also showed the same value for both sample types (i.e., E ≈ 64 GPa). Examination of the porous structures indicated that significantly larger sinter necks resulted in the AM-made samples which presumably resulted in the higher mechanical properties. The development of mechanical properties was attributed to the different sinter anneal procedures required to make 35 vol % porous samples by the two methods. A primary objective of the present study, in addition to reporting on bending strength and sample stiffness (elastic constant) characteristics, was to determine why the two processes resulted in the observed mechanical property differences for samples of equivalent volume percentage of porosity. An understanding of the fundamental reason(s) for the observed effect is considered important for developing improved processes for preparation of porous CPP implants as bone substitutes for use in high load-bearing skeletal sites.
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Sustitutos de Huesos/química , Compuestos de Calcio/química , Polifosfatos/química , Porosidad , Estrés MecánicoRESUMEN
Porous calcium polyphosphate (CPP) structures with 30 volume percent porosity and made by solid freeform fabrication (SFF) were implanted in rabbit femoral condyle sites for 6-wk periods. Two forms of SFF implants with different stacked layer orientation were made in view of prior studies reporting on anisotropic/orthotropic mechanical properties of structures so formed. In addition, porous CPP implants of equal volume percent porosity made by conventional sintering and machining methods were prepared. Bone ingrowth and in vivo degradation of the three different implant types were compared using back-scattered scanning electron microscopy (BS-SEM) of implant samples and quantitative analysis of the images. The results indicated bone ingrowth with all samples resulting in 30-40% fill of available porosity by bone within the 6-wk period. In the 6-wk in vivo period, approximately 7-9% loss of CPP by degradation had occurred.
Asunto(s)
Sustitutos de Huesos/química , Polifosfatos/química , Animales , Fosfatos de Calcio/química , Fémur/patología , Fémur/cirugía , Masculino , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Oseointegración , Porosidad , Prótesis e Implantes , ConejosRESUMEN
Porous calcium polyphosphate (CPP) is being investigated for fabrication of novel biodegradable bone substitutes. In this study, porous CPP implants formed by conventional CPP powder packing and using a two-step sinter/anneal process was used to form 20 and 30 vol % porous samples displaying relatively high strength. These were implanted in rabbit femoral condyle sites to study their ability for secure fixation in prepared sites through bone ingrowth. Porous implants of 20 and 30 vol % porosity and displaying compressive strengths ~80 and 35 MPa, respectively, were used. Bone ingrowth sufficient to allow secure implant fixation was observed by 6 weeks (~19% bone ingrowth per available pore space for the 30 vol % and 13% for the 20 vol % porous implants). The results of the in vivo study suggest the potential usefulness of porous CPP as biodegradable bone substitutes/augments in high load-bearing skeletal regions.
Asunto(s)
Sustitutos de Huesos , Fosfatos de Calcio , Animales , Microscopía Electrónica de Rastreo , ConejosRESUMEN
Clinical utilization of tissue-engineered cartilage constructs has been limited by their inferior mechanical properties compared to native articular cartilage. A number of strategies have been investigated to increase the accumulation of major extracellular matrix components within in vitro-formed cartilage, including the administration of growth factors and mechanical stimulation. In this study, the anabolic effect of inorganic polyphosphates, a linear polymer of orthophosphate residues linked by phosphoanhydride bonds, was demonstrated in both chondrocyte cultures and native articular cartilage cultured ex vivo. Compared to untreated controls, polyphosphate treatment of three-dimensional primary chondrocyte cultures induced increased glycosaminoglycan and collagen accumulation in a concentration- and chain length-dependent manner. This effect was transient, because chondrocytes express exopolyphosphatases that hydrolyze polyphosphate. The anabolic effect of polyphosphates was accompanied by a lower rate of DNA increase within the chondrocyte cultures treated with inorganic polyphosphate. Inorganic polyphosphate enhances cartilage matrix accumulation and is a promising approach to improve the quality of tissue-engineered cartilage constructs.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Cartílago Articular/fisiología , Condrogénesis/efectos de los fármacos , Polifosfatos/farmacología , Ingeniería de Tejidos , Ácido Anhídrido Hidrolasas/metabolismo , Animales , Bovinos , Células Cultivadas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Condrogénesis/genética , ADN/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Polifosfatos/administración & dosificación , Factores de TiempoRESUMEN
A major challenge for cartilage tissue engineering remains the proper integration of constructs with surrounding tissues in the joint. Biphasic osteochondral constructs that can be anchored in a joint through bone ingrowth partially address this requirement. In this study, a methodology was devised to generate a cell-mediated zone of calcified cartilage (ZCC) between the in vitro-formed cartilage and a porous calcium polyphosphate (CPP) bone substitute in an attempt to improve the mechanical integrity of that interface. To do so, a calcium phosphate (CaP) film was deposited on CPP by a sol-gel process to prevent the accumulation of polyphosphates and associated inhibition of mineralization as the substrate degrades. Cartilage formed in vitro on the top surface of CaP-coated CPP by deep-zone chondrocytes was histologically and biochemically comparable to that formed on uncoated CPP. Furthermore, the mineral in the ZCC was similar in crystal structure, morphology and length to that formed on uncoated CPP and native articular cartilage. The generation of a ZCC at the cartilage-CPP interface led to a 3.3-fold increase in the interfacial shear strength of biphasic constructs. Improved interfacial strength of these constructs may be critical to their clinical success for the repair of large cartilage defects.
Asunto(s)
Calcificación Fisiológica/efectos de los fármacos , Fosfatos de Calcio/farmacología , Cartílago/efectos de los fármacos , Cartílago/fisiología , Condrogénesis/efectos de los fármacos , Polifosfatos/farmacología , Resistencia al Corte/efectos de los fármacos , Animales , Sustitutos de Huesos/farmacología , Cartílago/citología , Cartílago/ultraestructura , Bovinos , Células Cultivadas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Microscopía Electrónica de Rastreo , SolucionesRESUMEN
BACKGROUND: We developed a tissue-engineered biphasic cartilage bone substitute construct which has been shown to integrate with host cartilage and differs from autologous osteochondral transfer in which integration with host cartilage does not occur. QUESTIONS/PURPOSES: (1) Develop a reproducible in vitro model to study the mechanisms regulating tissue-engineered cartilage integration with host cartilage, (2) compare the integrative properties of tissue-engineered cartilage with autologous cartilage and (3) determine if chondrocytes from the in-vitro formed cartilage migrate across the integration site. METHODS: A biphasic construct was placed into host bovine osteochondral explant and cultured for up to 8 weeks (n = 6 at each time point). Autologous osteochondral implants served as controls (n = 6 at each time point). Integration was evaluated histologically, ultrastructurally, biochemically and biomechanically. Chondrocytes used to form cartilage in vitro were labeled with carboxyfluorescein diacetate which allowed evaluation of cell migration into host cartilage. RESULTS: Histologic assessment demonstrated that tissue-engineered cartilage integrated over time, unlike autologous osteochondral implant controls. Biochemically there was an increase in collagen content of the tissue-engineered implant over time but was well below that for native cartilage. Integration strength increased between 4 and 8 weeks as determined by a pushout test. Fluorescent cells were detected in the host cartilage up to 1.5 mm from the interface demonstrating chondrocyte migration. CONCLUSIONS: Tissue-engineered cartilage demonstrated improved integration over time in contrast to autologous osteochondral implants. Integration extent and strength increased with culture duration. There was chondrocyte migration from tissue-engineered cartilage to host cartilage. CLINICAL RELEVANCE: This in vitro integration model will allow study of the mechanism(s) regulating cartilage integration. Understanding this process will facilitate enhancement of cartilage repair strategies for the treatment of chondral injuries.
Asunto(s)
Cartílago Articular/cirugía , Movimiento Celular , Condrocitos/trasplante , Condrogénesis , Ingeniería de Tejidos , Animales , Fenómenos Biomecánicos , Cartílago Articular/metabolismo , Bovinos , Técnicas de Cultivo de Célula , Células Cultivadas , Condrocitos/metabolismo , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Ingeniería de Tejidos/métodos , Trasplante AutólogoRESUMEN
This study addresses the mechanical properties of calcium polyphosphate (CPP) structures formed by stacked layers using a powder-based solid freeform fabrication (SFF) technique. The mechanical properties of the 35% porous structures were characterized by uniaxial compression testing for compressive strength determination and diametral compression testing to determine tensile strength. Fracture cleavage surfaces were analyzed using scanning electron microscopy. The effects of the fabrication process on the microarchitecture of the CPP samples were also investigated. Results suggest that the orientation of the stacked layers has a substantial influence on the mechanical behavior of the SFF-made CPP samples. The samples with layers stacked parallel to the mechanical compressive load are 48% stronger than those with the layers stacked perpendicular to the load. However, the samples with different stacking orientations are not significantly different in tensile strength. The observed anisotropic mechanical properties were analyzed based on the physical microstructural properties of the CPP structures.
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Fosfatos de Calcio/química , Fenómenos Mecánicos , Polifosfatos/química , Anisotropía , Fuerza Compresiva , Ensayo de Materiales/métodos , Microscopía Electrónica de Rastreo , Tamaño de la Partícula , Porosidad , Polvos , Propiedades de Superficie , Resistencia a la Tracción , Microtomografía por Rayos XRESUMEN
A single application of cyclic compression (1kPa, 1Hz, 30min) to bioengineered cartilage results in improved tissue formation through sequential catabolic and anabolic changes mediated via cell shape changes that are regulated by α5ß1 integrin and membrane-type metalloprotease (MT1-MMP). To determine if calcium was involved in this process, the role of calcium in regulating cell shape changes, MT1-MMP expression and integrin activity in response to mechanical stimulation was examined. Stimulation-induced changes in cell shape and MT1-MMP expression were abolished by chelation of extracellular calcium, and this effect was reversed by re-introduction of calcium. Spreading was inhibited by blocking stretch-activated channels (with gadolinium), while retraction was prevented by blocking the L-Type voltage-gated channel (with nifedipine); both compounds inhibited MT1-MMP upregulation. Calcium A23187 ionophore restored cellular response further supporting a role for these channels. Calcium regulated the integrin-mediated signalling pathway, which was facilitated through Src kinase. Both calcium- and integrin-mediated pathways converged on ERK-MAPK in response to stimulation. While both integrins and calcium signalling mediate chondrocyte mechanotransduction, calcium appears to play the major regulatory role. Understanding the underlying molecular mechanisms involved in chondrocyte mechanotransduction may lead to the development of improved bioengineered cartilage.
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Calcio/metabolismo , Cartílago/citología , Condrocitos/metabolismo , Estrés Mecánico , Animales , Bioingeniería , Calcimicina/farmacología , Canales de Calcio Tipo L/metabolismo , Bovinos , Forma de la Célula , Células Cultivadas , Condrocitos/citología , Condrocitos/enzimología , Fuerza Compresiva/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Gadolinio/farmacología , Integrina alfa5beta1/metabolismo , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Fosforilación , Regulación hacia ArribaRESUMEN
The formation of biodegradable implants for use in osteosynthesis has been a major goal of biomaterials research for the past 2-3 decades. Self-reinforced polylactide systems represent the most significant success of this research to date, however, with elastic constants up to 12-15 GPa at best, they fail to provide the initial stiffness required of devices for stabilizing fractures of major load-bearing bones. Our research has investigated the use of calcium polyphosphate (CPP), an inorganic polymer in combination with polyvinyl-urethane carbonate (PVUC) organic polymers for such applications. Initial studies indicated that composite samples formed as interpenetrating phase composites (IPC) exhibited suitable as-made strength and stiffness, however, they displayed a rapid loss of properties when exposed to in vitro aging. An investigation to determine the mechanism of this accelerated in vitro degradation for the IPCs as well as to identify possible design changes to overcome this drawback was undertaken using a model IPC system. It was found that strong interfacial strength and minimal swelling of the PVUC are very important for obtaining and maintaining appropriate mechanical properties in vitro.
Asunto(s)
Fosfatos de Calcio/química , Carbonatos , Polifosfatos , Polivinilos , Uretano , Implantes Absorbibles , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Fosfatos de Calcio/metabolismo , Carbonatos/química , Carbonatos/metabolismo , Elasticidad , Fijación Interna de Fracturas/métodos , Humanos , Ensayo de Materiales , Polifosfatos/química , Polifosfatos/metabolismo , Polivinilos/química , Polivinilos/metabolismo , Cementos de Resina/química , Cementos de Resina/metabolismo , Resistencia al Corte , Estrés Mecánico , Resistencia a la Tracción , Uretano/química , Uretano/metabolismo , Soporte de PesoRESUMEN
A major challenge to the successful clinical application of bioengineered cartilage remains its integration to surrounding tissues upon implantation. One way to address this consists of generating biphasic constructs composed of articular cartilage formed in vitro on the top surface and integrated with the porous sub-surface of a bone substitute material - in the case of this study, calcium polyphosphate (CPP). To improve the mechanical integrity of the cartilage-bone substitute interface, attempts have been made to generate a zone of calcified cartilage (ZCC) within the CPP-cartilage interface, thereby mimicking the native joint architecture. The purpose of this work was to establish the effects of the degradation products of CPP on cartilage calcification in order to explain the observed positioning of a ZCC away from the interface junction. It was determined that polyphosphate released from the CPP accumulates within in vitro-grown cartilage and inhibits cartilage calcification in a concentration and chain length (i.e. molecular weight) dependent manner. It was found that this effect is transient as chondrocytes express exopolyphosphatases which hydrolyze polyphosphate to release orthophosphate. Hence, the generation of biphasic constructs with a properly located ZCC will require tailoring of CPP substrates with lower degradation rates or the upregulation of exopolyphosphatases by chondrocytes.
Asunto(s)
Sustitutos de Huesos/farmacología , Calcificación Fisiológica/efectos de los fármacos , Cartílago/efectos de los fármacos , Cartílago/fisiología , Fosfatos/farmacología , Ácidos Fosfóricos/farmacología , Polímeros/farmacología , Animales , Calcio/metabolismo , Bovinos , Condrocitos/citología , Condrocitos/efectos de los fármacos , Ácidos Fosfóricos/química , Polímeros/químicaRESUMEN
Solid freeform fabrication (SFF) enables the fabrication of anatomically shaped porous components required for formation of tissue engineered implants. This article reports on the characterization of a three-dimensional-printing method, as a powder-based SFF technique, to create reproducible porous structures composed of calcium polyphosphate (CPP). CPP powder of 75-150 microm was mixed with 10 wt % polyvinyl alcohol (PVA) polymeric binder, and used in the SFF machine with appropriate settings for powder mesh size. The PVA binder was eliminated during the annealing procedure used to sinter the CPP particles. The porous SFF fabricated components were characterized using scanning electron microscopy, micro-CT scanning, X-ray diffraction, and mercury intrusion porosimetry. In addition, mechanical testing was conducted to determine the compressive strength of the CPP cylinders. The 35 vol % porous structures displayed compressive strength on average of 33.86 MPa, a value 57% higher than CPP of equivalent volume percent porosity made through conventional gravity sintering. Dimensional deviation and shrinkage analysis was conducted to identify anisotropic factors required for dimensional compensation during SFF sample formation and subsequent sintering. Cell culture studies showed that the substrate supported cartilage formation in vitro, which was integrated with the top surface of the porous CPP similar to that observed when chondrocytes were grown on CPP formed by conventional gravity sintering methods as determined histologically and biochemically.
Asunto(s)
Materiales Biocompatibles , Fosfatos de Calcio , Cartílago/citología , Condrocitos/citología , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Bovinos , Células Cultivadas , Ensayo de Materiales/métodos , Alcohol Polivinílico , PorosidadRESUMEN
We have shown previously that cyclic compression of newly forming bioengineered cartilage in vitro results in improved tissue formation via changes in expression of matrix metalloproteases, such as, MT1-MMP (membrane type metalloprotease), and increased synthesis of matrix molecules. Several studies have suggested an association between MT1-MMP and integrins, which are known to influence cell shape. Thus, the objectives of this study were to determine the effect of compressive mechanical stimulation on cell shape and the role of integrins and MT1-MMP in mediating these changes and influencing matrix accumulation. Bovine articular chondrocytes were grown on the surface of a porous ceramic substrate for 72 h and then cyclically compressed for 30 min. Scanning electron microscopy and morphometric analysis demonstrated that compression induced a rapid, transient increase in chondrocyte spreading by 10 min, followed by a retraction to prestimulated size within 6 h. This was associated with increased accumulation of newly synthesized proteoglycans, as determined by quantification of radioisotope incorporation. Blocking the alpha5beta1 integrin, or its beta1 subunit, inhibited cell spreading and resulted in a partial inhibition of compression-induced increase in matrix accumulation. Knockdown of MT1-MMP expression partially inhibited cell retraction and resulted in a reduced matrix accumulation as well. These results suggest that chondrocyte spreading and retraction following cyclic compression in vitro regulates matrix accumulation. Understanding the mechanisms that regulate chondrocyte mechanotransduction may ultimately lead to the design of improved repair tissue for cartilage damage. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.