RESUMEN

The use of cell therapies in bone reconstruction has been the subject of extensive research. It is known that human bone marrow stromal cell (HBMSC) cultures contain a population of progenitor cells capable of differentiation towards the osteogenic lineage. In the present study, the correlation between the in vitro osteogenic potential of HBMSC cultures and their capacity to form bone in vivo was investigated. HBMSC cultures were established from 14 different donors. Fourth passage cells were examined for the expression of alkaline phosphatase (ALP), procollagen I (PCI) and osteopontin (OP), through flow cytometry and the effect of the osteogenic differentiation factor dexamethasone (Dex) on this expression was evaluated. In addition, the capacity of the cultures to induce in vivo bone formation was analysed by culturing the cells on porous hydroxyapatite (HA) scaffolds followed by subcutaneous implantation of these constructs in nude mice. Results showed expression of PCI, OP and ALP in all cultures, irrespective of the presence of Dex in the culture medium. Dex failed to have a significant effect on the expression of PCI and OP but it induced a consistent increase in the relative amount of cells expressing ALP. Nevertheless, although in vitro testing clearly indicated osteogenic potential in all cultures, HBMSC from six of the 14 tested donors did not form bone in vivo. The results, therefore, demonstrate that neither the expression of PCI, OP and ALP nor the absolute increase in Dex-stimulated ALP expression can as yet be used as predictive markers for in vivo bone formation by HBMSC. However, preliminary data indicate that not the absolute, but the relative increase in the percentage of ALP expressing cells caused by Dex stimulation may be related to the ability of the HBMSC to form bone.

Asunto(s)

Células de la Médula Ósea/citología , Sustitutos de Huesos , Huesos/citología , Osteogénesis , Células del Estroma/citología , Adulto , Anciano , Anciano de 80 o más Años , Fosfatasa Alcalina/biosíntesis , Animales , Técnicas de Cultivo de Célula/métodos , Linaje de la Célula , Células Cultivadas , Técnicas de Cultivo , Dexametasona/farmacología , Durapatita/química , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Osteocalcina/química , Osteopontina , Procolágeno/biosíntesis , Sialoglicoproteínas/biosíntesis , Células Madre/citología , Factores de Tiempo , Ingeniería de Tejidos

RESUMEN

The aim of this study was to evaluate two biodegradable polymeric systems as scaffolds for bone tissue engineering. Rat bone marrow cells were seeded and cultured for 1 week on two biodegradable porous polymeric systems, one composed of poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) and the other composed of cornstarch blended with poly(epsilon-caprolactone) (SPCL). Porous hydroxyapatite granules were used as controls. The ability of cells to proliferate and form extracellular matrix on these scaffolds was assessed by a DNA quantification assay and by scanning electron microscopy examination; their osteogenic differentiation was screened by the expression of alkaline phosphatase. In addition, the in vivo osteogenic potential of the engineered constructs was evaluated through ectopic implantation in a nude mouse model. Results revealed that cells were able to proliferate, differentiate, and form extracellular matrix on all materials tested. Moreover, all constructs induced abundant formation of bone and bone marrow after 4 weeks of implantation. The extent of osteogenesis (approximately 30% of void volume) was similar in all types of implants. However, the amount of bone marrow and the degree of bone contact were higher on HA scaffolds, indicating that the polymers still need to be modulated for higher osteoconductive capacity. Nevertheless, the findings suggest that both PEGT/PBT and SPCL systems are excellent candidates to be used as scaffolds for a cell therapy approach in the treatment of bone defects.

Asunto(s)

Células de la Médula Ósea/metabolismo , Sustitutos de Huesos , Tereftalatos Polietilenos/análogos & derivados , Polímeros , Ingeniería de Tejidos , Fosfatasa Alcalina/metabolismo , Animales , Células de la Médula Ósea/citología , ADN/metabolismo , Microscopía Electrónica de Rastreo , Ácidos Ftálicos , Poliésteres , Polietilenglicoles , Ratas , Almidón , Factores de Tiempo

RESUMEN

The development of cell therapy methods to confer osteogenic potential to synthetic bone replacement materials has become common during the last years. At present, in the bone tissue engineering field, two different approaches use patient own cultured osteogenic cells in combination with a scaffold material to engineer autologous osteogenic grafts. One of the approaches consists of seeding cells on a suitable biomaterial, after which the construct is ready for implantation. In the other approach, the seeded cells are further cultured on the scaffold to obtain in vitro formed bone (extracellular matrix and cells), prior to implantation. In the present study, we investigated the in vivo osteogenic potential of both methods through the implantation of porous hydroxyapatite (HA) scaffolds coated with a layer of in vitro formed bone and porous HA scaffolds seeded with osteogenic cells. Results showed that as early as 2 days after implantation, de novo bone tissue was formed on scaffolds in which an in vitro bone-like tissue was cultured, while it was only detected on the cell seeded implants from 4 days onwards. In addition, after 4 days of implantation statistical analysis revealed a significantly higher amount of bone in the bone-like tissue containing scaffolds as compared to cell seeded ones.

RESUMEN

At present, it is well known that populations of human bone marrow stromal cells (HBMSCs) can differentiate into osteoblasts and produce bone. However, the amount of cells with osteogenic potential that is ultimately obtained will still be dependent on both patient physiological status and culture system. In addition, to use a cell therapy approach in orthopedics, large cell numbers will be required and, as a result, knowledge of the factors affecting the growth kinetics of these cells is needed. In the present study we investigated the effect of dexamethasone stimulation on the in vivo osteogenic potential of HBMSCs. After a proliferation step, the cells were seeded and cultured on porous calcium phosphate scaffolds for 1 week, and then subcutaneously implanted in nude mice for 6 weeks, in order to evaluate their in vivo bone-forming ability. Furthermore, the effect of donor age on the proliferation rate of the cultures and their ability to induce in vivo bone formation was studied. In 67% of the assayed patients (8 of 12), the presence of dexamethasone in culture was not required to obtain in vivo bone tissue formation. However, in cultures without bone-forming ability or with a low degree of osteogenesis, dexamethasone increased the bone-forming capacity of the cells. During cellular proliferation, a significant age-related decrease was observed in the growth rate of cells from donors older than 50 years as compared with younger donors. With regard to the effect of donor age on in vivo bone formation, HBMSCs from several donors in all age groups proved to possess in vivo osteogenic potential, indicating that the use of cell therapy in the repair of bone defects can be applicable irrespective of patient age. However, the increase in donor age significantly decreased the frequency of cases in which bone formation was observed.

Asunto(s)

Células de la Médula Ósea , Sustitutos de Huesos , Prótesis e Implantes , Factores de Edad , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/ultraestructura , Técnicas de Cultivo de Célula , Dexametasona/farmacología , Durapatita , Glucocorticoides/farmacología , Humanos , Ratones , Ratones Desnudos , Microscopía Electrónica de Rastreo , Células del Estroma/efectos de los fármacos , Células del Estroma/ultraestructura , Donantes de Tejidos

RESUMEN

This paper describes an extensive biocompatibility evaluation of biodegradable starch-based materials aimed at orthopaedic applications as temporary bone replacement/fixation implants. For that purpose, a polymer (starch/ethylene vinyl alcohol blend, SEVA-C) and a composite of SEVA-C reinforced with hydroxyapatite (HA) particles, were evaluated in both in vitro and in vivo assays. For the in vitro analysis cell culture methods were used. The in vivo tissue reactions were evaluated in an intramuscular and intracortical bone implantation model on goats, using light and scanning electron microscopy. A computerized image analysis system was used to obtain histomorphometric data regarding bone contact and remodelling after 6 and 12 weeks of implantation. In both in vitro and in vivo models, the SEVA-C-based materials did not induce adverse reactions, which in addition to their bone-matching mechanical properties makes them promising materials for bone replacement fixation.

Asunto(s)

Materiales Biocompatibles/química , Sustitutos de Huesos/química , Durapatita/química , Ortopedia , Polivinilos/química , Almidón/química , Animales , Materiales Biocompatibles/toxicidad , Biodegradación Ambiental , Sustitutos de Huesos/toxicidad , Huesos/cirugía , Línea Celular/efectos de los fármacos , Durapatita/toxicidad , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Reacción a Cuerpo Extraño/etiología , Cabras , Humanos , Células L/efectos de los fármacos , Ensayo de Materiales , Ratones , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Músculo Esquelético/cirugía , Oseointegración , Polivinilos/toxicidad , Prótesis e Implantes , Almidón/efectos adversos , Almidón/toxicidad

RESUMEN

It has been shown that blends of starch with a poly(ethylene-vinyl-alcohol) copolymer, EVOH, designated as SEVA-C, present an interesting combination of mechanical, degradation and biocompatible properties, specially when filled with hydroxyapatite (HA). Consequently, they may find a range of applications in the biomaterials field. This work evaluated the influence of HA fillers and of blowing agents (used to produce porous architectures) over the viscoelastic properties of SEVA-C polymers, as seen by dynamic mechanical analysis (DMA), in order to speculate on their performances when withstanding cyclic loading in the body. The composite materials presented a promising performance under dynamic mechanical solicitation conditions. Two relaxations were found being attributed to the starch and EVOH phases. The EVOH relaxation process may be very useful in vivo improving the implants performance under cyclic loading. DMA results also showed that it is possible to produce SEVA-C compact surface/porous core architectures with a mechanical performance similar to that of SEVA-C dense materials. This may allow for the use of these materials as bone replacements or scaffolds that must withstand loads when implanted.

RESUMEN

Bone marrow is known to contain a population of osteoprogenitor cells that can go through complete differentiation when cultured in a medium containing appropriate bioactive factors. In this study, porous particles of a calcium phosphate material were seeded with adult human bone marrow cells in the second passage. After an additional culture period of 1 wk in the particles, these hybrid constructs were subcutaneouslly implanted in nude mice with a survival period of 4 wk. The cell seeding densities range from 0-200 000 cells per particle and the cell culture system was designed to investigate the single and combined effects of dexamethasone and recombinant human bone morphogenetic protein 2 (rhBMP-2). The hybrid "material/tissue" constructs were processed for histology and the amount of de novo bone formation was quantified, for each culture condition, by histomorphometric techniques. The relative percentage of mineralized bone formation reached a maximal value of 19.77+/-5.06, for samples cultured in the presence of rhBMP-2 and with a seeding density of 200 000 cells/particle, compared to 0.52+/-0.45 for samples in which no cells had been cultured and had been incubated in culture medium supplemented with Dex and rhBMP-2. For the tested conditions and for the low cell numbers used in this study, rhBMP-2 proved to be an essential bioactive factor to obtain in vivo bone formation by our culture system. The results from this study prove the potential of cultured adult human bone marrow cells to initiate and accelerate de novo bone formation after transplantation into an ectopic site.