F.C. Oner

Bone Tissue-Engineered Implants Using Human Bone Marrow Stromal Cells: Effect of Culture Conditions and Donor Age

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.

Viable osteogenic cells are obligatory for tissue-engineered ectopic bone formation in goats

In this study we investigated the bone-forming capacity of tissue-engineered (TE) constructs implanted ectopically in goats. As cell survival is questionable in large animal models, we investigated the significance of vitality, and thus whether living cells instead of only the potentially osteoinductive extracellular matrix are required to achieve bone formation. Vital TE constructs of porous hydroxyapatite (HA) covered with differentiated bone marrow stromal cells (BMSCs) within an extracellular matrix (ECM) were compared with identical constructs that were devitalized before implantation. The devitalized implants did contain the potentially osteoinductive ECM. Furthermore, we evaluated HA impregnated with fresh bone marrow and HA only. Two different types of HA granules with a volume of approximately 40 microm were investigated: HA70/800, a microporous HA with 70% interconnected macroporosity and an average pore size of 800 microm, and HA60/400, a smooth HA with 60% interconnected macropores and an average size of 400 microm. Two granules of each type were combined and then treated as a single unit for cell seeding, implantation, and histology. The tissue-engineered samples were obtained by seeding culture-expanded goat BMSCs on the HA and subsequently culturing these constructs for 6 days to allow cell differentiation and ECM formation. To devitalize, TE constructs were frozen in liquid nitrogen according to a validated protocol. Fresh bone marrow impregnation was performed perioperatively (4 mL per implant unit). All study groups were implanted in bilateral paraspinal muscles. Fluorochromes were administered at three time points to monitor bone mineralization. After 12 weeks the units were explanted and analyzed by histology of nondecalcified sections. Bone formation was present in all vital tissue-engineered implants. None of the other groups showed any bone formation. Histomorphometry indicated that microporous HA70/800 yielded more bone than did HA60/400. Within the newly formed bone, the fluorescent labels showed that mineralization had occurred before 5 weeks of implantation and was directed from the HA surface toward the center of the pores. In conclusion, tissue-engineered bone formation in goats can be achieved only with viable constructs of an appropriate scaffold and sufficient BMSCs.

Relation between in vitro and in vivo osteogenic potential of cultured human bone marrow stromal cells

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.

Bone tissue engineering and spinal fusion: the potential of hybrid constructs by combining osteoprogenitor cells and scaffolds

In this paper, we discuss the current knowledge and achievements on bone tissue engineering with regard to spinal fusion and highlight the technique that employs hybrid constructs of porous scaffolds with bone marrow stromal cells. These hybrid constructs potentially function in a way comparable to the present golden standard, the autologous bone graft, which comprises besides many other factors, a construct of an optimal biological scaffold with osteoprogenitor cells. However, little is known about the role of the cells in autologous grafts, and especially survival of these cells is questionable. Therefore, more research will be needed to establish a level of functioning of hybrid constructs to equal the autologous bone graft. Spinal fusion models are relevant because of the increasing demand for graft material related to this procedure. Furthermore, they offer a very challenging environment to further investigate the technique. Anterior and posterolateral animal models of spinal fusion are discussed together with recommendations on design and assessment of outcome parameters.

Application and limitations of chloromethyl-benzamidodialkylcarbocyanine for tracing cells used in bone Tissue engineering.

Bone tissue engineering has the potential to provide us with an autologous bone substitute. Despite extensive research to optimize the technique, little is known about the survival and function of the cells after implantation. To monitor the cells, in vivo labeling is the method of choice. In this study we investigated the use of the fluorescent membrane marker chloromethyl-benzamidodialkylcarbocyanine (CM-Dil) to label cells used in bone tissue engineering. When applying label concentrations up to 50 microM, cells could be labeled efficiently without negative effects on cell vitality, proliferation, or bone-forming capacity. Porous hydroxyapatite scaffolds were seeded with labeled cells, and up to 6 weeks after implantation in nude mice cells could be traced inside tissue-engineered bone. However, contrary to other reports concerning intramembranous labels, transfer of the label from labeled to unlabeled cells was detected. Transfer occurred both in vitro and in vivo between vital cells and between dead and living cells. To determine when in vivo label transfer happened, devitalized, labeled constructs were implanted for various time periods in nude mice. The presence of vital labeled cells inside these constructs, when evaluated at different implantation periods, indicated transfer of the label. Transfer occurred at 7 days postimplantation when 40 microM label was applied, whereas 10 microM labeled constructs showed transfer 10 days after implantation. These findings indicate that CM-Dil label is useful for in vivo tracing of cells for follow-up periods up to 10 days. This makes the label particularly useful for cell survival studies in tissue-engineered implants.

Bone tissue engineering for spine fusion: An experimental study on ectopic and orthotopic implants in rats

Alternatives to the use of autologous bone as a bone graft in spine surgery are needed. The purpose of this study was to examine tissue-engineered bone constructs in comparison with control scaffolds without cells in a posterior spinal implantation model in rats. Syngeneic bone marrow cells were cultured in the presence of bone differentiation factors and seeded on porous hydroxyapatite particles. Seven rats underwent a posterior surgical approach, in which scaffolds with (five rats) or without cells (two rats) were placed on both sides of the lumbar spine. In addition, separate scaffolds were inserted intramuscularly and subcutaneously during the surgical procedure. After 4 weeks, all rats were killed and examined radiographically, by manual palpation of the excised spine and histologically for signs of bone formation or spine fusion. All rats that received cell-seeded scaffolds showed newly formed bone in all three locations, whereas none of the locations in the control rats showed bone formation. The results of this study support the concept of developing tissue-engineering techniques in posterior spine fusion as an alternative to autologous bone.

13 Het hip-spine-dilemma

Bij 10-15% van de patienten met lage rugpijn en pijn rondom het heupgebied kan er sprake zijn van gecombineerde pathologie van de lumbale wervelkolom en de heup, zodat we kunnen spreken van een ‘hip-spine’-dilemma. In de meeste gevallen leveren een grondige anamnese, lichamelijk onderzoek en aanvullende rontgendiagnostiek voldoende informatie op om tot een diagnose te komen. Zie voor de klassieke presentaties van de hip-spine-problematiek tabel 13.1.