Pierre Hardouin

Qualitative and quantitative study of human osteoblast adhesion on materials with various surface roughnesses

We quantitatively evaluated the adhesion of human osteoblasts on orthopedic metallic substrates (Ti6Al4V alloy) with various surface roughnesses at several times after inoculation and studied its correlation with qualitative changes in the expression of adhesion proteins and with parameters extensively describing the surface topographies. Cells were orientated in a parallel order on polished surfaces. This orientation was not affected by residual grooves after polishing. On sandblasted surfaces the cells never attained confluence and had a stellate shape, and the cell layer had no particular organization. Extracellular matrix (fibronectin, type I collagen, osteopontin) and cytoskeletal protein (actin, vinculin) orientation reflected the cell layer organization. In our experiment human osteoblasts expressed alpha3beta1 integrin but not alpha2beta1 integrin. In addition to currently analyzed roughness magnitude parameters, we calculated roughness organization parameters (fractal dimension parameters) of the substrates. We observed lower adhesion and proliferation on less organized surfaces (i.e., sandblasted ones). The significant statistical correlation observed between fractal dimension parameters (describing surface roughness organization) and cell parameters adds a new concept to the studies of substratum roughness influence on cell behavior. An attempt at modelization of the cell-surface interaction was made that includes the influence of fractal dimensions parameters.

ROLE OF INTERCONNECTIONS IN POROUS BIOCERAMICS ON BONE RECOLONIZATION IN VITRO AND IN VIVO

The interconnections in a porous biomaterial are the pathways between the pores. They conduct cells and vessels between pores. Thus they favour bone ingrowth inside ceramics. The aim of our study was to determine the effect on bone ingrowth of interconnections in two ceramics: hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) with the same porosity of about 50% and a mean pores size of 100–300 μm and a mean interconnection size of 30–100 μm. In vitro, four discs for osteoblast culture were studied after 14 and 28 days of incubation. The results show that human osteoblasts can penetrate interconnections over 20 μm in size, and colonize and proliferate inside macropores, but the most favourable size is over 40 μm. In vivo, eight cylinders were implanted in the middle shaft of both rabbit femurs for 12 or 24 weeks. The histomorphometric results show that interconnections in porous ceramics favour bone ingrowth inside the macropores. In the HA group the rate of calcification and bone ingrowth do not differ, and chondroid tissue is observed inside pores. But in β-TCP, the calcification rate and the bone ingrowth increased significantly. At week 12 significant correlation between new bone ingrowth and the size of the interconnections is observed between new bone ingrowth and the density of pores. In conclusion we notice that in vivo a 20 μm interconnection size only allows cell penetration and chondroid tissue formation; however the size of the interconnections must be over 50 μm to favour new bone ingrowth inside the pores. We propose the concept of “interconnection density” which expresses the quantity of links between pores of porous materials. It assures cell proliferation and differentiation with blood circulation and extracellular liquid exchange. In resorbable materials, pore density and interconnection density are more important than their size, contrary to unresorbable materials in which the sizes and the densities are equally important.

Comparative study of tissue reactions to calcium phosphate ceramics among cancellous, cortical, and medullar bone sites in rabbits

In order to understand the influence of the implantation site on bone biomaterial evaluation, we implanted cylinders of HA and beta-TCP ceramics in the femoral diaphysis and condyle of rabbits. After 3, 8, 12, and 24 weeks of implantation, histological investigation and histomorphometry were performed on undecalcified samples. Our results show that spontaneous bone healing in the empty cavities is significantly different (p < 0.05) between cortical (SBH > 80%) and cancellous bone sites (SBH < 31%) and that no new bone is formed in marrow tissue. For both porous ceramics, the highest osteogenesis was obtained in the cortical site. Osteogenesis was intermediate in the cancellous site and weak in the medullar site. The material biodegradation was the strongest in the medullar site and higher in the cancellous site than in the cortical site. Both activities were better in the beta-TCP than in the HA (p > 0.05). The marrow tissue presents a foreign-body reaction more reliable, sensitive, and durable than other bone tissues. Therefore, the cancellous bone site is a good site for evaluation of the biofunctionality of biomaterials because of the equilibrium of the osteogenesis and the biodegradation activities, but marrow tissue seems to be better for testing material biocompatibility in vivo.

Porous HA ceramic for bone replacement: role of the pores and interconnections - experimental study in the rabbit.

Hydroxyapatite (HA) porous ceramics are increasingly used in biomedical applications. Their physical characteristics, such as porous volume, require perfect control of the pore shape, as well as the number and the size of their interconnections.The aim of our study was to evaluate a new HA ceramic using polymethylmethacrylate microbeads (PMMA) as the porous agent. Four interconnection sizes (30, 60, 100 and 130 μm) with a 175–260 μm pore size and three pore sizes (175–260, 260–350 and 350–435 μm) for a 130 μm interconnection size were tested. Various HA implants were appraised by microscopic evaluation in a 4.6×10 mm rabbit femur cancellous bone defect 12 weeks after implantation. The best osteoconduction result was obtained in the center of the ceramic by means of a 130 μm interconnection size and a 175–260 μm mean pore size. Bone formation obtained within the pores was double that obtained in our previous study where naphtalen microbeads were used as the porous agents.© 2001 Kluwer Academic Publishers

Influence of Adipokines and Ghrelin on Bone Mineral Density and Fracture Risk: A Systematic Review and Meta-Analysis

CONTEXT Adipokines (leptin, adiponectin, resistin, visfatin) and ghrelin may be implicated in bone metabolism. OBJECTIVE The aim was to perform an overview of the influence of blood levels of adipokines or ghrelin on bone mineral density (BMD), osteoporotic status, and fracture risk in healthy men and women. DATA SOURCES We reviewed Medline, Embase, and Cochrane databases up to March 2010 and abstracts of international meetings from 2008 to 2009. STUDY SELECTION Fifty-nine studies meeting the inclusion criteria (healthy men or women evaluated for both BMD or fracture risk and at least one adipokine and/or ghrelin levels) were analyzed in the systematic review of the 931 references found in the electronic databases. DATA EXTRACTION We used a predefined extraction sheet. DATA SYNTHESIS We performed meta-analyses using the method of the inverse of the variance estimated pooled correlations between adipokines/ghrelin and BMD. Inverse correlations between adiponectin levels and BMD were highlighted (pooled r from -0.14 to -0.4). Leptin is positively associated to BMD, especially in postmenopausal women (pooled r from 0.18 to 0.33). High levels of leptin were reported to be predictive of low risk of fractures, whereas high levels of adiponectin may be predictive of high risk of vertebral fractures in men only. No discriminative capacity of osteoporotic status was reported. We found no convincing data to support an association between resistin, visfatin, or ghrelin and BMD. CONCLUSION Adiponectin is the most relevant adipokine negatively associated with BMD, independent of gender and menopausal status. Inconsistent associations between adipokines and BMD are probably confounded by body composition, in particular fat mass parameters.

TNF-α and IL-1β inhibit RUNX2 and collagen expression but increase alkaline phosphatase activity and mineralization in human mesenchymal stem cells

AIMS Joint inflammation leads to bone erosion in rheumatoid arthritis (RA), whereas it induces new bone formation in spondyloarthropathies (SpAs). Our aims were to clarify the effects of tumour necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) on osteoblast differentiation and mineralization in human mesenchymal stem cells (MSCs). MAIN METHODS In MSCs, expression of osteoblast markers was assessed by real-time PCR and ELISA. Activity of tissue-nonspecific alkaline phosphatase (TNAP) and mineralization were determined by the method of Lowry and alizarin red staining respectively. Involvement of RUNX2 in cytokine effects was investigated in osteoblast-like cells transfected with a dominant negative construct. KEY FINDINGS TNF-alpha (from 0.1 to 10 ng/ml) and IL-1beta (from 0.1 to 1 ng/ml) stimulated TNAP activity and mineralization in MSCs. Addition of 50 ng/ml of IL-1 receptor antagonist in TNF-alpha-treated cultures did not reverse TNF-alpha effects, indicating that IL-1 was not involved in TNF-alpha-stimulated TNAP activity. Both TNF-alpha and IL-1beta decreased RUNX2 expression and osteocalcin secretion, suggesting that RUNX2 was not involved in mineralization. This hypothesis was confirmed in osteoblast-like cells expressing a dominant negative RUNX2, in which TNAP expression and activity were not reduced. Finally, since mineralization may merely rely on increased TNAP activity in a collagen-rich tissue, we investigated cytokine effects on collagen expression, and observed that cytokines decreased collagen expression in osteoblasts from MSC cultures. SIGNIFICANCE The different effects of cytokines on TNAP activity and collagen expression may therefore help explain why inflammation decreases bone formation in RA whereas it induces ectopic ossification from collagen-rich entheses during SpAs.

In vitro control of human bone marrow stromal cells for bone tissue engineering.

For the clinical application of cultured human mesenchymal stem cells (MSCs), cells must have minimal contact with fetal calf serum (FCS) because it might be a potential vector for contamination by adventitious agents. The use of human plasma and serum for clinical applications also continues to give rise to considerable concerns with respect to the transmission of known and unknown human infectious agents. With the objective of clinical applications of cultured human MSCs, we tested the ability of autologous plasma, AB human serum, FCS, and artificial serum substitutes containing animal-derived proteins (Ultroser G) or vegetable-derived proteins (Prolifix S6) to permit their growth and differentiation in vitro. To conserve as much autologous plasma as possible, we attempted to mix it at decreasing concentrations with the serum substitute containing vegetable-derived mitogenic factors. Under control conditions, by day 10 all the fibroblast colony-forming units (CFU-Fs) were alkaline phosphatase (ALP) positive. However, their number and size were highly variable among donors. Better CFU-F formation was obtained with Ultroser G, and with human AB serum and autologous plasma mixed at, respectively, 5 and 1% with Prolifix S6. The effects of these mixtures on CFU-F formation demonstrate synergy, with the human serum or plasma supplying the factors that favor differentiation of MSCs while Prolifix S6 supplies the mitogenic factors. Finally, we demonstrated the possibility of controlling human MSC growth and differentiation in vitro. Notably, by means of a minimal quantity of human serum or human plasma mixed with a new serum substitute containing vegetable-derived proteins, we displayed growth and differentiation of human MSCs comparable to that obtained with FCS or serum substitutes containing animal-derived proteins. These results will have crucial significance for future applications of cultured human MSCs in bone tissue engineering.

Should percutaneous vertebroplasty be used to treat osteoporotic fractures? An update.

Acrylic cement vertebroplasty is being increasingly used to treat osteoporotic vertebral compression fractures (VCFs), although no controlled studies supporting this trend have been published. Vertebroplasty remains controversial as a treatment for osteoporotic fractures because it is a local response to a systemic disease and because the pain caused by osteoporotic fractures usually subsides within a few days or weeks. Current data suggest that pain severity may decrease by half, on average, in 90-100% of patients. Although vertebroplasty is usually well tolerated, serious neurological complications have been reported in a few patients. The most common adverse event is nerve root pain, usually caused by leakage of the cement into the intervertebral foramen. Whether vertebroplasty is followed by an increased risk of osteoporotic fractures in the adjacent vertebras remains unclear. Resorbable cements are being developed and may provide better results than the acrylic cements used today. At present, acrylic cement vertebroplasty to treat osteoporotic VCFs is appropriate in only a minority of patients selected carefully by a multidisciplinary team including a rheumatologist.

Human osteoblast adhesion on titanium alloy, stainless steel, glass and plastic substrates with same surface topography.

Osteoblast adhesion on materials will depend on the surface aspects of materials which may be described according to their surface chemistry, surface topography or surface energy. To separate the effects of roughness and composition of materials on osteoblast response, we chose to compare substrates with various surface composition but with the same smooth surface. Ti6Al4V alloy, stainless steel, glass and standard tissue culture polystyrene were tested. Adhesion was evaluated using specific antibodies against adhesion proteins and by a quantitative cell detachment assay. After 1, 7 and 14 days, cells expressed extracellularly fibronectin fibers, and intracellularly type I collagen and osteopontin. Vinculin-labeled focal contacts were visible on all materials but were more frequent on glass and stainless steel surfaces. β1-integrin subunit-labeled patches were visible on all surfaces at each delay. The quantitative cell detachment assay showed few differences between materials. Adhesion was higher on metallic substrates although cell proliferation was higher on glass and stainless steel compared to tissue culture polystyrene and Ti6Al4V alloy. Substrates with various surface composition but with the same surface topography did not induce significant differences of adhesion although cell proliferation was variable.

Bone Marrow Adipose Tissue: To Be or Not To Be a Typical Adipose Tissue?

Bone marrow adipose tissue (BMAT) emerges as a distinct fat depot whose importance has been proved in the bone–fat interaction. Indeed, it is well recognized that adipokines and free fatty acids released by adipocytes can directly or indirectly interfere with cells of bone remodeling or hematopoiesis. In pathological states, such as osteoporosis, each of adipose tissues – subcutaneous white adipose tissue (WAT), visceral WAT, brown adipose tissue (BAT), and BMAT – is differently associated with bone mineral density (BMD) variations. However, compared with the other fat depots, BMAT displays striking features that makes it a substantial actor in bone alterations. BMAT quantity is well associated with BMD loss in aging, menopause, and other metabolic conditions, such as anorexia nervosa. Consequently, BMAT is sensed as a relevant marker of a compromised bone integrity. However, analyses of BMAT development in metabolic diseases (obesity and diabetes) are scarce and should be, thus, more systematically addressed to better apprehend the bone modifications in that pathophysiological contexts. Moreover, bone marrow (BM) adipogenesis occurs throughout the whole life at different rates. Following an ordered spatiotemporal expansion, BMAT has turned to be a heterogeneous fat depot whose adipocytes diverge in their phenotype and their response to stimuli according to their location in bone and BM. In vitro, in vivo, and clinical studies point to a detrimental role of BM adipocytes (BMAs) throughout the release of paracrine factors that modulate osteoblast and/or osteoclast formation and function. However, the anatomical dissemination and the difficulties to access BMAs still hamper our understanding of the relative contribution of BMAT secretions compared with those of peripheral adipose tissues. A further characterization of the phenotype and the functional regulation of BMAs are ever more required. Based on currently available data and comparison with other fat tissues, this review addresses the originality of the BMAT with regard to its development, anatomy, metabolic properties, and response to physiological cues.