GLP-2 administration in ovariectomized mice enhances collagen maturity but did not improve bone strength.

Osteoporosis and bone fragility are progressing worldwide. Previous published literature reported a possible beneficial role of gut hormones, and especially glucagon-like peptide-2 (GLP-2), in modulating bone remodeling. As now (Gly2)GLP-2 is approved in the treatment of short bowel syndrome, we thought to investigate whether such molecule could be beneficial in bone fragility. MC3T3 and Raw 264.7 were cultured in presence of ascending concentrations of (Gly2)GLP-2. Collagen crosslinks, maturity, lysyl oxidase activity and osteoclastogenesis were then analyzed. Furthermore, (Gly2)GLP-2, at the clinical approved dose of 50 µg/kg/day, was also administered to ovariectomized Balb/c mice for 8 weeks. Hundred µg/kg zoledronic acid (once iv) was also used as a positive comparator. Bone strength, microarchitectures and bone tissue composition were analyzed by 3-point bending, compression test, microCT and Fourier transform infrared imaging, respectively. In vitro, (Gly2)GLP-2 was potent in enhancing bone matrix gene expression but also to dose-dependently enhanced collagen maturation and post-processing. (Gly2)GLP-2 was also capable of reducing dose-dependently the number of newly generated osteoclasts. However, in vivo, (Gly2)GLP-2 was not capable of improving neither bone strength, at the femur diaphysis or lumbar vertebrae, nor bone microarchitecture. On the other hand, at the tissue material level, (Gly2)GLP-2 significantly enhances collagen maturity and reduce phosphate/amide ratio. Overall, this study highlights that despite modification of bone tissue composition, (Gly2)GLP-2, at the clinical approved dose of 50 µg/kg/day, did not provide real beneficial effects in improving bone strength in a mouse model of bone fragility. Further studies are recommended to validate the best dose and regimen of administration to significantly enhance bone strength.

Polyhydroxyalkanoate (PHBV) fibers obtained by a wet spinning method: Good in vitro cytocompatibility but absence of in vivo biocompatibility when used as a bone graft.

Polyhydroxyalkanoates (PHAs) are biomaterials widely investigated for tissue-engineering applications. In this regard, we describe a method to prepare fibers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by a wet-spinning technique. Polymer fibers were used to test the cytocompatibility of the material in vitro. We have investigated their behavior in vitro in presence of the osteoblast-like (SaOs2) and macrophage (J774.2) cell lines. The PHBV fibers used were 100-200µm in diameter and offered a large surface for cell adhesion, similar to that they encounter when apposed onto a bone trabeculae. The fiber surface possessed a suitable roughness, a factor known to favor the adherence of cells, particularly osteoblasts. PHBV fibers were degraded in vitro by J774.2 cells as erosion pits were observable by transmission electron microscopy. The fibers were also colonisable by SaOs2 cells, which can spread and develop onto their surface. However, despite this good cytocompatibility observed in vitro, implantation in a bone defect drilled in rabbit femoral condyles showed that the material was only biotolerated without any sign of osteoconduction or degradation in vivo. We can conclude that PHBV is cytocompatible but is not suitable to be used as a bone graft as it does not favor osteoconduction and is not resorbed by bone marrow macrophages.

Metaplastic woven bone in bone metastases: A Fourier-transform infrared analysis and imaging of bone quality (FTIR).

Most osteolytic tumors are in fact mixed and contain an osteoblastic component associated with the predominant osteolytic areas. This metaplastic woven bone is always evidenced by histological analysis even in the absence of radiological expression. Metaplastic bone formation reflects the activation of new osteoblasts coming from the stimulation of the dormant lining cells. Twelve patients with secondary metastases of the iliac crest evidenced by hot spots on a 99Tc-MBP san were diagnosed by histomorphometry on bone biopsies. Fourier Transformed InfraRed analysis and Imaging (FTIRI) was used on 4µm thick sections of undecalcified bone. The mineralization degree, carbonate substitution, crystallinity and the cross-links ratio of collagen (1660/1690cm-1 bands) were determined. The matrix characteristics were analyzed and imaged in the pre-existing residual bone and in the metaplastic woven bone in the vicinity of the tumor cells. FTIRI provided images of the phosphate, amide and combination of peak ratio after having selected the peaks of interest. In addition, the matrix properties can be measured and compared between the old and newly-formed bones. Woven bone appeared poorly calcified with a low phosphate/amide ratio (P=0.03) crystallinity (P<0.0001) and carbonate substitution (P=0.003). Collagen was less mature as evidenced by lower cross-links (P=0.01). Woven bone associated with bone metastasis appears poorly mineralized and rapidly elaborated by osteoblasts. The collagenous phase of the bone matrix has a low level of reticulation. FTIRI is a powerful tool to measure and visualize the various components of the bone matrix in human diseases.

Strontium ranelate stimulates trabecular bone formation in a rat tibial bone defect healing process.

Strontium ranelate treatment is known to prevent fractures. Here, we showed that strontium ranelate treatment enhances bone healing and affects bone cellular activities differently in intact and healing bone compartments: Bone formation was increased only in healing compartment, while resorption was reduced in healing and normal bone compartments. INTRODUCTION: Systemic administration of strontium ranelate (SrRan) accelerates the healing of bone defects; however, controversy about its action on bone formation remains. We hypothesize that SrRan could affect bone formation differently in normal mature bone or in the bone healing process. METHODS: Proximal tibia bone defects were created in 6-month-old female rats, which orally received SrRan (625 mg/kg/day, 5/7 days) or vehicle (control groups) for 4, 8, or 12 weeks. Bone samples were analyzed by micro-computed tomography and histomorphometry in various regions, i.e., metaphyseal 2nd spongiosa, a region close to the defect, within the healing defect and in cortical defect bridging region. Additionally, we evaluated the quality of the new bone formed by quantitative backscattered electron imaging and by red picosirius histology. RESULTS: Healing of the bone defect was characterized by a rapid onset of bone formation without cartilage formation. Cortical defect bridging was detected earlier compared with healing of trabecular defect. In the healing zone, SrRan stimulated bone formation early and laterly decreased bone resorption improving the healing of the cortical and trabecular compartment without deleterious effects on bone quality. By contrast, in the metaphyseal compartment, SrRan only decreased bone resorption from week 8 without any change in bone formation, leading to little progressive increase of the metaphyseal trabecular bone volume. CONCLUSIONS: SrRan affects bone formation differently in normal mature bone or in the bone healing process. Despite this selective action, this led to similar increased bone volume in both compartments without deleterious effects on the newly bone-formed quality.

Interplay between bone and incretin hormones: A review.

Bone is a tissue with multiple functions that is built from the molecular to anatomical levels to resist and adapt to mechanical strains. Among all the factors that might control the bone organization, a role for several gut hormones called "incretins" has been suspected. The present review summarizes the current evidences on the effects of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) in bone physiology.

Aluminum and bone: Review of new clinical circumstances associated with Al(3+) deposition in the calcified matrix of bone.

Several decades ago, aluminum encephalopathy associated with osteomalacia has been recognized as the major complication of chronic renal failure in dialyzed patients. Removal of aluminum from the dialysate has led to a disappearance of the disease. However, aluminum deposit occurs in the hydroxyapatite of the bone matrix in some clinical circumstances that are presented in this review. We have encountered aluminum in bone in patients with an increased intestinal permeability (coeliac disease), or in the case of prolonged administration of aluminum anti-acid drugs. A colocalisation of aluminum with iron was also noted in cases of hemochromatosis and sickle cell anemia. Aluminium was also identified in a series of patients with exostosis, a frequent benign bone tumor. Corrosion of prosthetic implants composed of grade V titanium (TA6V is an alloy containing 6% aluminum and 4% vanadium) was also observed in a series of hip or knee revisions. Aluminum can be identified in undecalcified bone matrix stained by solochrome azurine, a highly specific stain allowing the detection of 0.03 atomic %. Colocalization of aluminum and iron does not seem to be the fruit of chance but the cellular and molecular mechanisms are still poorly understood. Histochemistry is superior to spectroscopic analyses (EDS and WDS in scanning electron microscopy).

Factors associated with an increased risk of vertebral fracture in monoclonal gammopathies of undetermined significance.

Monoclonal gammopathies of undetermined significance (MGUS) have been shown to be associated with an increased risk of fractures. This study describes prospectively the bone status of MGUS patients and determines the factors associated with vertebral fracture. We included prospectively 201 patients with MGUS, incidentally discovered, and with no known history of osteoporosis: mean age 66.6±12.5 years, 48.3% women, 51.7% immunoglobulin G (IgG), 33.3% IgM and 10.4% IgA. Light chain was kappa in 64.2% patients. All patients had spinal radiographs and bone mineral density measurement in addition to gammopathy assessment. At least one prevalent non-traumatic vertebral fracture was discovered in 18.4% patients and equally distributed between men and women. Fractured patients were older, had a lower bone density and had also more frequently a lambda light chain isotype. Compared with patients with κ light chain, the odds ratio of being fractured for patients with λ light chain was 4.32 (95% confidence interval 1.80-11.16; P=0.002). These results suggest a high prevalence of non-traumatic vertebral fractures in MGUS associated with lambda light chain isotype and not only explained by low bone density.

Comparison between quantitative X-ray imaging, dual energy X-ray absorptiometry and microCT in the assessment of bone mineral density in disuse-induced bone loss.

OBJECTIVES: We recently introduced a new methodology called quantitative X-ray imaging (qXRI) to investigate bone mineral density in isolated rodent bones. The aims of the present study were to compare DXA and microCT with qXRI in a rat model of disuse osteoporosis. METHODS: Fourteen Copenhagen rats were injected with a single dose of botulinum toxin (BTX - 2 UI) in the right Mus quadriceps femoris. The left hindlimb serves as control. Areal BMD and vBMD were determined with a Hologic Discovery-W device and a Skyscan 1172 microcomputed tomograph (microCT). Absorbing material density (AMD) was determined on digitized X-ray images obtained with a Faxitron M020 device. RESULTS: All three methods highlighted significant lower values for aBMD, vBMD and AMD in trabecular and cortical bone in the BTX-injected side. In trabecular bone, aBMD, vBMD and AMD were significantly correlated with BV/TV. In cortical bone, only aBMD and vBMD were significantly correlated with cortical bone mass On the other hand, only AMD was significantly correlated with the mechanical parameters bending strength and bending modulus. CONCLUSIONS: qXRI is a rapid and cheap method to assess trabecular bone mass in isolated rodent bones and can be used as a surrogate for the densitometry of small animals.

Double incretin receptor knock-out (DIRKO) mice present with alterations of trabecular and cortical micromorphology and bone strength.

UNLABELLED: A role for gut hormone in bone physiology has been suspected. We evidenced alterations of microstructural morphology (trabecular and cortical) and bone strength (both at the whole-bone--and tissue-level) in double incretin receptor knock-out (DIRKO) mice as compared to wild-type littermates. These results support a role for gut hormones in bone physiology. INTRODUCTION: The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), have been shown to control bone remodeling and strength. However, lessons from single incretin receptor knock-out mice highlighted a compensatory mechanism induced by elevated sensitivity to the other gut hormone. As such, it is unclear whether the bone alterations observed in GIP or GLP-1 receptor deficient animals resulted from the lack of a functional gut hormone receptor, or by higher sensitivity for the other gut hormone. The aims of the present study were to investigate the bone microstructural morphology, as well as bone tissue properties, in double incretin receptor knock-out (DIRKO) mice. METHODS: Twenty-six-week-old DIRKO mice were age- and sex-matched with wild-type (WT) littermates. Bone microstructural morphology was assessed at the femur by microCT and quantitative X-ray imaging, while tissue properties were investigated by quantitative backscattered electron imaging and Fourier-transformed infrared microscopy. Bone mechanical response was assessed at the whole-bone- and tissue-level by 3-point bending and nanoindentation, respectively. RESULTS: As compared to WT animals, DIRKO mice presented significant augmentations in trabecular bone mass and trabecular number whereas bone outer diameter, cortical thickness, and cortical area were reduced. At the whole-bone-level, yield stress, ultimate stress, and post-yield work to fracture were significantly reduced in DIRKO animals. At the tissue-level, only collagen maturity was reduced by 9 % in DIRKO mice leading to reductions in maximum load, hardness, and dissipated energy. CONCLUSIONS: This study demonstrated the critical role of gut hormones in controlling bone microstructural morphology and tissue properties.

Diversity of bone matrix adhesion proteins modulates osteoblast attachment and organization of actin cytoskeleton.

Interaction of cells with extracellular matrix is an essential event for differentiation, proliferation and activity of osteoblasts. In bone, binding of osteoblasts to bone matrix is required to determine specific activities of the cells and to synthesize matrix bone proteins. Integrins are the major cell receptors involved in the cell linkage to matrix proteins such as fibronectin, type I collagen and vitronectin, via the RGD-sequences. In this study, cultures of osteoblast-like cells (Saos-2) were done on coated glass coverslips in various culture conditions: DMEM alone or DMEM supplemented with poly-L-lysine (PL), fetal calf serum (FCS), fibronectin (FN), vitronectin (VN) and type I collagen (Col-I). The aim of the study was to determine the specific effect of these bone matrix proteins on cell adherence and morphology and on the cytoskeleton status. Morphological characteristics of cultured cells were studied using scanning electron microscopy and image analysis. The heterogeneity of cytoskeleton was studied using fractal analysis (skyscrapers and blanket algorithms) after specific preparation of cells to expose the cytoskeleton. FAK and MAPK signaling pathways were studied by western blotting in these various culture conditions. Results demonstrated that cell adhesion was reduced with PL and VN after 240 min. After 60 min of adhesion, cytoskeleton organization was enhanced with FN, VN and Col-I. No difference in FAK phosphorylation was observed but MAPK phosphorylation was modulated by specific adhesion on extracellular proteins. These results indicate that culture conditions modulate cell adhesion, cytoskeleton organization and intracellular protein pathways according to extracellular proteins present for adhesion.

Micro and macroarchitectural changes at the tibia after botulinum toxin injection in the growing rat.

The aim of this study was to analyze bone microarchitecture and macroarchitecture of tibia in a disuse model in growing rats. Eight-weeks-old Copenhagen rats were injected intramuscularly with 1.5 units BTX in the quadriceps muscle of the right hind limb. Saline injection was done at the left hind limb to serve as control. Five rats were killed at day 1 and represented the baseline group (D1), 5 rats were killed at day 14 (D14), 5 at day 21 (D21), 5 at day 28 (D28) and 5 at day 35 (35). For each group, muscle surface, parameters of bone microarchitecture and macroarchitecture (including length, width and curvature of the tibia) were measured using microtomography. Paralysis occurred as soon as day 2. At the left hind limb, muscle surface area, cortical thickness, cross sectional total area and growth in length significantly increased during the time study. At the right hind limb, muscle surface area, bone trabecular volume, and cortical thickness decreased as soon as day 14 associated with an increased cortical porosity. Growth in length did not differ from left side; cross sectional total area did not increase and the diaphyseal cross section acquired a more rounded shape. There was no modification of the curvature between right and left hind limbs during the time study. In this murine model of unilateral muscle paralysis in growing animals, we showed a rapid muscle loss leading to a decreased growth in width; however growth in length and curvature were unaltered.

Measurement by vertical scanning profilometry of resorption volume and lacunae depth caused by osteoclasts on dentine slices.

The resorption pit assay is classically used to evaluate osteoclast activity on bone or dentine slices that can be eroded by these cells. Two different types of cells were generated from peripheral blood mononuclear cells cultured in the presence of M-CSF + sRANKL or with M-CSF + LPS. At the end of the culture period (21 days), cells were discarded and the dentine slices stained with toluidine blue and examined with an NT9100 Wyco vertical scanning profilometer. The images of the dentine surface were corrected for tilt and the eroded volume was calculated on the whole images. The depth of the eroded pits was determined. The data files were used to reconstruct the surface of the slices by standardizing the ground level to compare both conditions. Osteoclasts generated with M-CSF + sRANKL were capable of resorbing a more important volume than those generated with M-CSF + LPS. In addition, the formers were able to resorb the dentine matrix more deeply. Data provided by the microscope were used to reconstruct three-dimensional images of the dentine slices with pseudo colours varying with the depth of erosion. Vertical scanning profilometry, a technique used to measure the roughness of polished or etched surfaces in metallurgic industry, can be used to accurately measure the eroded volume and the mean erosion depth done by osteoclasts in the resorption pit assay.

Thiazolidinediones induce osteocyte apoptosis and increase sclerostin expression.

AIMS: Thiazolidinediones (TZDs) are associated with a higher risk of bone fracture in women compared with men. The aim of the present study was to investigate whether TZDs could influence osteocyte behaviour and contribute to the skeletal phenotype observed in TZD-treated patients. METHODS: The murine MLO-Y4 cell line was used as a source of osteocytes. These cells were cultured for 24 h with 0, 10(-8) m, 10(-7) m, 10(-6) m, 10(-5) m or 10(-4) m of pioglitazone, rosiglitazone or troglitazone in the presence or absence of 17beta-oestradiol. The extent of osteocyte apoptosis was assessed, as was the expression of the bone formation inhibitor sclerostin and receptor activator for nuclear factor kappaB ligand (RANKL) also. RESULTS: In the absence of 17beta-oestradiol, pioglitazone, rosiglitazone and troglitazone induced osteocyte apoptosis dose-dependently even at the lowest concentration of 10(-8) m. Furthermore, the expression of sclerostin but not RANKL was significantly increased in TZD-treated cultures compared with untreated cultures. The presence of 17beta-oestradiol significantly reduced TZD-induced osteocyte apoptosis and also sclerostin up-regulation. CONCLUSIONS: These findings therefore raise the potential concern of using TZDs in post-menopausal women where the lack of oestrogen would not prevent osteocyte apoptosis and sclerostin up-regulation and may aggravate the reduction in bone mass in these patients.

Role of neuropathy on fracture healing in Charcot neuro-osteoarthropathy.

Charcot neuro-osteoarthropathy (CNO) is a devastating condition affecting most commonly the foot/ankle joint in diabetic patients and may lead to severe deformities and amputation. Peripheral sensory neuropathy seems to be a pre-requisite to the development of CNO. The aim of this review article is to summarise the skeletal effects of the nervous system on bone remodelling and fracture healing of normal and damaged joints and to describe how neuropathy, in the context of modern concept of neuro-osteopathology, is crucial in the predisposition of the patient to develop acute CNO.

Cobalt, chromium and nickel affect hydroxyapatite crystal growth in vitro.

Metals are widely used in orthopaedics and recent studies have reported that patients with metal implants have a significant increase of metal levels in serum and synovial fluid. Femoral neck fracture occurred in some patients with metal-on-metal implants for unknown reasons. Recently, bone quality has emerged as an important factor of bone strength and few studies have investigated the effects of metal ions on hydroxyapatite properties. In the present study, we investigated the effects of Co(2+), Cr(3+) and Ni(2+) on hydroxyapatite (HA) growth in vitro, using carboxymethylated poly(2-hydroxyethyl methacrylate) (pHEMA) as a biomaterial for calcification. We have demonstrated that metal ions reduced the quantity of mineral formed at the surface of the polymer and decreased the ratio Ca/P by 1.12-, 1.05- and 1.08-fold for Cr(2+), Cr(3+) and Ni(2+) respectively. Furthermore, the size of calcospherites was significantly increased in the metal-doped HA compared to the controls, indicating a possible effect of metal ions on the crystal lattice. Indeed, the presence of metal ions increased the crystal size as well as the crystallinity of HA and reduce the lattice parameter c of the HA framework. The information obtained from this work suggests that the quality of the mineral around metallic implants could be altered. However, further investigation should be conducted to further elucidate the effects of metal incorporation on bone mineral and the functional consequences.

Increased osteoclastic activity in acute Charcot's osteoarthropathy: the role of receptor activator of nuclear factor-kappaB ligand.

AIMS/HYPOTHESIS: Our aims were to compare osteoclastic activity between patients with acute Charcot's osteoarthropathy and diabetic and healthy controls, and to determine the effect of the receptor activator of nuclear factor-kappaB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG). METHODS: Peripheral blood monocytes isolated from nine diabetic Charcot patients, eight diabetic control and eight healthy control participants were cultured in the presence of macrophage-colony stimulating factor (M-CSF) alone, M-CSF and RANKL, and also M-CSF and RANKL with excess concentrations of OPG. Osteoclast formation was assessed by expression of tartrate-resistant acid phosphatase on glass coverslips and resorption on dentine slices. RESULTS: In cultures with M-CSF, there was a significant increase in osteoclast formation in Charcot patients compared with healthy and diabetic control participants (p=0.008). A significant increase in bone resorption was also seen in the former, compared with healthy and diabetic control participants (p<0.0001). The addition of RANKL to the cultures with M-CSF led to marked increase in osteoclastic resorption in Charcot (from 0.264+/-0.06% to 41.6+/-8.1%, p<0.0001) and diabetic control (0.000+/-0.00% to 14.2+/-16.5%, p<0.0001) patients, and also in healthy control participants (0.004+/-0.01% to 10.5+/-1.9%, p<0.0001). Although the addition of OPG to cultures with M-CSF and RANKL led to a marked reduction of resorption in Charcot patients (41.6+/-8.1% to 5.9+/-2.4%, p=0.001), this suppression was not as complete as in diabetic control patients (14.2+/-16.5% to 0.45+/-0.31%, p=0.001) and in healthy control participants (from 10.5+/-1.9% to 0.00+/-0.00%, p<0.0001). CONCLUSIONS/INTERPRETATION: These results indicate that RANKL-mediated osteoclastic resorption occurs in acute Charcot's osteoarthropathy. However, the incomplete inhibition of RANKL after addition of OPG also suggests the existence of a RANKL-independent pathway.

Influence of fluoride, hydrogen peroxide and lactic acid on the corrosion resistance of commercially pure titanium.

Titanium is widely used in dental implantology and orthopaedics due to its excellent corrosion resistance and mechanical properties. However, it has been reported that Ti is sensitive to F(-), H(2)O(2) and lactic acid. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to investigate the corrosion resistance of CP-Ti disks after 9 days immersion in different test solutions, based on artificial saliva containing F(-) (0.5% and 2.5%), H(2)O(2) (0.1% and 10%) and/or lactic acid. Because activated macrophages and bacteria can also release locally some of these oxidative compounds, we investigated the role of these cells when plated onto titanium disks. The surface roughness (R(a)) was highly increased when titanium disks were immersed in artificial saliva containing F(-), H(2)O(2) and lactic acid. After 21 days of cell culture, R(a) was significantly increased on disks incubated with activated-J774.2 cells or Streptococcus mitis. AFM appeared to be more sensitive than SEM in evaluating the corrosion of the titanium. Chemical species, either environmental or those released by macrophages and bacteria, can provoke a marked attack of the titanium surface.

Evaluation of surface roughness of hydrogels by fractal texture analysis during swelling.

The surface of a biomaterial reacts in contact with biological fluids. Hydrogels are used to prepare biomaterials. The surface roughness of materials can be explored by several techniques. However, when considering hydrogels, the surface examined in the dry state does not reflect the final conformation. How the surface roughness is affected by swelling has been little explored by quantitative methods. We have evaluated the surface roughness of poly(2-hydroxyethyl methacrylate) (i.e., pHEMA) by image analysis. Images of disks, prepared from linear pHEMA, were obtained on a light microscope after various incubation times in saline. Fractal texture analysis was done on images to determine the fractal dimension D. In this study, D exhibited a significant decrease during swelling and was highly correlated with the swelling ratio (r2 = 0.994, p < 0.00001). Water uptake by the surface of the polymer affected the surface roughness. Image analysis using fractal algorithms appears to be the most interesting technique for the quantitative exploration of surfaces of hydrated materials that cannot be measured by conventional methods.

Effects of the length of crosslink chain on poly(2-hydroxyethyl methacrylate) (pHEMA) swelling and biomechanical properties.

Polymers are widely used in medicine for vascular prostheses, bone substitutes, and devices for controlled release. Among these polymers, poly(2-hydroxyethyl methacrylate) (pHEMA) is the most employed. To confer particular properties, pHEMA can be copolymerized with other monomers or in the presence of plasticizers or crosslinking agents. The influence of the length of crosslink chains on swelling, surface rugosity, hardness, and stiffness of crosslinked pHEMA were studied by several techniques, including fractal analysis and AFM. Four crosslinking agents (divinyl benzene, DVB; ethylene glycol dimethacrylate, EGDMA; tetraethylene glycol diacrylate, TEGDA; and polyethylene glycol diacrylate, PEGDA) were added to the bulk polymerization mixture. Only linear and PEGDA-pHEMA presented a significant decrease in surface roughness confirmed by fractal analysis. Differences in hardness and biomechanical properties were evidenced on dried polymers but the highest differences were exhibited for hydrated pHEMA. Correlations between the length of the crosslink chain and hardness or stiffness of hydrated crosslinked pHEMA were evidenced. TEGDA and PEGDA appeared to be the two most suitable crosslinking agents for controlled release of bioactive molecules in bone.

Biodegradability of poly (2-hydroxyethyl methacrylate) in the presence of the J774.2 macrophage cell line.

The degradation of cross-linked and linear poly(2-hydroxyethyl methacrylate) (pHEMA), was examined in vitro with J774.2 cells. pHEMA microbeads were prepared with both types of polymers. Only cells in contact with the microbeads increased their production of lysosomal enzymes (TRAcP and ANAE) and released large amounts of reactive oxygen species with both types of pHEMA microbeads. Electron microscopy showed that macrophages were able to erode the surface of linear pHEMA but unable to erode the surface of the cross-linked polymer. Cells appeared wrapped by the linear pHEMA surface, but those cultured on the cross-linked polymer were only laying at the surface. After cell culture, the surface roughness of pHEMA slices was observed by atomic force microscopy (AFM). There was a significant increase in roughness (R(a)) of the surface of linear pHEMA slices cultured with J774.2 cells whereas no difference in R(a) between the surface of cross-linked pHEMA slices could be measured. AFM image of the hydrated materials were done: the surface of linear pHEMA swelled considerably in saline whereas the hydrated cross-linked polymer did not differ from the air-dried appearance. In conclusion, linear pHEMA swells in biological fluids, activates macrophages in close contact with the polymer and can be progressively eroded.