Effect of biomaterial properties on bone healing in a rabbit tooth extraction socket model

In this work we sought to understand the effect of biomaterial properties upon healing bone tissue. We hypothesized that a hydrophilic polymer gel implanted into a bone tissue defect would impede the healing process owing to the biomaterial's prevention of protein adsorption and thus cell adhesion. To test this hypothesis, healing bone was investigated within a rabbit incisor extraction socket, a subcritical size bone defect that resists significant soft tissue invasion by virtue of its conformity. After removal of the incisor teeth, one tooth socket was left as an empty control, one was filled with crosslinked polymer networks formed from the hydrophobic polymer poly(propylene fumarate) (PPF), and one was filled with a hydrogel formed from the hydrophilic oligomer oligo(poly(ethylene glycol) fumarate) (OPF). At five different times (4 days as well as 1, 2, 4, and 8 weeks), jaw bone specimens containing the tooth sockets were removed. We analyzed bone healing by histomorphometrical analysis of hematoxylin and eosin stained sections as well as immunohistochemically stained sections. The proposed hypothesis, that a hydrophilic material would hinder bone healing, was supported by the histomorphometrical results. In addition, the immunohistochemical results reflect molecular signaling indicative of the early invasion of platelets, the vascularization of wound-healing tissue, the differentiation of migrating progenitor cells, and the formation and remodeling of bone tissue. Finally, the results emphasize the need to consider biomaterial properties and their differing effects upon endogenous growth factors, and thus bone healing, during the development of tissue engineering devices.     

In vitro degradation of polymeric networks of poly(propylene fumarate) and the crosslinking macromer poly(propylene fumarate)-diacrylate

Polymeric networks of poly(propylene fumarate) (PPF) crosslinked with poly(propylene fumarate)-diacrylate (PPF-DA) are currently being investigated as an injectable, biodegradable bone cement. This study examined the effect of crosslinking density, medium pH, and the incorporation of a beta-tricalcium phosphate (beta-TCP) filler on the in vitro degradation of PPF/PPF-DA. Cylindrical specimens were submerged in buffered saline at 37 degrees C and the change in weight, geometry, and compressive mechanical properties were monitored over a 52-week period. All formulations showed an initial increase in modulus and yield strength over the first 12 weeks, achieving maxima of 1307+/-101 and 51+/-3MPa, respectively, for the beta-TCP composite. PPF/PPF-DA networks with the lower crosslinking density demonstrated the greatest degradation with a 17% mass loss. Samples in the lower buffer pH 5.0 compared to physiological pH 7.4 did not show any differences in mass loss, but exhibited a faster decrease in the compressive strength over time. The beta-TCP composites maintained their mechanical properties at the level following their initial increase. These results show that the degradation of PPF/PPF-DA networks can be controlled by the crosslinking density, accelerated at a lower pH, and prolonged with the incorporation of the beta-TCP filler.     

Increased frequency of the rare PstI allele (P2) in a population of CAD patients in Northern Greece

Restriction fragment length polymorphisms (RFLPs) at the apolipoprotein AI-CIII-AIV gene cluster and their association with coronary artery disease (CAD) and lipid levels were studied in a Northern Greek population. Ninety-five patients with CAD and fifty-four normal controls, angio-graphically proven, were included in this study. Using genomic hybridization techniques, three polymorphic restriction sites were identified at this locus: the PstI at the 3' end of the apoAI gene, the SacI at the 3' non-coding region of the apoCIII gene and the PvuII at the intergenic region between the apoCIII-AIV genes. The rare allele (P2) arising from the absence of the PstI restriction site was observed with a significantly higher frequency (p<0.01) in patients compared to normals (0.11 vs 0.02). In contrast, the rare allele for the SacI polymorphic site had a similar distribution among patients and controls (0.12 vs 0.16). The same was observed for the PvuII RFLP (0.04 vs 0.05). Correlation of lipid and apolipoprotein AI levels with the three RFLPs revealed no significant association, although apo AI and HDL were lower in patients with the P2 allele. Thus, in this Greek population, only the PstI polymorphism, among the polymorphic restriction sites examined, appears to be associated with CAD.     

Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

Proximal tubular epithelial cell integrins respond to high glucose by altered cell-matrix interactions and differentially regulate matrixin expression

Thickening of the tubular basement membrane (TBM) occurs in diabetic nephropathy, but the effects of high glucose on the functional aspects of proximal tubular epithelial cells are not clearly understood. In the present study, we examined the effects of elevated glucose concentrations on (a) integrin expression by human proximal tubular epithelial cells (HK-2) and integrin-mediated interactions with type IV collagen (colIV) and laminin, major components of TBM; (b) the expression of matrixins/matrix metalloproteinases (MMPs), which is regulated by integrins; and (c) the expression of tissue inhibitors of metalloproteinases (TIMPs). HK-2 cells cultured in 25 mM glucose underwent a reduction of the expression of alpha3, beta1, alpha(v)beta3, and alpha5 integrin subunits, with a concomitant increase of the alpha2 subunit, compared with cells grown in 5 mM glucose. Adhesion experiments demonstrated that high glucose led to increased cell adhesion on either colIV or laminin. Experiments of competition of adhesion using anti-integrin antibodies indicated that HK-2 cells in 5 mM glucose used mainly alpha(v)beta3 and alpha5beta1 integrins to adhere to colIV, whereas in 25 mM glucose they additionally used alpha2beta1. In the case of laminin, a beta1-mediated adhesion was observed when HK-2 cells were in 5 mM glucose, whereas in 25 mM glucose, alpha2beta1 and alpha(v)beta3 were also involved. Elevated glucose concentrations resulted in decreased expression of MMP-9 and MMP-2, whereas an increase in TIMP-1 and a decrease in TIMP-2 expression were observed. We also examined which integrins mediated the expression and secretion of matrixins MMP-2 and MMP-9. Ligation of alpha3beta1 with mAbs resulted in induction of MMP-2 expression and secretion, whereas antibody ligation of alpha(v)beta3 led to down-regulation of MMP-9. The above data implicate integrins of proximal tubular epithelial cells in the regulation of MMPs and in the development of TBM thickening in diabetic nephropathy.     

Effect of intravenous immunoglobulin treatment on the Th1/Th2 balance in women with recurrent spontaneous abortions

PROBLEM: The way by which intravenous immunoglobulin (IvIg) acts to prevent immunlogically mediated recurrent spontaneous abortions (RSA) has not been clarified. In the present study, a possible effect of IvIg on the T helper cell (Th1/Th2) balance was investigated in abortions of either alloimmune or autoimmune abnormalities. METHOD OF STUDY: The study included 21 women treated with IvIg before conception because of a history of RSA characterized by alloimmune abnormalities (n = 15) or associated with anti-phospholipid antibodies (APA) (n = 6). Peripheral blood samples, collected before and 5 days after the first IvIg infusion, were stimulated, and Th1 and Th2 cells were detected by flow-cytometric analysis using a combination of monoclonal antibodies against T-cell surface markers and intracellular interferon (IFN)-gamma and interleukin (IL)-4. The percentage of IFN-gamma-producing (Th1) and IL-4-producing (Th2) cells and the Th1/Th2 ratio were compared between pre- and post-infusion samples. RESULTS: A decrease of Th1 percentage in 66.6% of the cases and a concurrent Th2 percentage increase (47.61%) resulted in a decrease in the Th1/Th2 ratio in most of the cases (76.1%) (p < 0.01). Similar results were found in Group A (Th1/Th2 decreased in 60% of the cases, p < 0.05), while in Group B the effect of IvIg was not clear (Th1/Th2 increased in three and decreased in another three cases). CONCLUSION: Our finding suggests that IvIg administration in women with alloimmune RSA enhances Th2 polarization. This is not always the case with APA-associated abortions.