The effect of anisotropic collagen-GAG scaffolds and growth factor supplementation on tendon cell recruitment, alignment, and metabolic activity.

Current surgical and tissue engineering approaches for treating tendon injuries have shown limited success, suggesting the need for new biomaterial strategies. Here we describe the development of an anisotropic collagen-glycosaminoglycan (CG) scaffold and use of growth factor supplementation strategies to create a 3D platform for tendon tissue engineering. We fabricated cylindrical CG scaffolds with aligned tracks of ellipsoidal pores that mimic the native physiology of tendon by incorporating a directional solidification step into a conventional lyophilization strategy. By modifying the freezing temperature, we created a homologous series of aligned CG scaffolds with constant relative density and degree of anisotropy but a range of pore sizes (55-243 µm). Equine tendon cells showed greater levels of attachment, metabolic activity, and alignment as well as less cell-mediated scaffold contraction, when cultured in anisotropic scaffolds compared to an isotropic CG scaffold control. The anisotropic CG scaffolds also provided critical contact guidance cues for cell alignment. While tendon cells were randomly oriented in the isotropic control scaffold and the transverse (unaligned) plane of the anisotropic scaffolds, significant cell alignment was observed in the direction of the contact guidance cues in the longitudinal plane of the anisotropic scaffolds. Scaffold pore size was found to significantly influence tendon cell viability, proliferation, penetration into the scaffold, and metabolic activity in a manner predicted by cellular solids arguments. Finally, the addition of the growth factors PDGF-BB and IGF-1 to aligned CG scaffolds was found to enhance tendon cell motility, viability, and metabolic activity in dose-dependent manners. This work suggests a composite strategy for developing bioactive, 3D material systems for tendon tissue engineering.

Effect of local antimicrobial agents on excisional palatal wound healing: a clinical and histomorphometric study in rats.

AIM: To assess the effect of topically applied antimicrobial agents on palatal excisional wound in rats. MATERIALS AND METHODS: Excisional wounds, 5 mm in diameter, were made in the centre of the palate of 125 Wistar male rats. In four experimental groups, chlorhexidine digluconate (CHX) 0.12% solution, 1% CHX gel, phenolic compounds solution (Listerine), amine/stannous fluoride solution (Meridol) and saline solution as a control group were applied daily for 1 min. The wound area was measured photographically and the epithelialization rate was determined histologically at 3, 7, 14 and 21 days post-surgery. RESULTS: The mean wound area and mean distance between the epithelial margins decreased significantly with time (p<0.001) in experimental and control groups, with the greatest wound area reduction and rate of epithelialization on day 14. A significantly superior rate of wound epithelialization (p=0.03) was presented following use of 1% CHX gel and Listerine and a comparatively inferior one when the Meridol solution was applied. CONCLUSIONS: Each tested antimicrobial agent when applied on an excisional wound with epithelial and connective tissue deficiency did not have a negative effect on the rate of wound closure. The best results were achieved with 1%CHX gel and Listerine.

Calcification of human valve interstitial cells is dependent on alkaline phosphatase activity.

BACKGROUND AND AIM OF THE STUDY: The calcification of heart valves is associated with valve degeneration and failure, but the mechanisms involved are poorly understood. The presence of lamellar bone has been demonstrated in calcified aortic valves. Since osseous calcification is closely associated with alkaline phosphatase (ALP) activity, it was hypothesized that ALP activity might be implicated in the calcification of isolated leaflet interstitial cells (ICs). METHODS: Human valve leaflet ICs were isolated from transplant-explanted hearts at the time of transplantation (n = 12). RESULTS: Isolated leaflet ICs expressed the fibroblast-specific antigen (100% of cells) and smooth muscle (SM) alpha-actin (70-80% of cells), but osteoblastic markers were not expressed. Cultured ICs did not calcify spontaneously, however when the growth medium was supplemented with beta-glycerophosphate (an organic phosphate) it induced the formation of calcified nodules that expressed osteonectin and ALP, but not SM alpha-actin. Beta-glycerophosphate-induced calcification of ICs showed a time-dependent effect on the calcium content of treated cells over a 14-day period. ALP activity was considerably increased in beta-glycerophosphate-treated ICs, and this correlated with the calcium content (r = 0.5: p = 0.01). Levamisol (an ALP inhibitor) inhibited the beta-glycerophosphate-induced calcification process, as well as the expression of osteoblastic differentiation markers. CONCLUSION: Isolated and cultured leaflet ICs did not calcify spontaneously, though organic phosphate induced the formation of calcified nodules that expressed osteoblastic markers. The calcification of isolated ICs was seen to be dependent on ALP activity.

Phenotypic screening for pharmaceuticals using tissue constructs.

Compounds can be screened for pharmaceutical activity either by detecting interactions with specified target molecules such as receptors or enzymes (molecular screening) or observing effects on the structure or physiological activities of cells or tissues (phenotypic screening). Screening at the molecular level has been greatly enhanced by fluorescence methods. Especially the combination of confocal detection with measurements of the amplitudes and time courses of fluorescence fluctuations have reduced sample volumes to < microliters and have increased throughputs to >100000 compounds per day. Screening at the molecular level, however, does not provide information about the effects of test compounds on cellular functions. Phenotypic screening, although much slower than molecular screening, does provide information about effects on cell or tissue structure or function and therefore can be used to eliminate at an early stage compounds that are toxic or do not produce the desired cellular response. Tissue constructs reconstituted using cells of specified types and defined extracellular matrix components provide test systems for detecting the effects of test compounds on cellular mechanical functions such as the development of contractile force and on cell and matrix structure and stiffness. For example, constructs based on vascular smooth muscle cells provide information about effects on cellular contractile force that can be used to identify agents that control blood pressure. Tissue constructs that mimic skeletal, smooth and heart muscles and connective tissues have been produced and can be used to study mechanical and structural responses to active compounds.

Effects of alumina and zirconium dioxide particles on arachidonic acid metabolism and proinflammatory interleukin production in osteoarthritis and rheumatoid synovial cells.

We describe a model which can be used for in vitro biocompatibility assays of biomaterials. We studied the in vitro response of human osteoarthritis or rheumatoid arthritis fibroblast-like synoviocytes to Al2O3 or ZrO2 particles by analysing the production of interleukin-1 (IL-1) and interleukin-6 (IL-6) and the metabolism of arachidonic acid via lipoxygenase and cyclo-oxygenase pathways. Our results show that, in these cells and under our experimental conditions, Al2O3 and ZrO2 did not significantly modify the synthesis of IL-1 and IL-6 or the metabolism of arachidonic acid.

Evaluation of ceramics composed of different hydroxyapatite to tricalcium phosphate ratios as carriers for rhBMP-2.

We have investigated pellet-shaped implants prepared from biphasic calcium phosphate (BCP) ceramics with five different ratios of hydroxyapatite (HAP) to beta tricalcium phosphate (beta-TCP). The purpose of this study was to evaluate these BCP ceramics as carriers for rhBMP-2. BCP ceramics impregnated with the different doses of recombinant human bone morphogenetic protein 2 (rhBMP-2) (1, 5 and 10g) were used for the experimental purpose and the ceramics without rhBMP-2 were used as control. The pellets were placed into subcutaneous pockets on the dorsum of 4-week-old male Wistar rats. The animals were sacrificed 2 and 4 weeks after implantation. Bone induction was estimated by alkaline phosphatase (ALP) activity measured at 2 weeks after implantation. Pellets were also examined radiologically, histologically and histomorphometrically. The results showed that all experimental pellets exhibited new bone formation whereas the control pellets produced only fibrous connective tissue. Here, 100% HAP ceramic showed most amount of bone formation, whereas 25% HAP to 75% TCP ceramic produced the bone least in amount among different BCP ceramics at the end of 4 weeks. This study indicates that formation of new bone depends on the ceramic content with high HAP-TCP ratio and high dose of rhBMP-2.