Neelam Gurav

Optimizing HAPEX™ Topography Influences Osteoblast Response

HAPEX (hydroxyapatite-reinforced polyethylene composite) is a second-generation orthopedic biomaterial designed as a bone analog material, which has found clinical success. The use of topography in cell engineering has been shown to affect cell attachment and subsequent response. Thus, by combining bioactivity and enhancing osteoblast response to the implant surface, improved tissue repair and implant life span may be achieved. In this study a primary human osteoblast-like cell model has been used to study the influence of surface topography and chemistry produced by three different production methods. Scanning electron microscopy, fluorescence microscopy, and confocal scanning laser microscopy have been used to study cell adhesion; tritiated thymidine uptake has been used to observe cell proliferation; and the reverse transcriptase-polymerase chain reaction and biochemical methods have been used to study phenotypic expression. Transmission electron microscopy has also been used to look at more long-term morphology. The results show that topography significantly influences cell response, and may be a means of enhancing bone apposition on HAPEX.

Biodegradable microspheres: A new delivery system for growth hormone

A drug delivery system for biologically active agents targeted to specific cells could be used to improve tissue repair in orthopaedics. The system should be controllable and capable of drug release over an extended period of time. Biodegradable, membrane-moderated, monolithic microspheres for the controlled release of growth hormone (GH) were developed and the release of GH was monitored in vitro. Cross-linked gelatin microspheres were used as the vehicle, with the drug dispersed within the gelatin. The amount of GH released from the microspheres was increased following ultrasonication. The release of growth hormone was monitored in phosphate buffered saline and horse serum. Interestingly, a higher level of GH was detected in the phosphate buffered saline than in serum. In addition, both pH and enzyme-induced degradation had an effect on the swelling kinetics of the gelatin microspheres. The release of GH from the microspheres was diffusion controlled, during the time period studied.

In vitro proliferation and differentiation of human mesenchymal stem cells on hydroxyapatite versus human demineralised bone matrix with and without osteogenic protein-1.

Background: Allografting is currently used in lower limb reconstruction surgery. Demineralised bone matrix (DBM) is more osteoinductive compared with allografts but lacks mechanical strength. Osteogenic protein-1 (OP-1) can improve the osteoinductivity of the allograft, however recent reports indicate significant allograft resorption when it is combined with OP-1. Objectives: Our hypothesis was that hydroxyapatite (HA) with human-mesenchymal stem cells (h-MSCs) and OP-1 (HA+h-MSCs+OP-1) has similar osteoinductive properties to human-DBM+h-MSCs. The objective was to evaluate h-MSC proliferation (by tritiated thymidine incorporation, total DNA Hoechst 33258 and scanning electron microscopy) and osteogenic differentiation (from alkaline phosphatase activity) in human demineralised bone matrix (h-DBM) and HA, with or without OP-1. Results: H-MSC proliferation on HA+OP-1 was significantly higher compared with that on HA at all time points (p < 0.05) and compared with DBM alone [day 1, (198.4 vs 95.4) p = 0.042; day 14 (286.1 vs 119.9), p < 0.001]. H-MSC proliferation was higher in DBM+OP-1, at all time points compared with HA+OP-1 but the difference was not statistically significant (p > 0.05). H-MSC differentiation was significantly higher in HA+OP-1 compared with HA (p < 0.05) but not significantly different from diffferentiation on DBM alone (p > 0.05). Differentiation was significantly higher on DBM+OP-1 at all time points compared with HA (p < 0.05) and with HA+OP-1 [day 1, (21.1 vs 10.1) (p = 0.03); day 7 (39.4 vs 7.1) (p < 0.01); day 14 (40.2 vs 14.4) (p < 0.001)]. Conclusions: When HA+h-MSCs is combined with OP-1 in vitro its osteogenic potential is similar to that of DBM+h-MSCs alone which may be adequate for non-weight-bearing applications. Mechanical testing however is of great importance for weight-bearing applications and the in vivo testing of the composite graft HA+h-MSCs+OP-1 vs DBM+h-MSCs is recommended.

Combinatorial design of calcium meta phosphate poly(vinyl alcohol) bone-like biocomposites

The incidence of degenerative diseases and the ageing population have added to the growing demand for bone grafts. Although autologous bone continues to be the gold standard, limited yield and potential morbidity of the donor site pose considerable challenges. Currently, clinically used synthetic grafts based on calcium phosphates are mechanically brittle and not compliant hence composite scaffolds are expected to be provide viable solutions. In this study we report composites of calcium meta phosphate-poly (vinyl alcohol) with tunable mechanical properties, low swelling and excellent biocompatibility. The elastomeric nature of the composites resist brittle fracture and the scaffolds can be easily shaped to the bone defect by the surgeon. Testing on bone plug shaped specimens of the scaffolds, exhibited superior mechanical properties compared to currently commercially available bone plugs with additional advantages being the ability to increase porosity without compromising properties in compression and degree of swelling, which make these composites promising synthetic alternatives for bone grafts and bone tissue engineering.

Processing of Ca-P Ceramics, Surface Characteristics and Biological Performance

Surface pollution by Mg and carbon has been identified by X-Ray Photoele ctr n Spectroscopy (XPS) analysis on monophasic (100% stoichiometric hydroxyapa tite) or biphasic (70% hydroxyapatite, 30% β-tricalcium phosphate) ceramics obtained by sintering of Ca-P powder of standard purity. Magnesium was the main impurity, although it is pr e ent only as traces in synthesised Ca-P powders, it concentrates on the surface of the cer amic during the sintering process due to its poor solubility in HA. Carbon pollution is common on all materials and generally as signed to the adsorption of atmospheric volatile organic compounds, although other type s of ollution may occur. These surface species alter the surface energy of the c erami s which has been shown to be related to cell adhesion. The change in surface composition and physical properties can interfere with the sintering process as well as with the biological behaviour of the ceramics and a special effort should be made to control these events at all levels from proc essing to handling, storage and packaging.

3 – Biocompatibility of degradable polymers for tissue engineering

For several decades biodegradable polymers have been attracting interest as the materials for filling in defects or being used as stabilising materials. However, with the increasing pace in the developments in tissue engineering, the usage of these materials has also increased. The chapter presents some data on the biocompatibility testing of these materials and some of the challenges at the basic level. In determining the biocompatibility of materials and devices, we need to focus more specifically on the applications, which in turn will affect the type of cells and tests that will be used. We also need to determine the duration the material will be in-vivo and where appropriate consider breakdown products. The use of accelerated degradation using enzymes in-vitro can change the properties of material which can be used to ‘mimic’ degradation in-vivo. These and other aspects of biocompatibility will be discussed in this chapter.