L. Di Silvio

Initial in vitro interaction of osteoblasts with nano-porous alumina

In the present study we have used a characterised primary human cell culture model to investigate cellular interactions with nano-porous alumina. This material, prepared by anodisation, is being developed as a coating on titanium alloy implants. The structure of the alumina, as determined by X-ray diffraction and transmission electron microscopy, was amorphous. When studying cell/material interactions we used both biochemical and morphological parameters. Cell viability, proliferation and phenotype were assessed by measurement of redox reactions in the cells, cellular DNA, tritiated thymidine ([3H]-TdR) incorporation and alkaline phosphatase (ALP) production. Results showed a normal osteoblastic growth pattern with increasing cell numbers during the first 2 weeks. A peak in cell proliferation was seen on day 3, after which cell growth decreased, followed by an increase in ALP production, thus indicating that the osteoblastic phenotype was retained on the alumina. Cell adhesion was observed, the osteoblast-like cells having a flattened morphology with filipodia attached to the pores of the material. SDS-PAGE and western blot measurements showed that the nano-porous alumina was able to adsorb fibronectin. Trace amounts of aluminium ions were measured in the surrounding medium, but no adverse effect on cell activity was observed.

Increasing hydroxyapatite incorporation into poly(methylmethacrylate) cement increases osteoblast adhesion and response

Poly(methylmethacrylate) (PMMA) is the current standard for cement held prostheses. It forms a strong bond with the implant, but the bond between the cement and the bone is considered to be weak, with fibroblastic cells observed at the implant site, rather than direct bone contact, a contributing factor leading to implant failure. Incorporation of hydroxyapatite (HA) increases the biological response to the cement from tissue around the implant site, thus giving increased bone apposition. In this study, PMMA discs with 0, 4.6 and 8.8 vol%. HA were examined. Primary human osteoblast-like cells (HOBs) were used for the biological evaluation of the response to the cements in vitro. Morphology was observed using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Measurement of tritiated thymidine (3H-TdR) incorporation and alkaline phosphatase (ALP) activity were used to assess proliferation and differentiation. A synergy between increasing focal contact formation, cytoskeletal organisation, cell proliferation and expression of phenotype was observed with increasing HA volume. Preferential anchorage of HOBs to HA rather than PMMA was a prominent observation.

Initial interaction of osteoblasts with the surface of a hydroxyapatite-poly(methylmethacrylate) cement

Failure of the bone/cement interface in cemented joint prostheses is a contributor to implant loosening. The introduction of a bioactive phase, such as hydroxyapatite (HA), to cement may enhance fixation by encouraging direct bone apposition rather than encapsulation of the implant by fibrous tissue. The effect of poly(methylmethacrylate) (PMMA) bone cement (incorporating 17.5% HA wt.) on bioactivity has been investigated using primary human osteoblast-like cells (HOB). A significantly higher cell proliferation and differentiation was seen on the PMMA/HA cement compared to the PMMA cement alone, with retention of phenotype up to 21 days of culture on both materials.

In vitro mechanical and biological assessment of hydroxyapatite-reinforced polyethylene composite

In vitro performance of hydroxyapatite (HA)-reinforced polyethylene (PE) composite (HAPEX) has been characterized from both mechanical and biological aspects. The mechanical properties of HAPEX, such as tensile strength and Youngs modulus, showed little change after immersion in a physiological solution at 37 and 70 degrees C for various periods. In addition, the biological response of primary human osteoblast-like (HOB) cells in vitro on HAPEX was assessed by measuring DNA synthesis and osteoblast phenotype expression. Cell proliferation rate on HAPEX was demonstrated by an increase in DNA content with time. A high tritiated thymidine ([3H]-TdR) incorporation/DNA rate was observed on day 1 for HAPEX, indicating a stimulatory effect on cell proliferation. The alkaline phosphatase (ALP) activity was expressed earlier on HAPEX than on unfilled PE and increased with time, indicating that HOB cells had commenced differentiation. Furthermore, it was found that the HA particles in the composite provided favourable sites for cell attachment. It appears that the presence of HA particles in HAPEX may have the advantage of acting as microanchors for bone bonding in vivo.

Porosity variation in hydroxyapatite and osteoblast morphology: a scanning electron microscopy study

The biocompatibility of hydroxyapatite has been demonstrated by previous studies, with enhancement of osteointegration through the use of porous hydroxyapatite (pHA). Emphasis has been focused on the use of coralline hydroxyapatite or the introduction of macroporosity into synthetic hydroxyapatite. The current study investigates the role of macro‐ and microporosities in synthetic phase‐pure porous hydroxyapatite on the morphological aspects of human osteoblast‐like cells using scanning electron microscopy. Cells were seeded on four different types of porous hydroxyapatite (HA1, HA2, HA3 and HA4) and examined following 1, 2, 14 and 30 days of incubation in vitro. The results indicated that the cells had an affinity to micropores through filopodia extensions, at initial stage of attachment. Cellular proliferation and colonization was evident on all materials with cells forming cellular bridges across the macropores at day 14 with cellular canopy formation covering entire macropores observed by day 30. This study demonstrates that while the introduction of microporosity has no evident effect on cellular morphology at later time points, it seems to play a role in initial cellular anchorage and attachment.

Osteoblast behaviour on HA/PE composite surfaces with different HA volumes.

A hydroxyapatite (HA) reinforced polyethylene (PE) composite (designated HAPEX), with high mechanical specification and a bioactive HA phase, has been optimised as a bone analogue material. Manufacturing conditions and machining of the materials were carefully controlled to give a reproducible material surface roughness with minimal batch variation. The effect of surface composition was examined in vitro using primary human osteoblasts (HOB). HOBs were cultured in direct contact with the test materials containing 20% and 40% vol. HA. The results showed that 40% HA/PE enhanced cellular activity by increasing proliferation rate and differentiation compared to the 20% vol. HA composite. The cytoskeletal organisation of the cells was also examined and HOBs cultured on 40% HA/PE were flatter and had an enhanced rate of cytoskeletal organisation and an increase in focal contact points compared to the 20% HA/PE.

In vitro evaluation of a new polymethylmethacrylate cement reinforced with hydroxyapatite.

The nature of the orthopedic implant surface affects the interaction between cells and subsequent bone formation. The bone/cement interface in cement-held prostheses is considered to be the main cause of fracture leading to implant revision. It is thought that the introduction of a bioactive phase, such as hydroxyapatite (HA), to cement may permit a stronger implant by encouraging direct bone apposition rather than encapsulation of the implant by fibrous tissue. Thus, a poly(methylmethacrylate) (PMMA) cement incorporating 17.5% HA by weight has been investigated. In this study, in order to analyze the interaction at the cellular level, the in vitro biological response of the HA/PMMA to a similar PMMA without HA incorporation has been studied. Primary human osteoblast-like cells (HOB) were used as they are a model of the cell type the cements might encounter in vivo. Cell proliferation and growth were assessed by measurement of total cellular DNA and tritiated thymidine ([3H]-TdR) incorporation. Alkaline phosphatase (ALP) production was measured as an indicator of HOB phenotype upon the cements. The results showed that HA/PMMA was a better substrate for HOB cells, resulting in increased proliferation and ALP activity. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that HOB cells cultured on the HA-filled PMMA preferentially anchored to HA particles exposed at the cement surface, with a close intimacy observed between HA and HOB cells.

In vitro response of osteoblasts to hydroxyapatite-reinforced polyethylene composites

A primary human cell culture model was used to investigate a range of hydroxyapaptite (HA)-reinforced high-density polyethylene (HDPE) composites (HAPEXTM). These materials are being developed as potential bone-substitute materials. When designing and optimizing a second-generation biomaterial, it is important to achieve a balance between mechanical and biological properties without compromising either. Biochemical and histological parameters have been used to compare the biological response of 20% and 40% volume HA in HDPE. Cellular DNA and incorporation of tritiated thymidine was measured to assess cell proliferation. Alkaline phosphatase (ALP) production was used as a marker of osteoblast phenotype expression. In this preliminary study, osteoblasts cultured on the 20% HAPEXTM showed a greater increase in the rate of proliferation and osteoblast expression as indicated by an increase in ALP activity compared to the 40% HAPEXTM over the time period studied. Osteoblast-like cells showed a flattened morphology on both composites and in some cases a greater covering was observed on the 20% HAPEXTM. These results indicate that the composites may not be identical in terms of bioactivity and that further research on surface topography and physico-chemical properties is required to assess fully the biological response of these composites.

Initial attachment of osteoblasts to an optimised HAPEX topography

The interactions between an implant material and the surrounding tissue are of a complicated nature, and the initial attachment of cells to the surface is important in determining the implant success. HAPEX has been developed as a second-generation orthopaedic biomaterial, with both mechanical and biological characteristics that make it suitable for bone augmentation. Further optimisation of the material is being continued to increase the attachment of osteoblasts coupled with improving mechanical characteristics, so it may be used in load bearing applications. It has been previously observed that polishing followed by roughening the surface of HAPEX enhances osteoblast proliferation and phenotype. This article discusses the recruitment of primary human osteoblast cells onto the optimised surface, by examining morphology and cytoskeletal changes using scanning electron microscopy and confocal laser scanning microscopy. The results show that the cells attach in greater numbers to the optimised surface, and develop notably faster, than cells on machined 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.