The Characterization and Distribution of Magnesium Whitlockite Crystals in Human Articular Cartilage

The occurrence of crystals (previously termed 'cuboid crystals': 50-500nmsize range) not apparent by light microscopy, in human articular cartilage has been confirmed by transmission electron microscopy of tissue prepared by various techniques, including anhydrous and cryo processing. Earlier reports of such crystal deposition had been limited to osteoarthritic and elderly femoral head articular cartilage. In this study crystals have been reported in articular cartilage across an age range from five to ninety two years in normal and osteoarthritic tissue from a variety of joint sites. The distribution of crystal deposition within articular cartilage was described both qualitatively and quantitatively; normal femoral head tissue was investigated in most detail. Over 90 % of crystals were commonly deposited within the first 50μm below the articular surface; crystals appeared either in a band parallel to the surface or in a pericellular distribution. In deeper zones crystal deposition was restricted to pericellular distribution, and areas of chondrocyte necrosis. Quantitative analysis of crystal deposition distribution in articular cartilage at sites around the femoral head revealed a significantly greater deposition in the superior (zenith) region than the inferior (infrafoveal) region. Elemental analysis of crystals confirmed a calcium, phosphorus and magnesium content. It also demonstrated no variation in the mean calcium to phosphorus ratio with crystal size, specimen age, or between normal and osteoarthritic specimens. A crystal isolation technique involving collagenase digestion, centrifugation and sodium hypochlorite treatment was developed, enabling crystal characterization by electron and x-ray diffraction. Crystals were identified as magnesium whitlockite; the first report of this mineral in articular cartilage. The mode of formation and role of these crystals remain unknown, although histological and histochemical investigations revealed a consistent association with intramatrical lipid, containing a phospholipid component. The results of this study are most tenable with a concept of opportunistic crystal deposition

Impact of serum source on human mesenchymal stem cell osteogenic differentiation in culture

Human mesenchymal stem cells (MSCs) show promise for musculoskeletal repair applications. Animal-derived serum is extensively used for MSC culture as a source of nutrients, extracellular matrix proteins and growth factors. However, the routine use of fetal calf serum (FCS) is not innocuous due to its animal antigens and ill-defined composition, driving the development of alternatives protocols. The present study sought to reduce exposure to FCS via the transient use of human serum. Transient exposure to animal serum had previously proved successful for the osteogenic differentiation of MSCs but had not yet been tested with alternative serum sources. Here, human serum was used to support the proliferation of MSCs, which retained surface marker expression and presented higher alkaline phosphatase activity than those in FCS-based medium. Addition of osteogenic supplements supported strong mineralisation over a 3-week treatment. When limiting serum exposure to the first five days of treatment, MSCs achieved higher differentiation with human serum than with FCS. Finally, human serum analysis revealed significantly higher levels of osteogenic components such as alkaline phosphatase and 25-Hydroxyvitamin D, consistent with the enhanced osteogenic effect. These results indicate that human serum used at the start of the culture offers an efficient replacement for continuous FCS treatment and could enable short-term exposure to patient-derived serum in the future

Oxidation state of a polyurethane membrane after plasma etching

Low moduli cell culture substrates can be used to apply dynamic mechanical strain to cells, by surface deformation. Understanding the surface interaction with cells is critical to improving cell adhesion and normal growth. A medical grade polyurethane (PU), Chronoflex AL 80A, was modified by oxygen plasma etching and characterised by X-ray photoelectron spectroscopy. Etching resulted in increased cross-linking at the isocyanate bond and formation of new oxygen moieties. The model, derived from patent data and XPS data of the unetched PU, indicated that the additional oxygen was likely to be hydroxyl and carbonyl groups. Etched membranes enhanced protein adhesion, resulting in full surface coverage compared to unetched PU. The etched PU supported cell adhesion and spreading, while the unetched PU was not conducive to monolayer formation

Integrated multi-assay culture model for stem cell chondrogenic differentiation

Recent osteochondral repair strategies highlight the promise of mesenchymal progenitors, an accessible stem cell source with osteogenic and chondrogenic potential, used in conjunction with biomaterials for tissue engineering. For this, regenerative medicine approaches require robust models to ensure selected cell populations can generate the desired cell type in a reproducible and measurable manner. Techniques for in vitro chondrogenic differentiation are well-established but largely qualitative, relying on sample staining and imaging. To facilitate the in vitro screening of pro-chondrogenic treatments, a 3D micropellet culture combined with three quantitative GAG assays has been developed, with a fourth parallel assay measuring sample content to enable normalisation. The effect of transforming growth factor beta (TGF-β) used to validate this culture format produced a measurable increase in proteoglycan production in the parallel assays, in both 2D and 3D culture configurations. When compared to traditional micropellets, the monolayer format appeared less able to detect changes in cell differentiation, however in-well 3D cultures displayed a significant differential response. Effects on collagen 2 expression confirmed these observations. Based on these results, a microplate format was optimised for 3D culture, in a high-throughput in-well configuration. This model showed improved sensitivity and confirmed the 3D micropellet in-well quantitative assays as an effective differentiation format compatible with streamlined, high-throughput chondrogenic screens

Laser sintering of nano-hydroxyapatite coated polyamide 12 powders

As part of a larger study on the laser sintering (LS) of nano-composite structures for biomedical applications, a wet mixing method was used to coat Polyamide 12 (PA12) particles with nano-hydroxyapatite (nHA). The addition of nHA significantly affected powder processability due to laser absorption and heat transfer effects which led to part warping. This phenomenon has not been reported in other studies investigating LS of polymer/HA and nHA powders. Nano-composites containing 0.5–1.5 wt% nHA were successfully produced and tensile testing showed that 0.5 wt% nHA provided the greatest reinforcement with a 20% and 15% increase in modulus and strength respectively. However, the elongation at break had significantly declined which was likely due to the formation of nHA aggregates at the sintering borders following the processing of the coated powders despite being initially well dispersed on the particle surface

Effect of Q-switched laser surface texturing of titanium on osteoblast cell response

Titanium and its alloys are important biomedical materials. It is known that the surface texture of implanted medical devices affects cell response. Control of cell response has the potential to enhance fixation of implants into bone and, in other applications, to prevent undesired cell adhesion. The potential use of a 100W Q-switched YAG laser miller (DMG Lasertec 60 HSC) for texturing titanium is investigated. A series of regular features with dimensions of the order of tens of micrometers are generated in the surface of titanium samples and the cell response to these features is determined. Characterisation of the laser milled features reveals features with a lengthscale of a few microns superposed on the larger scale structures, this is attributed to resolidification of molten droplets generated and propelled over the surface by individual laser pulses. The laser textured samples are exposed to osteoblast cells and it is seen that cells do respond to the features in the laser textured surfaces

Topographical and chemical effects of electrochemically assisted deposited hydroxyapatite coatings on osteoblast-like cells

A recently commercialised hydroxyapatite electrochemically assisted chemical deposition technique (BoneMaster) has been shown to induce increased bone apposition; whether this response is caused by the surface topography or chemistry is unknown. An in-vitro examination using human osteoblast-like cells was performed on a series of BoneMaster-coated surfaces. The chemistry was separated from the topography using a thin gold coating; Thermanox coverslips were used as a control. BoneMaster surfaces showed significantly greater alkaline phosphatase activity and osteocalcin production compared with controls; however, no difference was found between the gold-coated and uncoated BoneMaster samples, indicating topography is the main contributing factor

Bioresorbable composite bone fracture repair plates: manufacture and characterisation

This study reports on Bioresorbable composites manufactured using PLA as matrix and phosphate-based glass fibres as reinforcement. Composites were manufactured with varying volume fraction (from 25% - 45%) and mechanically tested

Impact of Serum Source on Human Mesenchymal Stem Cell Osteogenic Differentiation in Culture

Human mesenchymal stem cells (MSCs) show promise for musculoskeletal repair applications. Animal-derived serum is extensively used for MSC culture as a source of nutrients, extracellular matrix proteins and growth factors. However, the routine use of fetal calf serum (FCS) is not innocuous due to its animal antigens and ill-defined composition, driving the development of alternatives protocols. The present study sought to reduce exposure to FCS via the transient use of human serum. Transient exposure to animal serum had previously proved successful for the osteogenic differentiation of MSCs, but had not yet been tested with alternative serum sources. Here, human serum supported proliferation of MSCs, which retained surface marker expression and presented higher alkaline phosphatase activity than those in FCS-based medium. Addition of osteogenic supplements supported strong mineralisation over a 3-week treatment. When limiting serum exposure to the first 5 days of treatment, MSCs achieved higher differentiation with human serum than FCS. Finally, human serum analysis revealed significantly higher levels of osteogenic components such as alkaline phosphatase and 25-Hydroxyvitamin D, consistent with the enhanced osteogenic effect. These results indicate that human serum used at the start of the culture offers an efficient replacement for continuous FCS treatment, and could enable short-term exposure to patient-derived serum in the future

Transient serum exposure regimes to support dual differentiation of human mesenchymal stem cells

Human mesenchymal stem cells (MSCs), which can generate both osteoblasts and chondrocytes, represent an ideal resource for orthopaedic repair using tissue-engineering approaches. One major difficulty for the development of osteochondral constructs using undifferentiated MSCs is that serum is typically used in culture protocols to promote differentiation of the osteogenic component, whereas existing chondrogenic differentiation protocols rely on the use of serum-free conditions. In order to define conditions which could be compatible with both chondrogenic and osteogenic differentiation in a single bioreactor, we have analysed the efficiency of new biphasic differentiation regimes based on transient serum exposure followed by serum-free treatment. MSC differentiation was assessed either in serum-free medium or with a range of transient exposure to serum, and compared to continuous serum-containing treatment. Although osteogenic differentation was not supported in the complete absence of serum, marker expression and extensive mineralization analyses established that 5 days of transient exposure triggered a level of differentiation comparable to that observed when serum was present throughout. This initial phase of serum exposure was further shown to support the successful chondrogenic differentiation of MSCs, comparable to controls maintained in serum-free conditions throughout. This study indicates that a culture based on temporal serum exposure followed by serum-free treatment is compatible with both osteogenic and chondrogenic differentiation of MSCs. These results will allow the development of novel strategies for osteochondral tissue engineering approaches using MSCs for regenerative medicine. © 2012 John Wiley & Sons, Ltd