Owen Davies

A comparison of the in vitro mineralisation and dentinogenic potential of mesenchymal stem cells derived from adipose tissue, bone marrow and dental pulp

Stem-cell-based therapies provide a biological basis for the regeneration of mineralised tissues. Stem cells isolated from adipose tissue (ADSCs), bone marrow (BMSCs) and dental pulp (DPSCs) have the capacity to form mineralised tissue. However, studies comparing the capacity of ADSCs with BMSCs and DPSCs for mineralised tissue engineering are lacking, and their ability to regenerate dental tissues has not been fully explored. Characterisation of the cells using fluorescence-activated cell sorting and semi-quantitative reverse transcription PCR for MSC markers indicated that they were immunophenotypically similar. Alizarin red (AR) staining and micro-computed tomography (µCT) analyses demonstrated that the osteogenic potential of DPSCs was significantly greater than that of BMSCs and ADSCs. Scanning electron microscopy and AR staining showed that the pattern of mineralisation in DPSC cultures differed from ADSCs and BMSCs, with DPSC cultures lacking defined mineralised nodules and instead forming a diffuse layer of low-density mineral. Dentine matrix components (DMCs) were used to promote dentinogenic differentiation. Their addition to cultures resulted in increased amounts of mineral deposited in all three cultures and significantly increased the density of mineral deposited in BMSC cultures, as determined by µCT analysis. Addition of DMCs also increased the relative gene expression levels of the dentinogenic markers dentine sialophosphoprotein and dentine matrix protein 1 in ADSC and BMSC cultures. In conclusion, DPSCs show the greatest potential to produce a comparatively high volume of mineralised matrix; however, both dentinogenesis and mineral volume was enhanced in ADSC and BMSC cultures by DMCs, suggesting that these cells show promise for regenerative dental therapies.

The effects of cryopreservation on cells isolated from adipose, bone marrow and dental pulp tissues☆

The effects of cryopreservation on mesenchymal stem cell (MSC) phenotype are not well documented; however this process is of increasing importance for regenerative therapies. This study examined the effect of cryopreservation (10% dimethyl-sulfoxide) on the morphology, viability, gene-expression and relative proportion of MSC surface-markers on cells derived from rat adipose, bone marrow and dental pulp. Cryopreservation significantly reduced the number of viable cells in bone marrow and dental pulp cell populations but had no observable effect on adipose cells. Flow cytometry analysis demonstrated significant increases in the relative expression of MSC surface-markers, CD90 and CD29/CD90 following cryopreservation. sqRT-PCR analysis of MSC gene-expression demonstrated increases in pluripotent markers for adipose and dental pulp, together with significant tissue-specific increases in CD44, CD73-CD105 following cryopreservation. Cells isolated from different tissue sources did not respond equally to cryopreservation with adipose tissue representing a more robust source of MSCs.

Isolation of adipose and bone marrow mesenchymal stem cells using CD29 and CD90 modifies their capacity for osteogenic and adipogenic differentiation

Mesenchymal stem cells isolated from rats are frequently used for tissue engineering research. However, considerable differences have been identified between rat mesenchymal stem cells and those derived from humans, and no defined panel of markers currently exists for the isolation of these cells. The aim of this study was to examine the effects of cell sorting for CD29+/CD90+ cells from rat adipose and bone marrow tissues on their differentiation and expression of stem cell–associated genes. Flow cytometry showed 66% and 78% CD29+/CD90+ positivity within passage 1 of adipose and bone marrow cultures, respectively. CD29+/CD90+ cells showed a reduction in both osteogenic and adipogenic differentiation when compared with unsorted cells, as determined by alizarin red and Oil Red-O staining, respectively. These findings could not entirely be explained by fluorescence-activated cell sorting–induced cell injury as sort recovery was only modestly affected in adipose-derived cells. Maintaining cells in fluorescence-activated cell sorting buffer did not affect adipose-derived cell viability, but a significant (p < 0.05) reduction was found in bone marrow–derived cell viability. Additionally, CD29+/CD90+ selection was associated with a significant decrease in the expression of Lin28, Sox2, Nanog and CD73 in adipose-derived cell cultures, whereas differences in stem cell–associated gene expression were not observed in sorted bone marrow–derived cell cultures. In summary, this study demonstrated that fluorescence-activated cell sorting had differential effects on adipose-derived cells and bone marrow–derived cells, and both CD29+/CD90+ cells displayed a significantly reduced capacity for osteogenic/adipogenic differentiation. In conclusion, we identify that maintaining heterogeneity within the mesenchymal stem cell population may be important for optimal differentiation.

Identifying the cellular mechanisms leading to heterotopic ossification

Heterotopic ossification (HO) is a debilitating condition defined by the de novo development of bone within non-osseous soft tissues, and can be either hereditary or acquired. The hereditary condition, fibrodysplasia ossificans progressiva is rare but life threatening. Acquired HO is more common and results from a severe trauma that produces an environment conducive for the formation of ectopic endochondral bone. Despite continued efforts to identify the cellular and molecular events that lead to HO, the mechanisms of pathogenesis remain elusive. It has been proposed that the formation of ectopic bone requires an osteochondrogenic cell type, the presence of inductive agent(s) and a permissive local environment. To date several lineage-tracing studies have identified potential contributory populations. However, difficulties identifying cells in vivo based on the limitations of phenotypic markers, along with the absence of established in vitro HO models have made the results difficult to interpret. The purpose of this review is to critically evaluate current literature within the field in an attempt identify the cellular mechanisms required for ectopic bone formation. The major aim is to collate all current data on cell populations that have been shown to possess an osteochondrogenic potential and identify environmental conditions that may contribute to a permissive local environment. This review outlines the pathology of endochondral ossification, which is important for the development of potential HO therapies and to further our understanding of the mechanisms governing bone formation.

Defining the Balance between Regeneration and Pathological Ossification in Skeletal Muscle Following Traumatic Injury

Heterotopic ossification (HO) is characterized by the formation of bone at atypical sites. This type of ectopic bone formation is most prominent in skeletal muscle, most frequently resulting as a consequence of physical trauma and associated with aberrant tissue regeneration. The condition is debilitating, reducing a patients range of motion and potentially causing severe pathologies resulting from nerve and vascular compression. Despite efforts to understand the pathological processes governing HO, there remains a lack of consensus regarding the micro-environmental conditions conducive to its formation, and attempting to define the balance between muscle regeneration and pathological ossification remains complex. The development of HO is thought to be related to a complex interplay between factors released both locally and systemically in response to trauma. It develops as skeletal muscle undergoes significant repair and regeneration, and is likely to result from the misdirected differentiation of endogenous or systemically derived progenitors in response to biochemical and/or environmental cues. The process can be sequentially delineated by the presence of inflammation, tissue breakdown, adipogenesis, hypoxia, neo-vasculogenesis, chondrogenesis and ossification. However, exactly how each of these stages contributes to the formation of HO is at present not well understood. Our previous review examined the cellular contribution to HO. Therefore, the principal aim of this review will be to comprehensively outline changes in the local tissue micro-environment following trauma, and identify how these changes can alter the balance between skeletal muscle regeneration and ectopic ossification. An understanding of the mechanisms governing this condition is required for the development and advancement of HO prophylaxis and treatment, and may even hold the key to unlocking novel methods for engineering hard tissues.

Annexin-enriched osteoblast-derived vesicles act as an extracellular site of mineral nucleation within developing stem cell cultures

The application of extracellular vesicles (EVs) as natural delivery vehicles capable of enhancing tissue regeneration could represent an exciting new phase in medicine. We sought to define the capacity of EVs derived from mineralising osteoblasts (MO-EVs) to induce mineralisation in mesenchymal stem cell (MSC) cultures and delineate the underlying biochemical mechanisms involved. Strikingly, we show that the addition of MO-EVs to MSC cultures significantly (P < 0.05) enhanced the expression of alkaline phosphatase, as well as the rate and volume of mineralisation beyond the current gold-standard, BMP-2. Intriguingly, these effects were only observed in the presence of an exogenous phosphate source. EVs derived from non-mineralising osteoblasts (NMO-EVs) were not found to enhance mineralisation beyond the control. Comparative label-free LC-MS/MS profiling of EVs indicated that enhanced mineralisation could be attributed to the delivery of bridging collagens, primarily associated with osteoblast communication, and other non-collagenous proteins to the developing extracellular matrix. In particular, EV-associated annexin calcium channelling proteins, which form a nucleational core with the phospholipid-rich membrane and support the formation of a pre-apatitic mineral phase, which was identified using infrared spectroscopy. These findings support the role of EVs as early sites of mineral nucleation and demonstrate their value for promoting hard tissue regeneration.

PDGF is a potent initiator of bone formation in a tissue engineered model of pathological ossification

Heterotopic ossification (HO) is a debilitating condition defined by the rapid formation of bone in soft tissues. What makes HO fascinating is first the rate at which bone is deposited, and second the fact that this bone is structurally and compositionally similar to that of a healthy adult. If the mechanisms governing HO are understood, they have the potential to be exploited for the development of potent osteoinductive therapies. With this aim, a tissue‐engineered skeletal muscle was used model to better understand the role of inflammation on this debilitating phenomenon. It was shown that myoblasts could be divided into two distinct populations: myogenic cells and undifferentiated ‘reserve’ cells. Gene expression analysis of myogenic and osteoregulatory markers confirmed that ‘reserve’ cells were primed for osteogenic differentiation but had a reduced capacity for myogenesis. Osteogenic differentiation was significantly enhanced in the presence of platelet‐derived growth factor (PDGF)‐BB and bone morphogenetic protein 2 (BMP2), and correlated with conversion to a Sca‐1+/CD73+ phenotype. Alizarin red staining showed that PDGF‐BB promoted significantly more mineral deposition than BMP2. Finally, it was shown that PDGF‐induced mineralization was blocked in the presence of the pro‐inflammatory cytokines tumour necrosis factor‐α and interleukin 1. In conclusion, the present study identified that PDGF‐BB is a potent osteoinductive factor in a model of tissue‐engineered skeletal muscle, and that the osteogenic capacity of this protein was modulated in the presence of pro‐inflammatory cytokines. These findings reveal a possible mechanism by which HO develops following trauma. Importantly, these findings have implications for the induction and control of bone formation for regenerative medicine.

Interfacial Mineral Fusion and Tubule Entanglement as a Means to Harden a Bone Augmentation Material

A new bone augmenting material is reported, which is formed from calcium-loaded hydrogel-based spheres. On immersion of these spheres in a physiological medium, they become surrounded with a sheath of precipitate, which ruptures due to a build-up in osmotic pressure. This results in the formation of mineral tubes that protrude from the sphere surface. When brought into close contact with one another, these spheres become fused through the entanglement and subsequent interstitial mineralization of the mineral tubules. The tubular calcium phosphate induces the expression of osteogenic genes (runt-related transcription factor 2 (RUNX2), transcription factor SP7 (SP7), collagen type 1 alpha 1 (COL1A1), and bone gamma-carboxyglutamic acid-containing protein (BGLAP)) and promotes the formation of mineral nodules in preosteoblast cultures comparable to an apatitic calcium phosphate phase. Furthermore, alkaline phosphatase (ALP) is significantly upregulated in the presence of tubular materials after 10 d in culture compared with control groups (p < 0.001) and sintered apatite (p < 0.05). This is the first report of a bioceramic material that is formed in its entirety in situ and is therefore likely to provide a better proxy for biological mineral than other existing synthetic alternatives to bone grafts.

Isolation and Cryopreservation of Stem Cells from Dental Tissues

One of the principal aims of cell-based therapies is to deliver personalised medicine for the repair and regeneration of tissues lost to accidents or disease. To achieve this aim sub-zero temperatures (−196°) are applied that halt biological activity, thus preserving the cells for future clinical applications. The idea of banking stem cells as a means of ‘biological insurance’ has seen a recent rise in popularity that is at least in part due to increased media attention and a greater public awareness of regenerative medicine. Consequently, several companies now exist offering individuals the opportunity to store their own multipotent cells, with the aim of future therapeutic application to restore or regenerate a multitude of tissues throughout the body.