Rahul S. Tare

Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency

There is currently an unmet need for the supply of autologous, patient-specific stem cells for regenerative therapies in the clinic. Mesenchymal stem cell differentiation can be driven by the material/cell interface suggesting a unique strategy to manipulate stem cells in the absence of complex soluble chemistries or cellular reprogramming. However, so far the derivation and identification of surfaces that allow retention of multipotency of this key regenerative cell type have remained elusive. Adult stem cells spontaneously differentiate in culture, resulting in a rapid diminution of the multipotent cell population and their regenerative capacity. Here we identify a nanostructured surface that retains stem-cell phenotype and maintains stem-cell growth over eight weeks. Furthermore, the study implicates a role for small RNAs in repressing key cell signalling and metabolomic pathways, demonstrating the potential of surfaces as non-invasive tools with which to address the stem cell niche.

BMP2 Commitment to the Osteogenic Lineage Involves Activation of Runx2 by DLX3 and a Homeodomain Transcriptional Network

Several homeodomain (HD) proteins are critical for skeletal patterning and respond directly to BMP2 as an early step in bone formation. RUNX2, the earliest transcription factor proven essential for commitment to osteoblastogenesis, is also expressed in response to BMP2. However, there is a gap in our knowledge of the regulatory cascade from BMP2 signaling to the onset of osteogenesis. Here we show that BMP2 induces DLX3, a homeodomain protein that activates Runx2 gene transcription. Small interfering RNA knockdown studies in osteoblasts validate that DLX3 is a potent regulator of Runx2. Furthermore in Runx2 null cells, DLX3 forced expression suffices to induce transcription of Runx2, osteocalcin, and alkaline phosphatase genes, thus defining DLX3 as an osteogenic regulator independent of RUNX2. Our studies further show regulation of the Runx2 gene by several homeodomain proteins: MSX2 and CDP/cut repress whereas DLX3 and DLX5 activate endogenous Runx2 expression and promoter activity in non-osseous cells and osteoblasts. These HD proteins exhibit distinct temporal expression profiles during osteoblast differentiation as well as selective association with Runx2 chromatin that is related to Runx2 transcriptional activity and recruitment of RNA polymerase II. Runx2 promoter mutagenesis shows that multiple HD elements control expression of Runx2 in relation to the stages of osteoblast maturation. Our studies establish mechanisms for commitment to the osteogenic lineage directly through BMP2 induction of HD proteins DLX3 and DLX5 that activate Runx2, thus delineating a transcriptional regulatory pathway mediating osteoblast differentiation. We propose that the three homeodomain proteins MSX2, DLX3, and DLX5 provide a key series of molecular switches that regulate expression of Runx2 throughout bone formation.

Fabrication of pillar-like titania nanostructures on titanium and their interactions with human skeletal stem cells.

Surface nanotopography is known to influence the interaction of human skeletal (mesenchymal) stem cells (hMSC) with a material surface. While most surface nanopatterning has been performed on polymer-based surfaces there is a need for techniques to produce well-defined topography features with tuneable sizes on relevant load-bearing implant materials such as titanium (Ti). In this study titania nanopillar structures with heights of either 15, 55 or 100 nm were produced on Ti surfaces using anodization through a porous alumina mask. The influence of the surface structure heights on hMSC adhesion, spreading, cytoskeletal formation and differentiation was examined. The 15 nm high topography features resulted in the greatest cell response with bone matrix nodule forming on the Ti surface after 21 days.

Induction of human osteoprogenitor chemotaxis, proliferation, differentiation, and bone formation by osteoblast stimulating factor-1/pleiotrophin: osteoconductive biomimetic scaffolds for tissue engineering.

The process of bone growth, regeneration, and remodeling is mediated, in part, by the immediate cell‐matrix environment. Osteoblast stimulating factor‐1 (OSF‐1), more commonly known as pleiotrophin (PTN), is an extracellular matrix‐associated protein, present in matrices, which act as targets for the deposition of new bone. However, the actions of PTN on human bone progenitor cells remain unknown. We examined the effects of PTN on primary human bone marrow stromal cells chemotaxis, differentiation, and colony formation (colony forming unit‐fibroblastic) in vitro, and in particular, growth and differentiation on three‐dimensional biodegradable porous scaffolds adsorbed with PTN in vivo. Primary human bone marrow cells were cultured on tissue culture plastic or poly(DL‐lactic acid‐co‐glycolic acid) (PLGA; 75:25) porous scaffolds with or without addition of recombinant human PTN (1 pg‐50 ng/ml) in basal and osteogenic conditions. Negligible cellular growth was observed on PLGA scaffold alone, generated using a super‐critical fluid mixing method. PTN (50 μg/ml) was chemotactic to human osteoprogenitors and stimulated total colony formation, alkaline phosphatase‐positive colony formation, and alkaline phosphatase‐specific activity at concentrations as low as 10 pg/ml compared with control cultures. The effects were time‐dependent. On three‐dimensional scaffolds adsorbed with PTN, alkaline phosphatase activity, type I collagen formation, and synthesis of cbfa‐1, osteocalcin, and PTN were observed by immunocytochemistry and PTN expression by in situ hybridization. PTN‐adsorbed constructs showed morphologic evidence of new bone matrix and cartilage formation after subcutaneous implantation as well as within diffusion chambers implanted into athymic mice. In summary, PTN has the ability to promote adhesion, migration, expansion, and differentiation of human osteoprogenitor cells, and these results indicate the potential to develop protocols for de novo bone formation for skeletal repair that exploit cell‐matrix interactions.

HOXA10 controls osteoblastogenesis by directly activating bone regulatory and phenotypic genes

ABSTRACT HOXA10 is necessary for embryonic patterning of skeletal elements, but its function in bone formation beyond this early developmental stage is unknown. Here we show that HOXA10 contributes to osteogenic lineage determination through activation of Runx2 and directly regulates osteoblastic phenotypic genes. In response to bone morphogenic protein BMP2, Hoxa10 is rapidly induced and functions to activate the Runx2 transcription factor essential for bone formation. A functional element with the Hox core motif was characterized for the bone-related Runx2 P1 promoter. HOXA10 also activates other osteogenic genes, including the alkaline phosphatase, osteocalcin, and bone sialoprotein genes, and temporally associates with these target gene promoters during stages of osteoblast differentiation prior to the recruitment of RUNX2. Exogenous expression and small interfering RNA knockdown studies establish that HOXA10 mediates chromatin hyperacetylation and trimethyl histone K4 (H3K4) methylation of these genes, correlating to active transcription. HOXA10 therefore contributes to early expression of osteogenic genes through chromatin remodeling. Importantly, HOXA10 can induce osteoblast genes in Runx2 null cells, providing evidence for a direct role in mediating osteoblast differentiation independent of RUNX2. We propose that HOXA10 activates RUNX2 in mesenchymal cells, contributing to the onset of osteogenesis, and that HOXA10 subsequently supports bone formation by direct regulation of osteoblast phenotypic genes.

Biofabrication of bone tissue: approaches, challenges and translation for bone regeneration.

The rising incidence of bone disorders has resulted in the need for more effective therapies to meet this demand, exacerbated by an increasing ageing population. Bone tissue engineering is seen as a means of developing alternatives to conventional bone grafts for repairing or reconstructing bone defects by combining biomaterials, cells and signalling factors. However, skeletal tissue engineering has not yet achieved full translation into clinical practice as a consequence of several challenges. The use of additive manufacturing techniques for bone biofabrication is seen as a potential solution, with its inherent capability for reproducibility, accuracy and customisation of scaffolds as well as cell and signalling factor delivery. This review highlights the current research in bone biofabrication, the necessary factors for successful bone biofabrication, in addition to the current limitations affecting biofabrication, some of which are a consequence of the limitations of the additive manufacturing technology itself.

Genomic expression of mesenchymal stem cells to altered nanoscale topographies

The understanding of cellular response to the shape of their environment would be of benefit in the development of artificial extracellular environments for potential use in the production of biomimetic surfaces. Specifically, the understanding of how cues from the extracellular environment can be used to understand stem cell differentiation would be of special interest in regenerative medicine. In this paper, the genetic profile of mesenchymal stem cells cultured on two osteogenic nanoscale topographies (pitted surface versus raised islands) are compared with cells treated with dexamethasone, a corticosteroid routinely used to stimulate bone formation in culture from mesenchymal stem cells, using 19k gene microarrays as well as 101 gene arrays specific for osteoblast and endothelial biology. The current studies show that by altering the shape of the matrix a cell response (genomic profile) similar to that achieved with chemical stimulation can be elicited. Here, we show that bone formation can be achieved with efficiency similar to that of dexamethasone with the added benefit that endothelial cell development is not inhibited. We further show that the mechanism of action of the topographies and dexamethasone differs. This could have an implication for tissue engineering in which a simultaneous, targeted, development of a tissue, such as bone, without the suppression of angiogenesis to supply nutrients to the new tissue is required. The results further demonstrate that perhaps the shape of the extracellular matrix is critical to tissue development.

Characterization and multipotentiality of human fetal femur-derived cells: Implications for skeletal tissue regeneration

To date, the plasticity, multipotentiality, and characteristics of progenitor cells from fetal skeletal tissue remain poorly defined. This study has examined cell populations from human fetal femurs in comparison with adult‐derived mesenchymal cell populations. Real‐time quantitative polymerase chain reaction demonstrated expression of mesenchymal progenitor cell markers by fetal‐derived cells in comparison with unselected adult‐derived and immunoselected STRO‐1–enriched adult populations. Multipotentiality was examined using cells derived from femurs and single‐cell clones, culture‐expanded from explants, and maintained in basal medium prior to exposure to adipogenic, osteogenic, and chondrogenic conditions. Adipocyte formation was confirmed by Oil Red O lipid staining and aP2 immunocytochemistry, with expression of peroxisome proliferation‐activated receptor‐γ detected only in adipogenic conditions. In chondrogenic pellets, chondrocytes lodged within lacunae and embedded within dense proteoglycan matrix were observed using Alcian blue/Sirius red staining and type II collagen immunocytochemistry. Osteogenic differentiation was confirmed by alkaline phosphatase staining and type I collagen immunocytochemistry as well as by gene expression of osteopontin and osteocalcin. Single‐cell clonal analysis was used to demonstrate multipotentiality of the fetal‐derived populations with the formation of adipogenic, chondrogenic, and osteogenic populations. Mineralization and osteoid formation were observed after culture on biomimetic scaffolds with extensive matrix accumulation both in vitro and in vivo after subcutaneous implantation in severely compromised immunodeficient mice. These studies demonstrate the proliferative and multipotential properties of fetal femur–derived cells in comparison with adult‐derived cells. Selective differentiation and immunophenotyping will determine the potential of these fetal cells as a unique alternative model and cell source in the restoration of damaged tissue.

Versatile Biocompatible Polymer Hydrogels: Scaffolds for Cell Growth

A three-dimensional, biocompatible hydrogel (see picture) was generated by combining two cationic polymers, chitosan and poly(ethylenimine). The hydrogels were stable under cell-culture conditions and facilitated cell proliferation, yet prevented dedifferentiation of primary human skeletal cells into fibroblasts. A variety of materials such as DNA, proteins, and peptides can be stably incorporated into the gel network

Pleiotrophin/Osteoblast‐Stimulating Factor 1: Dissecting Its Diverse Functions in Bone Formation

OSF‐1, more commonly known as pleiotrophin (PTN) or heparin‐binding growth‐associated molecule (HB‐GAM), belongs to a new family of secreted HB proteins, which are structurally unrelated to any other growth factor family. The aims of this study were to dissect the diverse functions of PTN in bone formation. The study showed that PTN was synthesized by osteoblasts at an early stage of osteogenic differentiation and was present at sites of new bone formation, where PTN was stored in the new bone matrix. Low concentrations (10 pg/ml) of PTN stimulated osteogenic differentiation of mouse bone marrow cells and had a modest effect on their proliferation, whereas higher concentrations (ng/ml) had no effect. However, PTN did not have the osteoinductive potential of bone morphogenetic proteins (BMPs) because it failed to convert C2C12 cells, a premyoblastic cell line, to the osteogenic phenotype, whereas recombinant human BMP‐2 (rhBMP‐2) was able to do so. When PTN was present together with rhBMP‐2 during the osteoinductive phase, PTN inhibited the BMP‐mediated osteoinduction in C2C12 cells at concentrations between 0.05 pg/ml and 100 ng/ml. However, when added after osteoinduction had been achieved, PTN enhanced further osteogenic differentiation. An unusual effect of PTN (50 ng/ml) was the induction of type I collagen synthesis by chondrocytes in organ cultures of chick nasal cartilage and rat growth plates. Thus, PTN had multiple effects on bone formation and the effects were dependent on the concentration of PTN and the timing of its presence. To explain these multiple effects, we propose that PTN is an accessory signaling molecule, which is involved in a variety of processes in bone formation. PTN enhances or inhibits primary responses depending on the prevailing concentrations, the primary stimulus, and the availability of appropriate receptors.