A microarray approach to the identification of polyurethanes for the isolation of human skeletal progenitor cells and augmentation of skeletal cell growth

The present study has examined the efficacy of a polymer microarray platform to screen a library of polyurethanes for applications such as human skeletal progenitor cell isolation and surface modification of tissue engineering scaffolds to enhance skeletal cell growth and differentiation. Analysis of polyurethane microarrays incubated with adult human bone marrow-derived STRO-1+ skeletal progenitor cells identified 31 polyurethanes (from the entire library of 120 polyurethanes) capable of binding to the STRO-1+ cells. Four polyurethanes (out of the 31 identified in the previous screen) were able to selectively immobilise cells of the STRO-1+ fraction from the heterogeneous human bone marrow mononuclear cell population. These four polyurethanes were highly selective for the STRO-1+ fraction of human bone marrow as they failed to bind STRO-1+ immature osteoblast-like MG63 cells, the STRO-1+ fraction of human fetal skeletal cells and differentiated osteoblast-like SaOs cells. Culture of human bone marrow-derived STRO-1+ cells on fibres of Polyglycolic acid (PGA) fleece surface modified by polyurethane adsorption, in osteogenic conditions, enhanced the expression of early osteogenic genes. Similarly, surface modification of PGA fleece fibres by polyurethane adsorption increased the responsiveness of MG63 cells, cultured on this scaffold, to 1,25 dihydroxy Vitamin D3, as demonstrated by enhanced Osteocalcin expression.     

Whole proteome analysis of osteoprogenitor differentiation induced by disordered nanotopography and mediated by ERK signalling

Topographic features can modulate cell behaviours such as proliferation, migration, differentiation and apoptosis. Biochemical mechanotransduction implies the conversion of mechanical forces (e.g. changes in cell spreading and morphology from changing surface topography) into biochemical signal via biomolecules. Still, little is known concerning which pathways may be directly involved in cell response to changes in the material surface. A number of pathways have been implicated using focused studies of ‘selected’ biomolecules rather than a global analysis of signal pathways. This study used a controlled disorder nanopit topography (NSQ50, fabricated by electron beam lithography) to direct osteoblast differentiation of progenitor cells. This topography is unique as it represents a middle route (from absolute order or random roughness) that allows osteoconversion with similar efficiency as dexamethasone and ascorbate treatment. Two direct-comparison proteomics techniques, firstly gel-based and then chromatography-based, were used to analyse progenitor proteome changes in response to the nanotopography. Many of the changed proteins form part of the Extracellular Signal-regulated Kinase (ERK1/2) pathway.     

Augmentation of skeletal tissue formation in impaction bone grafting using vaterite microsphere biocomposites

The development of particulate bone void fillers with added biological function to augment skeletal tissue formation will lead to improved efficacy in bone replacement surgery. We demonstrate the potential for vaterite microsphere biocomposites to augment bone matrix formation within an in vivo model for impaction bone grafting seeded with human bone marrow stromal cells. In vitro tests demonstrate the significance of vaterite microspheres in the activation and promotion of 3D skeletal tissue formation. Further in vitro experiments using functionalized microspheres with surface integrated RGD peptide activate co-cultured skeletal populations in pellets and promote secretion of extracellular matrix collagens and human osteocalcin. Specific temporal release of entrapped RNase A was successfully demonstrated using these specialized microspheres with integrated magnetic beads, which physically disrupted the inorganic macrostructure. These studies demonstrate that bio-inspired calcium carbonate microspheres augment in vivo bone formation in impaction bone grafting. Such microspheres with added biological functionality offer innovative therapeutic approaches to activate skeletal populations and enhance bone formation with reparative implications for hard tissues.     

Development of in vivo muCT evaluation of neovascularisation in tissue engineered bone constructs

Due to an increasing aging population the need for innovative approaches to aid skeletal repair and reconstruction is a significant socio-economic increasing problem. The emerging discipline of tissue engineering has sort to augment the growth and repair of bone loss particularly in areas of trauma, degeneration and revision surgery. However, the initiation and development of a fully functional vascular network are critical for bioengineered bone to repair large osseous defects, whether the material is osteosynthetic (poly (d,l)-lactic acid, PLA) or natural bone allograft. Quantification and three-dimensional visualization of new vessel networks remain a problem in bone tissue engineering constructs. A novel technique utilising a radio-opaque dye and micro-computed tomography (muCT) has been developed and applied to study angiogenesis in an impaction bone graft model. Tissue-engineered constructs combining human bone marrow stromal cells (HBMSC) with natural allograft and synthetic grafts (PLA) were impacted and implanted into the subcutis of MF-1 nu/nu mice for a period of 28 days. Microfil consisting of radio-opaque polymer was perfused through the mice and scanned using a Bench Top CT system for micro-computed tomography. Analysis of three-dimensional muCT reconstructions demonstrated an increase in vessel volume and vessel number in the impacted scaffolds/HBMC compared to scaffolds alone. Vessel volume: allograft/HBMSC=0.57 mm(3)+/-0.19; allograft=0.04 mm(3)+/-0.04; PLA/HBMSC=1.19 mm(3)+/-0.31; and PLA=0.12 mm(3)+/-0.01. Penetrating vessel number: allograft/HBMSC=22.33+/-3.21; allograft=3.67+/-1.153; PLA/HBMSC=32.67+/-8.33; and PLA=7.67+/-3.06. Type 1 collagen and von Willebrand factor immunohistochemistry in scaffold/HBMSC constructs indicated the osteogenic cell phenotype, and new blood vessel formation respectively. Contrast-enhanced 3D reconstructions facilitated the visualization and quantification of neovascularisation. This novel technique has been used to demonstrate neovascularisation in impacted tissue engineered constructs providing a facile approach with wide experimental application.     

From bench to clinic and back: skeletal stem cells and impaction bone grafting for regeneration of bone defects

Tissue engineering offers enormous potential for bone regeneration. Despite extensive in vitro and in vivo work, few strategies translate into clinical practice. This paper describes the combination of skeletal stem cells (SSCs) and impaction bone grafting (IBG) for the treatment of patients with bone defects associated with avascular necrosis of the femoral head. SSCs and milled allograft were impacted into necrotic bone in the femoral heads of four patients. Three patients remained asymptomatic at 22–44 month follow‐up, but one patient has required total hip replacement (both hips). This has allowed retrieval of the femoral heads, which were analysed structurally and functionally by μCT, histology and mechanical testing. A central channel of impacted bone was found in the femoral heads, which displayed a mature trabecular micro‐architecture. The impacted bone was denser than the surrounding trabecular bone, as strong in compression and with histological micro‐architecture comparable to that of trabecular bone. Analysis of the retrieved femoral head samples has demonstrated that this tissue‐engineering strategy regenerates bone that is both structurally and functionally analogous to normal trabecular bone. SSCs, together with IBG, have proved an effective treatment for avascular necrosis of the femoral head and offer significant potential for the broader spectrum of bone defects. Copyright © 2012 John Wiley & Sons, Ltd.     

Intrauterine programming of bone. Part 1: Alteration of the osteogenic environment

Summary Osteoporosis is believed to partly be programmed in utero. Rat dams were given a low protein diet during pregnancy and 135 offspring studied at different ages. Bone biochemistry showed altered characteristics. Altered in utero diet has consequences for later life. Introduction Epidemiological studies suggest skeletal growth is programmed during intrauterine and early postnatal life. We have investigated this in a rat model of maternal protein insufficiency. Methods Dams received either 18% w/w (control) or 9% w/w (low protein) diet during pregnancy, and the offspring were studied at selected time points (4, 8, 12, 16, 20, 47 weeks). Results Alkaline phosphatase activity in controls reached peak levels from 8 to 20 weeks of age. In contrast, restricted diet offspring were at peak levels from 4 weeks of age. Peak levels were similar in both groups. Serum IGF-1 levels were lower in female restricted diet offspring at 4 weeks of age, and serum osteocalcin was significantly higher at 4 weeks of age in male and female offspring from mothers fed the restricted diet, whereas serum 25-OH vitamin D was significantly lower in restricted diet males at 8, 12, and 20 weeks of age. Conclusions These data indicate that a low protein diet in utero affected the osteogenic environment in the offspring with effects that persist into late adulthood. These results indicate the key role of the nutritional environment in early development on programming of skeletal development with implicit consequences in later life. This work was supported by Research into Ageing.     

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 microg/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.     

Remodelling of human bone on the chorioallantoic membrane of the chicken egg: De novo bone formation and resorption

Traditionally used as an angiogenic assay, the chorioallantoic membrane (CAM) assay of the chick embryo offers significant potential as an in vivo model for xenograft organ culture. Viable human bone can be cultivated on the CAM and increases in bone volume are evident; however, it remains unclear by what mechanism this change occurs and whether this reflects the physiological process of bone remodelling. In this study we tested the hypothesis that CAM‐induced bone remodelling is a consequence of host and graft mediated processes. Bone cylinders harvested from femoral heads post surgery were placed on the CAM of green fluorescent protein (GFP)‐chick embryos for 9 days, followed by micro computed tomography (μCT) and histological analysis. Three‐dimensional registration of consecutive μCT‐scans showed newly mineralised tissue in CAM‐implanted bone cylinders, as well as new osteoid deposition histologically. Immunohistochemistry demonstrated the presence of bone resorption and formation markers (Cathepsin K, SOX9 and RUNX2) co‐localising with GFP staining, expressed by avian cells only. To investigate the role of the human cells in the process of bone formation, decellularised bone cylinders were implanted on the CAM and comparable increases in bone volume were observed, indicating that avian cells were responsible for the bone mineralisation process. Finally, CAM‐implantation of acellular collagen sponges, containing bone morphogenetic protein 2, resulted in the deposition of extracellular matrix and tissue mineralisation. These studies indicate that the CAM can respond to osteogenic stimuli and support formation or resorption of implanted human bone, providing a humanised CAM model for regenerative medicine research and a novel short‐term in vivo model for tissue engineering and biomaterial testing.     

Human mesenchymal factors induce rat hippocampal‐ and human neural stem cell dependent oligodendrogenesis

The generation of new oligodendrocytes is essential for adult brain repair in diseases such as multiple sclerosis. We previously identified the multifunctional p57kip2 protein as a negative regulator of myelinating glial cell differentiation and as an intrinsic switch of glial fate decision in adult neural stem cells (aNSCs). In oligodendroglial precursor cells (OPCs), p57kip2 protein nuclear exclusion was recently found to be rate limiting for differentiation to proceed. Furthermore, stimulation with mesenchymal stem cell (MSC)‐derived factors enhanced oligodendrogenesis by yet unknown mechanisms. To elucidate this instructive interaction, we investigated to what degree MSC secreted factors are species dependent, whether hippocampal aNSCs respond equally well to such stimuli, whether apart from oligodendroglial differentiation also tissue integration and axonal wrapping can be promoted and whether the oligodendrogenic effect involved subcellular translocation of p57kip2. We found that CC1 positive oligodendrocytes within the hilus express nuclear p57kip2 protein and that MSC dependent stimulation of cultured hippocampal aNSCs was not accompanied by nuclear p57kip2 exclusion as observed for parenchymal OPCs after spontaneous differentiation. Stimulation with human MSC factors was observed to equally promote rat stem cell oligodendrogenesis, axonal wrapping and tissue integration. As forced nuclear shuttling of p57kip2 led to decreased CNPase‐ but elevated GFAP expression levels, this indicates heterogenic oligodendroglial mechanisms occurring between OPCs and aNSCs. We also show for the first time that dominant pro‐oligodendroglial factors derived from human fetal MSCs can instruct human induced pluripotent stem cell‐derived NSCs to differentiate into O4 positive oligodendrocytes.     

Development of specific collagen scaffolds to support the osteogenic and chondrogenic differentiation of human bone marrow stromal cells

Type I Collagen matrices of defined porosity, incorporating carbonate substituted hydroxyapatite (HA) crystals, were assessed for their ability to support osteo- and chondrogenic differentiation of human bone marrow stromal cells (HBMSCs). Collagen-HA composite scaffolds supported the osteogenic differentiation of HBMSCs both in vitro and in vivo as demonstrated by histological and micro-CT analyses indicating the extensive penetration of alkaline phosphatase expressing cells and new matrix synthesis with localised areas immunologically positive for osteocalcin. In vivo, extensive new osteoid formation of implant origin was observed in the areas of vasculature. Chondrogenic matrix synthesis was evidenced in the peripheral regions of pure collagen systems by an abundance of Sox9 expressing chondrocytes embedded within a proteoglycan and collagen II rich ECM. The introduction of microchannels to the scaffold architecture was seen to enhance chondrogenesis. Tissue specific gene expression and corresponding matrix synthesis indicate that collagen matrices support the growth and differentiation of HBMSCs and suggest the potential of this platform for understanding the ECM cues necessary for osteogenesis and chondrogenesis.