Mechano-Chemical Aspects of Organ Formation in Arabidopsis thaliana: The Relationship between Auxin and Pectin

How instructive signals are translated into robust and predictable changes in growth is a central question in developmental biology. Recently, much interest has centered on the feedback between chemical instructions and mechanical changes for pattern formation in development. In plants, the patterned arrangement of aerial organs, or phyllotaxis, is instructed by the phytohormone auxin; however, it still remains to be seen how auxin is linked, at the apex, to the biochemical and mechanical changes of the cell wall required for organ outgrowth. Here, using Atomic Force Microscopy, we demonstrate that auxin reduces tissue rigidity prior to organ outgrowth in the shoot apex of Arabidopsis thaliana, and that the de-methyl-esterification of pectin is necessary for this reduction. We further show that development of functional organs produced by pectin-mediated ectopic wall softening requires auxin signaling. Lastly, we demonstrate that coordinated localization of the auxin transport protein, PIN1, is disrupted in a naked-apex produced by increasing cell wall rigidity. Our data indicates that a feedback loop between the instructive chemical auxin and cell wall mechanics may play a crucial role in phyllotactic patterning.

Stimulation of new bone formation by direct transfer of osteogenic plasmid genes.

Degradable matrices containing expression plasmid DNA [gene-activated matrices (GAMs)] were implanted into segmental gaps created in the adult rat femur. Implantation of GAMs containing beta-galactosidase or luciferase plasmids led to DNA uptake and functional enzyme expression by repair cells (granulation tissue) growing into the gap. Implantation of a GAM containing either a bone morphogenetic protein-4 plasmid or a plasmid coding for a fragment of parathyroid hormone (amino acids 1-34) resulted in a biological response of new bone filling the gap. Finally, implantation of a two-plasmid GAM encoding bone morphogenetic protein-4 and the parathyroid hormone fragment, which act synergistically in vitro, caused new bone to form faster than with either factor alone. These studies demonstrate for the first time that repair cells (fibroblasts) in bone can be genetically manipulated in vivo. While serving as a useful tool to study the biology of repair fibroblasts and the wound healing response, the GAM technology may also have wide therapeutic utility.

Repair of calvarial defects with customized tissue-engineered bone grafts - I. Evaluation of osteogenesis in a three-dimensional culture system

Bone generation by autogenous cell transplantation in combination with a biodegradable scaffold is one of the most promising techniques being developed in craniofacial surgery. The objective of this combined in vitro and in vivo study was to evaluate the morphology and osteogenic differentiation of bone marrow derived mesenchymal progenitor cells and calvarial osteoblasts in a two-dimensional (2-D) and three-dimensional (3-D) culture environment (Part I of this study) and their potential in combination with a biodegradable scaffold to reconstruct critical-size calvarial defects in an autologous animal model [Part II of this study; see Schantz, J.T., et al. Tissue Eng. 2003;9(Suppl. 1):S-127-S-139; this issue]. New Zealand White rabbits were used to isolate osteoblasts from calvarial bone chips and bone marrow stromal cells from iliac crest bone marrow aspirates. Multilineage differentiation potential was evaluated in a 2-D culture setting. After amplification, the cells were seeded within a fibrin matrix into a 3-D polycaprolactone (PCL) scaffold system. The constructs were cultured for up to 3 weeks in vitro and assayed for cell attachment and proliferation using phase-contrast light, confocal laser, and scanning electron microscopy and the MTS cell metabolic assay. Osteogenic differentiation was analyzed by determining the expression of alkaline phosphatase (ALP) and osteocalcin. The bone marrow-derived progenitor cells demonstrated the potential to be induced to the osteogenic, adipogenic, and chondrogenic pathways. In a 3-D environment, cell-seeded PCL scaffolds evaluated by confocal laser microscopy revealed continuous cell proliferation and homogeneous cell distribution within the PCL scaffolds. On osteogenic induction mesenchymal progenitor cells (12 U/L) produce significantly higher (p < 0.05) ALP activity than do osteoblasts (2 U/L); however, no significant differences were found in osteocalcin expression. In conclusion, this study showed that the combination of a mechanically stable synthetic framework (PCL scaffolds) and a biomimetic hydrogel (fibrin glue) provides a potential matrix for bone tissue-engineering applications. Comparison of osteogenic differentiation between the two mesenchymal cell sources revealed a similar pattern.

Strategy in developing cell based therapy for large bone

Regeneration of osseous defects by tissue-engineering approach provides a novel means of treatment utilizing cell biology, materials science, and molecular biology. The concept of in vitro cultured osteoblasts having an ability to induce new bone formation has been demonstrated in the critical size defects using small animal models. The bone derived cells can be incorporated into bioengineered scaffolds and synthesize bone matrix, which on implantation can induce new bone formation. In search of optimal cell delivery materials, the extracellular matrix as cell carriers for the repair and regeneration of tissues is receiving increased attention. We have investigated extracellular matrix formed by osteoblasts in vitro as a scaffold for osteoblasts transplantation and found a mineralized matrix, formed by human osteoblasts in vitro, can initiate bone formation by activating endogenous mesenchymal cells. To repair the large bone defects, osteogenic or stem cells need to be prefabricated in a large three dimensional scaffold usually made of synthetic biomaterials, which have inadequate interaction with cells and lead to in vivo foreign body reactions. The interstitial extracellular matrix has been applied to modify biomaterials surface and identified vitronectin, which binds the heparin domain and RGD (Arg-Gly-Asp) sequence can modulate cell spreading, migration and matrix formation on biomaterials. We also synthesized a tri-block copolymer, methoxy-terminated poly(ethylene glycol)(MPEG)-polyL-lactide(PLLA)-polylysine(PLL) for human osteoblasts delivery. We identified osteogenic activity can be regulated by the molecular weight and composition of the triblock copolymers. Due to the sequential loss of lineage differentiation potential during the culture of bone marrow stromal cells that hinderers their potential clinical application, we have developed a clonal culture system and established several stem cell clones with fast growing and multi-differentiation properties. Using proteomics and subtractive immunization, several differential proteins have been identified and verified their potential application in stem cell characterization and tissue regeneration

Tissue engineering bone - reconstruction of critical sized segmental bone defects in a large animal model

Currently, well established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, their application, however, is associated with disadvantages. These include limited access and availability, donor site morbidity and haemorrhage, increased risk of infection, and insufficient transplant integration. As a result, recent research focuses on the development of complementary therapeutic concepts. The field of tissue engineering has emerged as an important alternative approach to bone regeneration. Tissue engineering unites aspects of cellular biology, biomechanical engineering, biomaterial sciences and trauma and orthopaedic surgery. To obtain approval by regulatory bodies for these novel therapeutic concepts the level of therapeutic benefit must be demonstrated rigorously in well characterized, clinically relevant animal models. Therefore, in this PhD project, a reproducible and clinically relevant, ovine, critically sized, high load bearing, tibial defect model was established and characterized as a prerequisite to assess the regenerative potential of a novel treatment concept in vivo involving a medical grade polycaprolactone and tricalciumphosphate based composite scaffold and recombinant human bone morphogenetic proteins.

Nano- to macroscale remodeling of functional tissue-engineered bone

A higher degree of mineralization is found within scaffold groups implanted with cells compared to scaffold alone demonstrating greater bone regenerative potential of cell-scaffold constructs Tissue engineered bone analysed using ESEM and SAXS demonstrates bone formation within the scaffold to be preferentially aligned around the scaffold struts. The mineral particles are not shown to orientate around the osteons within the native bone.

Species-specific homing mechanisms of human prostate cancer metastasis in tissue engineered bone

The development of effective therapeutic strategies against prostate cancer bone metastases has been impeded by the lack of adequate animal models that are able to recapitulate the biology of the disease in humans. Bioengineered approaches allow researchers to create sophisticated experimentally and physiologically relevant in vivo models to study interactions between cancer cells and their microenvironment under reproducible conditions. The aim of this study was to engineer a morphologically and functionally intact humanized organ bone which can serve as a homing site for human prostate cancer cells. Transplantation of biodegradable tubular composite scaffolds seeded with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone construct including a large number of human mesenchymal cells which were shown to be metabolically active and capable of producing extracellular matrix components. Micro-CT analysis demonstrated that the newly formed ossicle recapitulated the morphological features of a physiological organ bone with a trabecular network surrounded by a cortex-like outer structure. This microenvironment was supportive of the lodgement and maintenance of murine haematopoietic cell clusters, thus mimicking a functional organ bone. Bioluminescence imaging demonstrated that luciferase-transduced human PC3 cells reproducibly homed to the humanized tissue engineered bone constructs, proliferated, and developed macro-metastases. This model allows the analysis of interactions between human prostate cancer cells and a functional humanized bone organ within an immuno-incompetent murine host. The system can serve as a reproducible platform to study effects of therapeutics against prostate cancer bone metastases within a humanized microenvironment.

Mathematical modelling of intramembranous bone formation during fracture healing

During fracture healing, many complex and cryptic interactions occur between cells and bio-chemical molecules to bring about repair of damaged bone. In this thesis two mathematical models were developed, concerning the cellular differentiation of osteoblasts (bone forming cells) and the mineralisation of new bone tissue, allowing new insights into these processes. These models were mathematically analysed and simulated numerically, yielding results consistent with experimental data and highlighting the underlying pattern formation structure in these aspects of fracture healing.

Inflammation-driven bone formation in a mouse model of ankylosing spondylitis: Sequential not parallel processes

Background Ankylosing spondylitis (AS) is an immune-mediated arthritis particularly targeting the spine and pelvis and is characterised by inflammation, osteoproliferation and frequently ankylosis. Current treatments that predominately target inflammatory pathways have disappointing efficacy in slowing disease progression. Thus, a better understanding of the causal association and pathological progression from inflammation to bone formation, particularly whether inflammation directly initiates osteoproliferation, is required. Methods The proteoglycan-induced spondylitis (PGISp) mouse model of AS was used to histopathologically map the progressive axial disease events, assess molecular changes during disease progression and define disease progression using unbiased clustering of semi-quantitative histology. PGISp mice were followed over a 24-week time course. Spinal disease was assessed using a novel semi-quantitative histological scoring system that independently evaluated the breadth of pathological features associated with PGISp axial disease, including inflammation, joint destruction and excessive tissue formation (osteoproliferation). Matrix components were identified using immunohistochemistry. Results Disease initiated with inflammation at the periphery of the intervertebral disc (IVD) adjacent to the longitudinal ligament, reminiscent of enthesitis, and was associated with upregulated tumor necrosis factor and metalloproteinases. After a lag phase, established inflammation was temporospatially associated with destruction of IVDs, cartilage and bone. At later time points, advanced disease was characterised by substantially reduced inflammation, excessive tissue formation and ectopic chondrocyte expansion. These distinct features differentiated affected mice into early, intermediate and advanced disease stages. Excessive tissue formation was observed in vertebral joints only if the IVD was destroyed as a consequence of the early inflammation. Ectopic excessive tissue was predominantly chondroidal with chondrocyte-like cells embedded within collagen type II- and X-rich matrix. This corresponded with upregulation of mRNA for cartilage markers Col2a1, sox9 and Comp. Osteophytes, though infrequent, were more prevalent in later disease. Conclusions The inflammation-driven IVD destruction was shown to be a prerequisite for axial disease progression to osteoproliferation in the PGISp mouse. Osteoproliferation led to vertebral body deformity and fusion but was never seen concurrent with persistent inflammation, suggesting a sequential process. The findings support that early intervention with anti-inflammatory therapies will be needed to limit destructive processes and consequently prevent progression of AS.

The effect of simvastatin on bone formation and ceramic resorption in a peri-implant defect model

Experimental use of statins as stimulators of bone formation suggests they may have widespread applicability in the field of orthopaedics. With their combined effects on osteoblasts and osteoclasts, statins have the potential to enhance resorption of synthetic materials and improve bone ingrowth. In this study, the effect of oral and local administration of simvastatin to a 0 tricalcium phosphate (beta TCP)-filled defect around an implant was compared with recombinant human bone morphogenetic protein 2 (rhBMP2). On hundred and sixty-two Sprague-Dawley rats were assigned to treatment groups: local application of 0.1, 0.9 or 1.7 mg of simvastatin, oral simvastatin at 5, 10 or 50 mg kg(-1) day(-1) for 20 days, local delivery of I or 10 mu g of rhBMP2, or control. At 6 weeks rhBMP2 increased serum tartrate-resistant acid phosphatase 5b levels and reduced PTCP area fraction, particle size and number compared with control, suggesting increased osteoclast activity. There was reduced stiffness and increased mechanical strength with this treatment. Local simvastatin resulted in a decreased mineral apposition rate at 6 weeks and increased fibrous area fraction, PTCP area fraction, particle size and number at 26 weeks. Oral simvastatin had no effect compared with control. Local application of rhBMP2 increased resorption and improved mechanical strength whereas simvastatin was detrimental to healing. Oral simvastatin was ineffective at promoting either ceramic resorption or bone formation. The effect of statins on the repair of bone defects with graft substitute materials is influenced by its bioavailability. Thus, further studies on the optimal delivery system are needed. (C) 2007 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Ectopic bone formation in rapidly fabricated acellular injectable dense collagen-Bioglass hybrid scaffolds via gel aspiration-ejection

Gel aspiration-ejection (GAE) has recently been introduced as an effective technique for the rapid production of injectable dense collagen (IDC) gel scaffolds with tunable collagen fibrillar densities (CFDs) and microstructures. Herein, a GAE system was applied for the advanced production and delivery of IDC and IDC-Bioglass® (IDC-BG) hybrid gel scaffolds for potential bone tissue engineering applications. The efficacy of GAE in generating mineralizable IDC-BG gels (from an initial 75-25 collagen-BG ratio) produced through needle gauge numbers 8G (3.4 mm diameter and 6 wt% CFD) and 14G (1.6 mm diameter and 14 wt% CFD) was investigated. Second harmonic generation (SHG) imaging of as-made gels revealed an increase in collagen fibril alignment with needle gauge number. In vitro mineralization of IDC-BG gels was confirmed where carbonated hydroxyapatite was detected as early as day 1 in simulated body fluid, which progressively increased up to day 14. In vivo mineralization of, and host response to, acellular IDC and IDC-BG gel scaffolds were further investigated following subcutaneous injection in adult rats. Mineralization, neovascularization and cell infiltration into the scaffolds was enhanced by the addition of BG and at day 21 post injection, there was evidence of remodelling of granulation tissue into woven bone-like tissue in IDC-BG. SHG imaging of explanted scaffolds indicated collagen fibril remodelling through cell infiltration and mineralization over time. In sum, the results suggest that IDC-BG hybrid gels have osteoinductive properties and potentially offer a novel therapeutic approach for procedures requiring the injectable delivery of a malleable and dynamic bone graft that mineralizes under physiological conditions

The Bone Formation Capabilities of the Anorganic Bone Matrix-Synthetic Cell-Binding Peptide 15 Grafts in an Animal Periodontal Model: A Histologic and Histomorphometric Study in Dogs

Background: The aim of this study is to verify the regenerative potential of particulate anorganic bone matrix synthetic peptide-15 (ABM-P-15) in class III furcation defects associated or not with expanded polytetrafluoroethylene membranes. Methods: Class III furcation defects were produced in the mandibular premolars (P2, P3, and P4) of six dogs and filled with impression material. The membranes and the bone grafts were inserted into P3 and P4, which were randomized to form the test and control groups, respectively; P2 was the negative control group. The animals were sacrificed 3 months post-treatment. Results: Histologically, the complete closure of class III furcation defects was not observed in any of the groups. Partial periodontal regeneration with similar morphologic characteristics among the groups was observed, however, through the formation of new cementum, periodontal ligament, and bone above the notch. Histologic analysis showed granules from the bone graft surrounded by immature bone matrix and encircled by newly formed tissue in the test group. The new bone formation area found in the negative control group was 2.28 +/- 2.49 mm(2) and in the test group it was 6.52 +/- 5.69 mm(2), which showed statistically significant differences for these groups considering this parameter (Friedman test P <0.05). There was no statistically significant difference among the negative control, control, and test groups for the other parameters. Conclusions: The regenerative potential of ABM-P-15 was demonstrated through new bone formation circumscribing and above the graft particles. The new bone also was accompanied by the formation of new cementum and periodontal ligament fibers. J Periodontol 2010;81:594-603.

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

Oxytocin promotes bone formation during the alveolar healing process in old acyclic female rats

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