Four years follow-up after clavicle reconstruction in a child: a case report.

Clavicle reconstruction is a rare operation. In most cases a mid-shaft defect of the clavicle is bridged by using different grafting techniques or musculo-osteous flaps. In some clinical situations where reconstruction is not a suitable option claviculectomy as a salvation procedure has proven to be an acceptable solution. In the paediatric population the challenge of both the cosmetic and the functional result attempting reconstruction of large bone defects is of higher demand. To our knowledge, this is the first case of a successful clavicle reconstruction with a sufficient follow-up using a free vascularised fibula graft in a child. This case provides a technique description, considerations in the paediatric population, an overview of other techniques used, and a long-term follow-up.

Development and transplantation of a mineralized matrix formed by osteoblasts in vitro for bone regeneration

The use of extracellular matrix materials as scaffolds for the repair and regeneration of tissues is receiving increased attention. The current study was undertaken to test whether extracellular matrix formed by osteoblasts in vitro could be used as a scaffold for osteoblast transplantation and induce new bone formation in critical size osseous defects in vivo. Human osteoblasts derived from alveolar bone were cultured in six-well plates until confluent and then in mineralization media for a further period of 3 weeks to form an osteoblast-mineralized matrix complex. Histologically, at this time point a tissue structure with a connective tissue-like morphology was formed. Type I collagen was the major extracellular component present and appeared to determine the matrix macrostructure. Other bone-related proteins such as alkaline phosphatase (ALP), bone morphogenetic protein (BMP)-2 and -4, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) also accumulated in the matrix. The osteoblasts embedded in this matrix expressed mRNAs for these bone-related proteins very strongly. Nodules of calcification were detected in the matrix and there was a correlation between calcification and the distribution of BSP and OPN. When this matrix was transplanted into a critical size bone defect in skulls of inummodeficient mice (SCID), new bone formation occurred. Furthermore, the cells inside the matrix survived and proliferated in the recipient sites, and were traceable by the human-specific Alu gene sequence using in situ hybridization. It was found that bone-forming cells differentiated from both transplanted human osteoblasts and activated endogenous mesenchymal cells. This study indicates that a mineralized matrix, formed by human osteoblasts in vitro, can be used as a scaffold for osteoblast transplantation, which subsequently can induce new bone formation.

Characterizing the hierarchical structures of bioactive sol-gel silicate glass and hybrid scaffolds for bone regeneration

Bone is the second most widely transplanted tissue after blood. Synthetic alternatives are needed that can reduce the need for transplants and regenerate bone by acting as active temporary templates for bone growth. Bioactive glasses are one of the most promising bone replacement/regeneration materials because they bond to existing bone, are degradable and stimulate new bone growth by the action of their dissolution products on cells. Sol-gel-derived bioactive glasses can be foamed to produce interconnected macropores suitable for tissue ingrowth, particularly cell migration and vascularization and cell penetration. The scaffolds fulfil many of the criteria of an ideal synthetic bone graft, but are not suitable for all bone defect sites because they are brittle. One strategy for improving toughness of the scaffolds without losing their other beneficial properties is to synthesize inorganic/organic hybrids. These hybrids have polymers introduced into the sol-gel process so that the organic and inorganic components interact at the molecular level, providing control over mechanical properties and degradation rates. However, a full understanding of how each feature or property of the glass and hybrid scaffolds affects cellular response is needed to optimize the materials and ensure long-term success and clinical products. This review focuses on the techniques that have been developed for characterizing the hierarchical structures of sol-gel glasses and hybrids, from atomicscale amorphous networks, through the covalent bonding between components in hybrids and nanoporosity, to quantifying open macroporous networks of the scaffolds. Methods for non-destructive in situ monitoring of degradation and bioactivity mechanisms of the materials are also included. © 2012 The Royal Society.

Amniotic fluid stem cells produce robust mineral deposits on biodegradable scaffolds

Insufficient availability of osteogenic cells limits bone regeneration through cell-based therapies. This study investigated the potential of amniotic fluid–derived stem (AFS) cells to synthesize mineralized extracellular matrix within porous medical-grade poly-e-caprolactone (mPCL) scaffolds. The AFS cells were initially differentiated in two-dimensional (2D) culture to determine appropriate osteogenic culture conditions and verify physiologic mineral production by the AFS cells. The AFS cells were then cultured on 3D mPCL scaffolds (6-mm diameter9-mm height) and analyzed for their ability to differentiate to osteoblastic cells in this environment. The amount and distribution of mineralized matrix production was quantified throughout the mPCL scaffold using nondestructive micro computed tomography (microCT) analysis and confirmed through biochemical assays. Sterile microCT scanning provided longitudinal analysis of long-term cultured mPCL constructs to determine the rate and distribution of mineral matrix within the scaffolds. The AFS cells deposited mineralized matrix throughout the mPCL scaffolds and remained viable after 15 weeks of 3D culture. The effect of predifferentiation of the AFS cells on the subsequent bone formation in vivo was determined in a rat subcutaneous model. Cells that were pre-differentiated for 28 days in vitro produced seven times more mineralized matrix when implanted subcutaneously in vivo. This study demonstrated the potential of AFS cells to produce 3D mineralized bioengineered constructs in vitro and in vivo and suggests that AFS cells may be an effective cell source for functional repair of large bone defects

Addressing issues in sparseness, ecological bias and formulation of the adjacency matrix in Bayesian spatio-temporal analysis of disease counts

The main objective of this PhD was to further develop Bayesian spatio-temporal models (specifically the Conditional Autoregressive (CAR) class of models), for the analysis of sparse disease outcomes such as birth defects. The motivation for the thesis arose from problems encountered when analyzing a large birth defect registry in New South Wales. The specific components and related research objectives of the thesis were developed from gaps in the literature on current formulations of the CAR model, and health service planning requirements. Data from a large probabilistically-linked database from 1990 to 2004, consisting of fields from two separate registries: the Birth Defect Registry (BDR) and Midwives Data Collection (MDC) were used in the analyses in this thesis. The main objective was split into smaller goals. The first goal was to determine how the specification of the neighbourhood weight matrix will affect the smoothing properties of the CAR model, and this is the focus of chapter 6. Secondly, I hoped to evaluate the usefulness of incorporating a zero-inflated Poisson (ZIP) component as well as a shared-component model in terms of modeling a sparse outcome, and this is carried out in chapter 7. The third goal was to identify optimal sampling and sample size schemes designed to select individual level data for a hybrid ecological spatial model, and this is done in chapter 8. Finally, I wanted to put together the earlier improvements to the CAR model, and along with demographic projections, provide forecasts for birth defects at the SLA level. Chapter 9 describes how this is done. For the first objective, I examined a series of neighbourhood weight matrices, and showed how smoothing the relative risk estimates according to similarity by an important covariate (i.e. maternal age) helped improve the model’s ability to recover the underlying risk, as compared to the traditional adjacency (specifically the Queen) method of applying weights. Next, to address the sparseness and excess zeros commonly encountered in the analysis of rare outcomes such as birth defects, I compared a few models, including an extension of the usual Poisson model to encompass excess zeros in the data. This was achieved via a mixture model, which also encompassed the shared component model to improve on the estimation of sparse counts through borrowing strength across a shared component (e.g. latent risk factor/s) with the referent outcome (caesarean section was used in this example). Using the Deviance Information Criteria (DIC), I showed how the proposed model performed better than the usual models, but only when both outcomes shared a strong spatial correlation. The next objective involved identifying the optimal sampling and sample size strategy for incorporating individual-level data with areal covariates in a hybrid study design. I performed extensive simulation studies, evaluating thirteen different sampling schemes along with variations in sample size. This was done in the context of an ecological regression model that incorporated spatial correlation in the outcomes, as well as accommodating both individual and areal measures of covariates. Using the Average Mean Squared Error (AMSE), I showed how a simple random sample of 20% of the SLAs, followed by selecting all cases in the SLAs chosen, along with an equal number of controls, provided the lowest AMSE. The final objective involved combining the improved spatio-temporal CAR model with population (i.e. women) forecasts, to provide 30-year annual estimates of birth defects at the Statistical Local Area (SLA) level in New South Wales, Australia. The projections were illustrated using sixteen different SLAs, representing the various areal measures of socio-economic status and remoteness. A sensitivity analysis of the assumptions used in the projection was also undertaken. By the end of the thesis, I will show how challenges in the spatial analysis of rare diseases such as birth defects can be addressed, by specifically formulating the neighbourhood weight matrix to smooth according to a key covariate (i.e. maternal age), incorporating a ZIP component to model excess zeros in outcomes and borrowing strength from a referent outcome (i.e. caesarean counts). An efficient strategy to sample individual-level data and sample size considerations for rare disease will also be presented. Finally, projections in birth defect categories at the SLA level will be made.

Bioactive mesopore-glass microspheres with controllable protein-delivery properties by biomimetic surface modification

Microsphere systems with the ideal properties for bone regeneration need to be bioactive, and at the same time possess the capacity for controlled protein/drug-delivery; however, the current crop of microsphere system fails to fulfill these properties. The aim of this study was to develop a novel protein-delivery system of bioactive mesoporous glass (MBG) microspheres by a biomimetic method through controlling the density of apatite on the surface of microspheres, for potential bone tissue regeneration. MBG microspheres were prepared by using the method of alginate cross-linking with Ca2+ ions. The cellular bioactivity of MBG microspheres was evaluated by investigating the proliferation and attachment of bone marrow stromal cell (BMSC). The loading efficiency and release kinetics of bovine serum albumin (BSA) on MBG microspheres were investigated after coprecipitating with biomimetic apatite in simulated body fluids (SBF). The results showed that MBG microspheres supported BMSC attachment and the Si containing ionic products from MBG microspheres stimulated BMSCs proliferation. The density of apatite on MBG microspheres increased with the length of soaking time in SBF. BSA-loading efficiency of MBG was significantly enhanced by co-precipitating with apatite. Furthermore, the loading efficiency and release kinetics of BSA could be controlled by controlling the density of apatite formed on MBG microspheres. Our results suggest that MBG microspheres are a promising protein-delivery system as a filling material for bone defect healing and regeneration.

Multifunctional magnetic mesoporous bioactive glass scaffolds with a hierarchical pore structure

Hyperthermia and local drug delivery have been proposed the potential therapeutic approaches for bone defects resulting from malignant bone tumors. Development of bioactive materials with magnetic and drug-delivery properties may potentially meet this target. The aim of this study is to develop a multifunctional mesoporous bioactive glass (MBG) scaffold system for both hyperthermia and local-drug delivery application potentially. For this aim, Iron (Fe) containing MBG (Fe-MBG) scaffolds with hierarchically large pores (300-500 µm) and fingerprint-like mesopores (4.5 nm) have been successfully prepared. The effect of Fe on the mesopore structure, physiochemical, magnetism, drug delivery and biological properties of MBG scaffolds has been systematically investigated. The results showed that the morphology of the mesopore varied from straight channels to curved fingerprint-like channels after incorporated parts of Fe into MBG scaffolds. The magnetism magnitude of MBG scaffolds can be tailored by controlling Fe contents. Furthermore, the incorporating of Fe into mesoporous MBG glass scaffolds enhanced the mitochondrial activity and bone-relative gene (ALP and OCN) expression of human bone marrow mesenchymal stem cells (BMSCs) on the scaffolds. The obtained Fe-MBG scaffolds also possessed high specific surface areas and sustained drug delivery. Therefore, Fe-MBG scaffolds are magnetic, degradable and bioactive. The multifunction of Fe-MBG scaffolds indicates that there is a great potential for Fe-MBG scaffolds to be used for the therapy and regeneration of large-bone defects caused by malignant bone tumors through the combination of hyperthermia, local drug delivery and their osteoconductivity.

Porous scaffold architecture guides tissue formation

Critical-sized bone defect regeneration is a remaining clinical concern. Numerous scaffold-based strategies are currently being investigated to enable in vivo bone defect healing. However, a deeper understanding of how a scaffold influences the tissue formation process and how this compares to endogenous bone formation or to regular fracture healing is missing. It is hypothesized that the porous scaffold architecture can serve as a guiding substrate to enable the formation of a structured fibrous network as a prerequirement for later bone formation. An ovine, tibial, 30-mm critical-sized defect is used as a model system to better understand the effect of the scaffold architecture on cell organization, fibrous tissue, and mineralized tissue formation mechanisms in vivo. Tissue regeneration patterns within two geometrically distinct macroscopic regions of a specific scaffold design, the scaffold wall and the endosteal cavity, are compared with tissue formation in an empty defect (negative control) and with cortical bone (positive control). Histology, backscattered electron imaging, scanning small-angle X-ray scattering, and nanoindentation are used to assess the morphology of fibrous and mineralized tissue, to measure the average mineral particle thickness and the degree of alignment, and to map the local elastic indentation modulus. The scaffold proves to function as a guiding substrate to the tissue formation process. It enables the arrangement of a structured fibrous tissue across the entire defect, which acts as a secondary supporting network for cells. Mineralization can then initiate along the fibrous network, resulting in bone ingrowth into a critical-sized defect, although not in complete bridging of the defect. The fibrous network morphology, which in turn is guided by the scaffold architecture, influences the microstructure of the newly formed bone. These results allow a deeper understanding of the mode of mineral tissue formation and the way this is influenced by the scaffold architecture. Copyright © 2012 American Society for Bone and Mineral Research.

Copper-containing mesoporous bioactive glass scaffolds with multifunctional properties of angiogenesis capacity, osteostimulation and antibacterial activity

It is of great importance to develop multifunctional bioactive scaffolds, which combine angiogenesis capacity, osteostimulation, and antibacterial properties for regenerating lost bone tissues. In order to achieve this aim, we prepared copper (Cu)-containing mesoporous bioactive glass (Cu-MBG) scaffolds with interconnective large pores (several hundred micrometer) and well-ordered mesopore channels (around 5 nm). Both Cu-MBG scaffolds and their ionic extracts could stimulate hypoxia-inducible factor (HIF)-1a and vascular endothelial growth factor(VEGF) expression in human bone marrow stromal cells(hBMSCs). In addition, both Cu-MBG scaffolds and their ionic extracts significantly promoted the osteogenic differentiation of hBMSCs by improving their bone-related gene expression (alkaline phosphatase (ALP), osteopontin(OPN) and osteocalcin (OCN)). Furthermore, Cu-MBG scaffolds could maintain a sustained release of ibuprofen and significantly inhibited the viability of bacteria. This study indicates that the incorporation of Cu2þ ions into MBG scaffolds significantly enhances hypoxia-like tissue reaction leading to the coupling of angiogenesis and osteogenesis. Cu2þ ions play an important role to offer the multifunctional properties of MBG scaffold system. This study has demonstrated that it is possible to develop multifunctional scaffolds by combining enhanced angiogenesis potential, osteostimulation, and antibacterial properties for the treatment of large bone defects.

Advances and future directions for management of trauma patients with musculoskeletal injuries

Musculoskeletal injuries are the most common reason for operative procedures in severely injured patients and are major determinants of functional outcomes. In this paper, we summarise advances and future directions for management of multiply injured patients with major musculoskeletal trauma. Improved understanding of fracture healing has created new possibilities for management of particularly challenging problems, such as delayed union and non union of fractures and large bone defects. Optimum timing of major orthopaedic interventions is guided by increased knowledge about the immune response after injury. Individual treatment should be guided by trading off the benefits of early definitive skeletal stabilisation, and the potentially life-threatening risks of systemic complications such as fat embolism, acute lung injury, and multiple organ failure. New methods for measurement of fracture healing and function and quality of life outcomes pave the way for landmark trials that will guide the future management of musculoskeletal injuries.

A simple method for fabricating 3-D multilayered composite scaffolds

One limitation of electrospinning stems from the charge build-up that occurs during processing, preventing further fibre deposition and limiting the scaffold overall thickness and hence their end-use in tissue engineering applications targeting large tissue defect repair. To overcome this, we have developed a technique in which thermally induced phase separation (TIPS) and electrospinning are combined. Thick three-dimensional, multilayered composite scaffolds were produced by simply stacking individual polycaprolactone (PCL) microfibrous electrospun discs into a cylindrical holder that was filled with a 3% poly(lactic-co-glycolic acid) (PLGA) solution in dimethylsulfoxide (a good solvent for PLGA but a poor one for PCL). The construct was quenched in liquid nitrogen and the solvent removed by leaching out in cold water. This technique enables the fabrication of scaffolds composed principally of electrospun membranes that have no limit to their thickness. The mechanical properties of these scaffolds were assessed under both quasi-static and dynamic conditions. The multilayered composite scaffolds had similar compressive properties to 5% PCL scaffolds fabricated solely by the TIPS methodology. However, tensile tests demonstrated that the multilayered construct outperformed a scaffold made purely by TIPS, highlighting the contribution of the electrospun component of the composite scaffold to enhancing the overall mechanical property slate. Cell studies revealed cell infiltration principally from the scaffold edges under static seeding conditions. This fabrication methodology permits the rapid construction of thick, strong scaffolds from a range of biodegradable polymers often used in tissue engineering, and will be particularly useful when large dimension electrospun scaffolds are required.

Polycaprolactone scaffold and reduced rhBMP-7 dose for the regeneration of critical-sized defects in sheep tibiae

The transplantation of autologous bone graft as a treatment for large bone defects has the limitation of harvesting co-morbidity and limited availability. This drives the orthopaedic research community to develop bone graft substitutes. Routinely, supra-physiological doses of bone morphogenetic proteins (BMPs) are applied perpetuating concerns over undesired side effects and cost of BMPs. We therefore aimed to design a composite scaffold that allows maintenance of protein bioactivity and enhances growth factor retention at the implantation site. Critical-sized defects in sheep tibiae were treated with the autograft and with two dosages of rhBMP-7, 3.5 mg and 1.75 mg, embedded in a slowly degradable medical grade poly(ε-caprolactone) (PCL) scaffold with β-tricalcium phosphate microparticles (mPCL-TCP). Specimens were characterised by biomechanical testing, microcomputed tomography and histology. Bridging was observed within 3 months for the autograft and both rhBMP-7 treatments. No significant difference was observed between the low and high rhBMP-7 dosages or between any of the rhBMP-7 groups and autograft implantation. Scaffolds alone did not induce comparable levels of bone formation compared to the autograft and rhBMP-7 groups. In summary, the mPCL-TCP scaffold with the lower rhBMP-7 dose led to equivalent results to autograft transplantation or the high BMP dosage. Our data suggest a promising clinical future for BMP application in scaffold-based bone tissue engineering, lowering and optimising the amount of required BMP.

Stimulation of osteogenesis and angiogenesis of hBMSCs by delivering Si ions and functional drug from mesoporous silica nanospheres

Multifunctional bioactive materials with the ability to stimulate osteogenesis and angiogenesis of stem cells play an important role in the regeneration of bone defects. However, how to develop such biomaterials remains a significant challenge. In this study, we prepared mesoporous silica nanospheres (MSNs) with uniform sphere size (∼90 nm) and mesopores (∼2.7 nm), which could release silicon ions (Si) to stimulate the osteogenic differentiation of human bone marrow stromal cells (hBMSCs) via activating their ALP activity, bone-related gene and protein (OCN, RUNX2 and OPN) expression. Hypoxia-inducing therapeutic drug, dimethyloxaloylglycine (DMOG), was effectively loaded in the mesopores of MSNs (D-MSNs). The sustained release of DMOG from D-MSNs could stabilize HIF-1α and further stimulated the angiogenic differentiation of hBMSCs as indicated by the enhanced VEGF secretion and protein expression. Our study revealed that D-MSNs could combine the stimulatory effect on both osteogenic and angiogenic activity of hBMSCs. The potential mechanism of D-MSN-stimulated osteogenesis and angiogenesis was further elucidated by the supplementation of cell culture medium with pure Si ions and DMOG. Considering the easy handling characteristics of nanospheres, the prepared D-MSNs may be applied in the forms of injectable spheres for minimally invasive surgery, or MSNs/polymer composite scaffolds for bone defect repair. The concept of delivering both stimulatory ions and functional drugs may offer a new strategy to construct a multifunctional biomaterial system for bone tissue regeneration.

Graphene-oxide-modified β-tricalcium phosphate bioceramics stimulate in vitro and in vivo osteogenesis

Graphene oxide (GO) has attracted much interest for applications in bone tissue engineering; however, until now the interaction between GO and stem cells, and the in vivo bone-forming ability of GO has not been explored. The aim of this study was to produce a GO-modified β-tricalcium phosphate (β-TCP-GRA) biceramics and then explore the material’s osteogenic capacity in vitro and in vivo, as well as unravel some of the molecular mechanisms behind this. β-TCP-GRA disks and scaffolds were successfully prepared by a simple GO/water suspension soaking method in combination with heat treatment. These scaffolds were found to significantly enhance the proliferation, alkaline phosphatase activity and osteogenic gene expression of human bone marrow stromal cells (hBMSCs), when compared to β-TCP without GO modification (controls). Activation of the Wnt/β-catenin signaling pathway in hBMSCs appears to be the mechanism behind this osteogenic induction by β-TCP-GRA. β-TCP-GRA scaffolds led to an increased rate of in vivo new bone formation compared to β-TCP controls, indicative of the stimulatory effect of GO on in vivo osteogenesis, making GO modification of β-TCP a very promising method for applications in bone tissue engineering, in particular for the regeneration of large bone defects.

In vitro cytotoxicity and in vivo osseointergration properties of compression-molded HDPE-HA-Al2O3 hybrid biocomposites

The aim of this study was to investigate the in vivo biocompatibility in terms of healing of long segmental bone defect in rabbit model as well as in vitro cytotoxicity of eluates of compression-molded High density polyethylene (HDPE)hydroxyapatite (HA)-aluminum oxide (Al2O3) composite-based implant material. Based on the physical property in terms of modulus and strength properties, as reported in our recent publication, HDPE-40 wt % HA and HDPE-20 wt % HA-20 wt % Al2O3 hybrid composites were used for biocompatibility assessment. Osteoblasts cells were cultured in conditioned media, which contains varying amount of composite eluate (0.01, 0.1, and 1.0 wt %). In vitro, the eluates did not exhibit any significant negative impact on proliferation, mineralization or on morphology of human osteoblast cells. In vivo, the histological assessment revealed neobone formation at the bone/implant interface, characterized by the presence of osteoid and osteoblasts. The observation of osteoclastic activity indicates the process of bone remodeling. No inflammation to any noticeable extent was observed at the implantation site. Overall, the combination of in vitro and in vivo results are suggestive of potential biomedical application of compression-molded HDPE- 20 wt % HA- 20 wt % Al2O3 composites to heal long segmental bone defects without causing any toxicity of bone cells.