Homogentisic acid is not only eliminated by glomerular filtration and tubular secretion but also produced in the kidney in alkaptonuria.

The clinical effects of alkaptonuria (AKU) are delayed and ageing influences disease progression. Morbidity of AKU is secondary to high circulating homogentisic acid (HGA) and ochronosis. It is not known whether HGA is produced by or processed in the kidney in AKU. Data from AKU patients from four studies were merged to form a single AKU group. A control group of non-AKU subjects was generated by merging data from two non-AKU studies. Data were used to derive renal clearance and fractional excretion (FE) ratios for creatinine, HGA, phenylalanine (PHE) and tyrosine (TYR) using standard calculations, for comparison between the AKU and the control groups. There were 225 AKU patients in the AKU group and 52 in the non-AKU control group. Circulating HGA increased with age (P < 0.001), and was significantly associated with decreased HGA clearance (CLHGA ) (P < 0.001) and FEHGA (P < 0.001). CLHGA and FEHGA were increased beyond the theoretical maximum renal plasma flow, confirming renal production and emphasising the greater contribution of net tubular secretion than glomerular filtration to renal elimination of HGA. The kidneys are crucial to elimination of HGA. Elimination of HGA is impaired with age resulting in worsening disease over time. The kidney is an important site for production of HGA. Tubular secretion of HGA contributes more to elimination of HGA in AKU than glomerular filtration.

Defining the Properties of an Array of -NH2-Modified Substrates for the Induction of a Mature Osteoblast/Osteocyte Phenotype from a Primary Human Osteoblast Population Using Controlled Nanotopography and Surface Chemistry.

Accelerating the integration of a joint replacement or the healing of a bone fracture, particularly a complicated non-union fracture, would improve patient welfare and decrease healthcare costs. Currently, an autologous bone graft is the gold standard method for the treatment of complicated non-union fractures, but it is not always possible to harvest such a graft. A proactive highly inductive so-called smart material approach is pertinent in these cases. In this study, the surface chemistry of a previously approved material with desirable bulk material properties was modified to investigate its potential as an economical and effective alternative. The objective was to create stable synthetic chemical coatings that could guide cells along the osteogenic lineage required to generate mineralised tissue that would induce and accelerate bone healing. Primary human osteoblast-like cells were cultured in vitro for 7, 14 and 28 days on amine-terminated (chain length in the range 3-11) silane-modified glass surfaces with controlled nanotopography, to determine how surface chemistry and nanotopography change osteoblast function. The materials were characterised using atomic force microscopy (AFM), scanning electron microscopy (SEM), water contact angle (WCA) and a novel ninhydrin assay. The cells were analysed using qRT-PCR, von Kossa tinctural staining for mineralisation, and visualised using both transmitted white light and electron microscopy. Bone-like nodules, quantified using microscopy, only formed on the short-chain (chain length 3 and 4) amines after 7 days, as did the up-regulation of sclerostin, suggestive of a more mature osteoblast phenotype. In this paper, we report more rapid nodule formation than has previously been observed, without the addition of exogenous factors in the culture medium. This suggests that the coating would improve the integration of implants with bone or be the basis of a smart biomaterial that would accelerate the bone regeneration process.

In vivo characterization of Hyalonect, a novel biodegradable surgical mesh.

BACKGROUND: Musculoskeletal reconstructive surgery often requires removal of significant quantities of bone tissue, such as the periosteum, causing critical problems following surgery like friction between different tissues and adhesion of soft tissues to the underlying bone. We studied the long-term host response and closure of large bone defects for periosteal reconstruction using Hyalonect, a novel membrane comprising knitted fibers of esterified hyaluronan, (HYAFF11). MATERIALS AND METHODS: For biological characterization, 162 rats were used in a defect model in which a section of the dorsal muscular fascia was removed, and the membrane behavior observed over 540 d using conventional histology, with sham operated rats as controls. In addition, Hyalonect was used to cover defects made in the humeri of 7 dogs, filled with a variety of conventional bone filling compounds, and the regeneration process observed after 6 wks using histology. RESULTS: Low levels of inflammation were observed in the dorsal muscle fascia defect model, with cellular colonization of the mesh by 30 d, vascularization by 120 days, matrix fiber organization by 270 d, and the appearance of connective tissue identical to the surrounding tissue between 365 and 540 d, without the formation of fibrotic tissue. In addition, Hyalonect was shown to allow the regeneration of bone within the humeral defects whilst preventing fibrotic tissue in-growth, and allowing regeneration of tissue which, by 6 wk, had begun to resemble natural periosteal tissue. CONCLUSION: Hyalonect is suitable for improving the outcome of the final phases of orthopedic and trauma reconstructive surgical procedures, especially in the reconstruction of periosteal tissue.

Clinical development innovation in rare diseases: lessons learned and best practices from the DevelopAKUre consortium.

New opportunities have arisen for development of therapies for rare diseases with the increased focus and progress in the field. However, standardised framework integrating individual initiatives has not been formed. We present lessons learned and best practice from a collaborative success case in developing a treatment for a rare genetic disease. Our unique consortium model incorporated several of the identified developments under one project, DevelopAKUre, truly bringing together academia, industry and patient organisations in clinical drug development. We found that the equal partnership between all parties in our consortium was a key success factor creating a momentum based on a strong organisational culture where all partners had high engagement and taking ownership of the entire programme. With an agreed mutual objective, this provided synergies through connecting the strengths of the individual parties. Another key success factor was the central role of the patient organisation within the management team, and their unique study participants' advocacy role securing the understanding and meeting the needs of the clinical study participants in real-time. This resulted in an accelerated enrolment into the clinical studies with a high retention rate allowing for delivery of the programme with significantly improved timelines. Our project was partly funded through an external EU research grant, enabling our model with equal partnership. Further attention within the community should be given to establishing a functional framework where sustainable funding and risk sharing between private and public organisations allow for our model to be replicated.

Efficacy and safety of once-daily nitisinone for patients with alkaptonuria (SONIA 2): an international, multicentre, open-label, randomised controlled trial.

BACKGROUND: Alkaptonuria is a rare, genetic, multisystem disease characterised by the accumulation of homogentisic acid (HGA). No HGA-lowering therapy has been approved to date. The aim of SONIA 2 was to investigate the efficacy and safety of once-daily nitisinone for reducing HGA excretion in patients with alkaptonuria and to evaluate whether nitisinone has a clinical benefit. METHODS: SONIA 2 was a 4-year, open-label, evaluator-blind, randomised, no treatment controlled, parallel-group study done at three sites in the UK, France, and Slovakia. Patients aged 25 years or older with confirmed alkaptonuria and any clinical disease manifestations were randomly assigned (1:1) to receive either oral nitisinone 10 mg daily or no treatment. Patients could not be masked to treatment due to colour changes in the urine, but the study was evaluator-blinded as far as possible. The primary endpoint was daily urinary HGA excretion (u-HGA24) after 12 months. Clinical evaluation Alkaptonuria Severity Score Index (cAKUSSI) score was assessed at 12, 24, 36, and 48 months. Efficacy variables were analysed in all randomly assigned patients with a valid u-HGA24 measurement at baseline. Safety variables were analysed in all randomly assigned patients. The study was registered at ClinicalTrials.gov (NCT01916382). FINDINGS: Between May 7, 2014, and Feb 16, 2015, 139 patients were screened, of whom 138 were included in the study, with 69 patients randomly assigned to each group. 55 patients in the nitisinone group and 53 in the control group completed the study. u-HGA24 at 12 months was significantly decreased by 99·7% in the nitisinone group compared with the control group (adjusted geometric mean ratio of nitisinone/control 0·003 [95% CI 0·003 to 0·004], p<0·0001). At 48 months, the increase in cAKUSSI score from baseline was significantly lower in the nitisinone group compared with the control group (adjusted mean difference -8·6 points [-16·0 to -1·2], p=0·023). 400 adverse events occurred in 59 (86%) patients in the nitisinone group and 284 events occurred in 57 (83%) patients in the control group. No treatment-related deaths occurred. INTERPRETATION: Nitisinone 10 mg daily was well tolerated and effective in reducing urinary excretion of HGA. Nitisinone decreased ochronosis and improved clinical signs, indicating a slower disease progression. FUNDING: European Commission Seventh Framework Programme.

Inflammatory signals in the development of tissue-engineered soft tissue.

Scaffolds with 400 microm pores constructed from hyaluronan modified by benzyl esterification of the carboxylic acid groups (HYAFF-11) and viscous gels created from dodecyl-amidation of hyaluronan (HYADD-3) were implanted subcutaneously into rats for periods of up to 26 and 12 weeks, respectively. Tissue explants were infiltrated with methacrylate resin, sectioned and stained with a broad panel of inflammatory markers in addition to conventional histological stains. Both gels and sponges became rapidly infiltrated by cells that, in the case of HYAFF sponges, did not differentiate, whilst mature adipocytes were only observed at the margins of the sponges. This was combined with sustained inflammatory antigen expression. Conversely, in the HYADD gels, only moderate inflammatory staining was observed at 4 weeks which had diminished completely by 8 weeks. Mature and maturing adipocytes were observed deep within the gels. It is hypothesised that the gels present an excellent inflammatory cytokine profile which induces macrophage infiltration, proliferation then differentiation into adipocytes and is responsible for the generation of neoadipogenesis.

The effect of gas plasma modification on platelet and contact phase activation processes.

Medical-grade polytetrafluoroethylene (PTFE), polydimethylsiloxane (PDMS), polyetherurethane (PEU) and ultrahigh molecular weight polyethylene (UHMWPE) were plasma treated with O2, Ar, N2 and NH3. Their surface properties were characterised using X-ray photoelectron spectroscopy (XPS), static secondary ion mass spectroscopy (SSIMS), atomic force microscopy (AFM) and dynamic contact angle (DCA) analysis. Platelet adhesion, aggregation, activation and release of microparticles were determined after contact with whole blood in a cone and plate viscometer. Activation of the coagulation system was quantified in a static environment using a partial thromboplastin time (PTT) assay. The chemical compositions of the untreated surfaces were found to be very similar to those of the bulk material except for PEU, whose surface was comprised almost entirely of soft ether segments. For all materials, the different plasma treatments resulted in moderate etching with the incorporation of functional groups and removal of side groups: defluorination, dehydrogenation, cleavage of methyl side groups and soft segments for PTFE, UHMWPE, PDMS and PEU, respectively. Consequently, plasma treatment resulted in increased wettability in all cases. Blood contact with the virgin materials resulted in activation of platelets and the clotting cascade. Plasma treatment resulted in a significant reduction in platelet adhesion for all materials and all treatments. In the case of PTFE and PEU, the activation status of these cells was also reduced. Plasma treatment of all materials reduced fluid-phase CD62P expression. Platelet aggregate size correlated well with degree of aggregate formation, but many treatments increased the degree of aggregation, as was the case for microparticle shedding. There was no correlation between CD62P expression, aggregate formation and platelet microparticle (PMP) shedding. It is concluded that despite incorporation of the same chemical groups, the pattern of response to blood in vitro is not the same across different polymers.

In vitro cellular response to oxidized collagen-PLLA hybrid scaffolds designed for the repair of muscular tissue defects and complex incisional hernias.

Unique poly(l-lactic acid) (PLLA)-based scaffolds were constructed by embedding knitted PLLA yarns within a bioresorbable and differentially crosslinked three-dimensional (3D) oxidized collagen scaffold. The scaffolds were designed specifically for the repair of complex incisional abdominal wall hernias and the repair of defects within planar muscular tissues, such as the bladder. The chemical composition of the collagen matrix and the percentage of scaffold infiltration were compared for the different scaffold compositions. The results demonstrate that the incorporation of the collagen sponge within the PLLA scaffold facilitated bladder smooth muscle cell (bSMC) adhesion and proliferation. The highest dose of oxidized collagen (Oxicol) demonstrated better cell adhesion, resulting in the largest cell densities and most uniform distribution throughout the 3D collagen sponge. This formulation promoted the greatest α-smooth muscle actin (αSMA) expression detected through immunohistochemical staining and western blotting. For abdominal wall repair applications, the proliferation and differentiation of C2C12 myoblasts and myotube formation were studied. Following 7 days of myogenic induction, the greatest expression of mRNA of the myogenic markers myogenin and MRF4 was observed within the scaffolds with the highest dose of oxidized collagen, 1.5- and 3.85-fold greater expressions, respectively, compared to PLLA with unmodified collagen. Furthermore, in vitro myotube formation and MyMC expression were enhanced in the Oxicol scaffolds. We conclude that the Oxicol scaffold formulation with a high-dose oxidized collagen ratio provides enhanced myogenesis and αSMA, and the biological induction cues necessary to achieve better tissue integration, than standard PLLA scaffolds in the treatment of complex abdominal wall hernias. Copyright © 2013 John Wiley & Sons, Ltd.

The osteogenic response of mesenchymal stem cells to an injectable PLGA bone regeneration system.

The enrichment of substrates/surfaces with selected functional groups, methyl (-CH3), allyl amine (-NH2), allyl alcohol (-OH) and acrylic acid (-COOH), can be used to trigger mesenchymal stem (MSC) cell differentiation into specified lineages, minimising the need for exogenous biological supplementation. We present the successful translation of this research phenomenon to an injectable two phase injectable PLGA system, utilising plasma techniques, for the repair of bone defects. Modified microspheres were characterised using water contact angel (WCA), X-ray Photon Spectroscopy (XPS) and scanning electron microscopy (SEM). When cultured in contact with MSCs in vitro, the ability of the modified particles, within the 2 phase system, to induce differentiation was characterised using quantitative assays for cell viability and histological analysis for key markers of differentiation throughout the entirety of the three dimensional scaffold. Biological analysis proved that selected modified microspheres have the ability to induce MSC osteogenic (-NH2 modified scaffolds) and chondrogenic (-OH modified scaffolds) differentiation throughout the entirety of the formed scaffold. Therefore optimised plasma modification of microspheres is an effective tool for the production of injectable systems for the repair of bone and cartilage defects.

Elucidating the contribution of the elemental composition of fetal calf serum to antigenic expression of primary human umbilical-vein endothelial cells in vitro.

One of the major obstacles to obtaining human cells of a defined and reproducible standard suitable for use as medical therapies is the necessity for FCS (fetal calf serum) media augmentation in routine cell culture applications. FCS has become the supplement of choice for cell culture research, as it contains an array of proteins, growth factors and essential ions necessary for cellular viability and proliferation in vitro. It is, however, a potential route for the introduction of zoonotic pathogens and makes defining the cell culture milieu impossible in terms of reproducibility, as the precise composition of each batch of serum not only changes but is in fact extremely variable. The present study determined the magnitude of donor variations in terms of elemental composition of FCS and the effect these variations had on the expression of a group of proteins associated with the antigenicity of primary human umbilical-vein endothelial cells, using a combination of ICPMS (inductively coupled plasma MS) and flow cytometry. Statistically significant differences were demonstrated for a set of trace elements in FCS, with correlations made to variations in antigenic expression during culture. The findings question in detail the suitability of FCS for the in vitro supplementation of cultures of primary human cells due to the lack of reproducibility and modulations in protein expression when cultured in conjunction with sera from xenogeneic donors.

The use of flow perfusion culture and subcutaneous implantation with fibroblast-seeded PLLA-collagen 3D scaffolds for abdominal wall repair.

Highly cellularised 3D-tissue constructs designed to repair large, complex abdominal wall defects were prepared using poly (lactic acid) (PLLA)-collagen scaffolds in vitro using a flow perfusion bioreactor. The PLLA-collagen scaffolds had a unique structure consisting of a collagen sponge formed within the pores of a mechanically stable knitted mesh of PLLA. The effect of the flow perfusion bioreactor culturing conditions was investigated in vitro for 0, 7, 14 and 28 days on scaffolds seeded with dermal fibroblasts. The cultured constructs were subsequently studied subcutaneously (SC) in an in vivo animal model. The results of in vitro studies demonstrated that the perfusion system facilitated increased cell proliferation and homogenous distribution in the PLLA-collagen scaffolds compared to static conditions. A highly cellularised 3D-tissue construct was formed by 7 days incubation under perfusion conditions, with increased cellularity by the 28 day time point. The in vivo model demonstrated that implanting constructs with high cellularity resulted in exceptional cell stabilisation, with the survival of implanted cells and expression of the phenotypically-relevant extracellular matrix proteins collagen types I and III, studied by fluorescence in situ hybridisation (FISH) and immunohistochemistry. The implantation of this porous PPLA-collagen scaffold seeded with dermal fibroblasts following in vitro maturation using a flow perfusion bioreactor system suggests a significant advance over current state-of-the-art procedures for the reconstruction of large, complex abdominal wall tissue defects.

Derivation and performance of an entirely autologous injectable hydrogel delivery system for cell-based therapies.

A host-derived hydrogel has been designed and validated as an entirely autologous, injectable delivery system for cells with potential for cell-based therapies and tissue engineering applications. Each individual has components in their blood from which can be formed a mechanically stable hydrogel having the capacity to maintain cellular phenotype and support cellular proliferation of multiple cell types through several culture passages ex vivo. The hydrogel can be triggered to gel at the time of implantation into the patient through an injection system that facilitates a liquid injection of components of the donor plasma and cells into the site of interest. This results in stable ectopic tissue formation at the site of implantation. Our studies have demonstrated excellent integration of the neotissue with host tissues with maintenance of the phenotype of implanted cells whilst observing minimal host innate immune cell recruitment. These findings could provide the fundamental basis for new hydrogel-based biomaterial therapies, overcoming the histocompatibility factors associated with implantable biomaterials whilst providing a stable three dimensional medium for cellular growth both in vivo and ex vivo.

Autologous in vivo adipose tissue engineering in hyaluronan-based gels--a pilot study.

BACKGROUND: There is a major clinical need for strategies for adequately reconstructing the soft tissue defects found after deep burns, tumor resection, or trauma. A promising solution is adipose tissue engineering with preadipocytes, stem-cell derived precursors of the adipose tissue, implanted within biomaterials. This pilot study evaluated hyaluronan gels mixed with autologous undifferentiated preadipocytes in a pig model for their potency to generate new fat. MATERIALS AND METHODS: Preadipocytes were isolated from intra-abdominal pig fat by collagenase digestion, plated on fibronectin-coated culture dishes in Dulbecco's modified Eagle medium/Ham's F12 (Biochrom, Berlin, Germany) combined with 10% pig serum, expanded, and mixed with hyaluronan gel. Two types of gels with varying degrees of amidation of the carboxyl groups were tested (HYADD3, HYADD4). Cell-loaded gels and unseeded controls were injected subcutaneously into the ears of three pigs, explanted at 6 wk, and analyzed histologically. RESULTS: Both cell-loaded specimens were detected macroscopically. They demonstrated a slight volume effect with limited stability after 6 wk. Unloaded HYADD3 and HYADD4 controls could not be identified at the time of explantation. Histology of HYADD3 revealed islets of mature adipocytes and vessels embedded in fat tissue surrounded by gel. In contrast, no fat formation was found in HYADD4 gels when implanted in the ear. CONCLUSIONS: Histological findings demonstrate that HYADD3 is a promising gel for generating adipose tissue. Even though HYADD3 might be a potential material for the reconstruction of small tissue defects, the question remains as to whether the adipose tissue within the gel is attributable to preadipocyte maturation or ingrowth from neighboring tissue.

Analysis of the cellular infiltration of benzyl-esterified hyaluronan sponges implanted in rats.

Unseeded sponges of benzyl-esterified hyaluronan (HYAFF11) and HYAFF11 coated with unmodified hyaluronan were implanted subcutaneously and intramuscularly in adult rats for 1, 2, 4, 8, 12, and 26 weeks. Explanted samples were stained tincturally using Van Geison, von Kossa, and hematoxylin and eosin, enzyme histochemically by chloroacetate esterase, and by immunohistochemistry for the specific identification of cell types and subpopulations, targeting immature (ED1) and mature macrophages (ED2), MHC-I subset, MHC-II subset, CD54, T-cell alpha-beta receptor, T-cell gamma-delta receptor, CD2, CD4, CD8, natural killer cells, B-cells, vimentin, and TGFbeta. Little or no fibrous tissue formation was observed in any sample in either sponge type at any implantation site. Little degradation was observed in either location until 26 weeks. Little neovascularization occurred at early time periods but was in evidence at 26 weeks. Complete cellular infiltration was observed after 4 weeks, with some mature adipocytes observed within the center of the subcutaneous implants, but these cells were mainly observed around the periphery of the sponges. At 26 weeks, cells were mostly macrophages, with small numbers of T-lymphocytes present. No natural killer cells, B-cells, helper/inducer, or cytotoxic/suppressor T-cells were observed in any sample. Most infiltrating cells were MHC-II positive, and discrete pockets of TGFbeta protein were observed within the sponges. While a sustained inflammatory response was observed within both sponge types at 26 weeks, it was relatively benign and nonspecific immunologically, and inflammatory markers such as MHC-II were declining after 12 weeks. No fibrous capsule was observed, and sponge degradation was only observed at 26 weeks, an event essential for induction of neovasculargenesis. At 26 weeks, there was significant staining for vimentin and ED2 on macrophages. Taken with the pattern of other macrophage activation markers, angiogenic environment and absence of inhibitory matrix proteins, the conditions were consistent with the onset of neoadipogenesis, although this would need to be confirmed by longer term studies. For the generation of neoadipose tissue for clinical therapy, we hypothesize that macrophages require an inflammatory stimulus for infiltration, then a reduction in proinflammatory cytokine secretion simultaneous with angiogenic conditions allowing macrophage differentiation into adipocytes.

Intervertebral disc cell-mediated mesenchymal stem cell differentiation.

Low back pain is one of the largest health problems in the Western world today, and intervertebral disc degeneration has been identified as a main cause. Currently, treatments are symptomatic, but cell-based tissue engineering methods are realistic alternatives for tissue regeneration. However, the major problem for these strategies is the generation of a suitable population of cells. Adult bone marrow-derived mesenchymal stem cells (MSCs) are undifferentiated, multipotent cells that have the ability to differentiate into a number of cell types, including the chondrocyte-like cells found within the nucleus pulposus (NP) of the intervertebral disc; however, no method exists to differentiate these cells in an accessible monolayer environment. We have conducted coculture experiments to determine whether cells from the human NP can initiate the differentiation of human MSCs with or without cell-cell contact. Fluorescent labeling of the stem cell population and high-speed cell sorting after coculture with cell-cell contact allowed examination of individual cell populations. Real-time quantitative polymerase chain reaction showed significant increases in NP marker genes in stem cells when cells were cocultured with contact for 7 days, and this change was regulated by cell ratio. No significant change in NP marker gene expression in either NP cells or stem cells was observed when cells were cultured without contact, regardless of cell ratio. Thus, we have shown that human NP and MSC coculture with contact is a viable method for generating a large population of differentiated cells that could be used in cell-based tissue engineering therapies for regeneration of the degenerate intervertebral disc.

Inflammatory response to a novel series of siloxane-crosslinked polyurethane elastomers having controlled biodegradation.

A series of polyurethane polymers was synthesized with increasing proportions of silicone in the form of polydimethylsiloxane (PDMS) utilised as a cross-linking agent, based on an aromatic, non-biostable polyetherurethane (PEtU). Eight formulations ranging from 0-50% PDMS were constructed into porous and non-porous films. These were implanted subcutaneously in rats, both unstrained and 100% strained, for 3 and 6 months. Degradation was determined by FTIR-ATR. Porous films were implanted for 6 and 12 months intramuscularly in both rats and rabbits. These were explanted and examined for inflammatory cell markers by immunohistochemistry. Both low and high percentages of siloxane gave rise to increased degradation, with 20-40% PDMS resulting in the least degradation. Infrared spectral changes correlated well with both visual examination and observation by SEM. Changes to the concentration of siloxane gave rise to differences in the thickness of fibroblastic capsule and infiltration of inflammatory cells in both films & scaffolds. Cellular infiltration was greatest in the films with lower siloxane concentrations. Macrophage activation (MHC-I & MHC-II expression) was least in the higher siloxane variants. It is concluded that by varying the siloxane content in the PEtU matrix we can obtain an acceptable inflammatory response with a relatively short degradation time.