A new mRNA structure prediction based approach to identifying improved signal peptides for bone morphogenetic protein 2.

BACKGROUND: Signal peptide (SP) engineering has proven able to improve production of many proteins yet is a laborious process that still relies on trial and error. mRNA structure around the translational start site is important in translation initiation and has rarely been considered in this context, with recent improvements in in silico mRNA structure potentially rendering it a useful predictive tool for SP selection. Here we attempt to create a method to systematically screen candidate signal peptide sequences in silico based on both their nucleotide and amino acid sequences. Several recently released computational tools were used to predict signal peptide activity (SignalP), localization target (DeepLoc) and predicted mRNA structure (MXFold2). The method was tested with Bone Morphogenetic Protein 2 (BMP2), an osteogenic growth factor used clinically for bone regeneration. It was hoped more effective BMP2 SPs could improve BMP2-based gene therapies and reduce the cost of recombinant BMP2 production. RESULTS: Amino acid sequence analysis indicated 2,611 SPs from the TGF-ß superfamily were predicted to function when attached to BMP2. mRNA structure prediction indicated structures at the translational start site were likely highly variable. The five sequences with the most accessible translational start sites, a codon optimized BMP2 SP variant and the well-established hIL2 SP sequence were taken forward to in vitro testing. The top five candidates showed non-significant improvements in BMP2 secretion in HEK293T cells. All showed reductions in secretion versus the native sequence in C2C12 cells, with several showing large and significant decreases. None of the tested sequences were able to increase alkaline phosphatase activity above background in C2C12s. The codon optimized control sequence and hIL2 SP showed reasonable activity in HEK293T but very poor activity in C2C12. CONCLUSIONS: These results support the use of peptide sequence based in silico tools for basic predictions around signal peptide activity in a synthetic biology context. However, mRNA structure prediction requires improvement before it can produce reliable predictions for this application. The poor activity of the codon optimized BMP2 SP variant in C2C12 emphasizes the importance of codon choice, mRNA structure, and cellular context for SP activity.

Cost-effectiveness of decellularised bone allograft compared with fresh-frozen bone allograft for acetabular impaction bone grafting during a revision hip arthroplasty in the UK.

OBJECTIVES: Fresh-frozen allograft is the gold-standard bone graft material used during revision hip arthroplasty. However, new technology has been developed to manufacture decellularised bone with potentially better graft incorporation. As these grafts cost more to manufacture, the aim of this cost-effectiveness study was to estimate whether the potential health benefit of decellularised bone allograft outweighs their increased cost. STUDY DESIGN: A Markov model was constructed to estimate the costs and the quality-adjusted life years of impaction bone grafting during a revision hip arthroplasty. SETTING: This study took the perspective of the National Health Service in the UK. PARTICIPANTS: The Markov model includes patients undergoing a revision hip arthroplasty in the UK. INTERVENTION: Impaction bone grafting during a revision hip arthroplasty using either decellularised bone allograft or fresh-frozen allograft. MEASURES: Outcome measures included: total costs and quality-adjusted life years of both interventions over the lifetime of the model; and incremental cost-effectiveness ratios for both graft types, using base case parameters, univariate sensitivity analysis and probabilistic analysis. RESULTS: The incremental cost-effectiveness ratio for the base case model was found to be £270 059 per quality-adjusted life year. Univariate sensitivity analysis found that changing the discount rate, the decellularised bone graft cost, age of the patient cohort and the revision rate all had a significant effect on the incremental cost-effectiveness ratio. CONCLUSIONS: As there are no clinical studies of impaction bone grafting using a decellularised bone allograft, there is a high level of uncertainty around the costs of producing a decellularised bone allograft and the potential health benefits. However, if a decellularised bone graft was manufactured for £2887 and lowered the re-revision rate to less than 64 cases per year per 10 000 revision patients, then it would most likely be cost-effective compared with fresh-frozen allograft.

Characterisation of native and decellularised porcine tendon under tension and compression: A closer look at glycosaminoglycan contribution to tendon mechanics.

Decellularised porcine superflexor tendon (pSFT) has been characterised as a suitable scaffold for anterior cruciate ligament replacement, with dimensions similar to hamstring tendon autograft. However, decellularisation of tissues may reduce or damage extracellular matrix components, leading to undesirable biomechanical changes at a whole tissue scale. Although the role of collagen in tendons is well established, the mechanical contribution of glycosaminoglycans (GAGs) is less evident and could be altered by the decellularisation process. In this study, the contribution of GAGs to the tensile and compressive mechanical properties of pSFT was determined and whether decellularisation affected these properties by reducing GAG content or functionality. PSFTs were either enzymatically treated using chondroitinase ABC to remove GAGs or decellularised using previously established methods. Native, GAG-depleted and decellularised pSFT groups were then subjected to quantitative assays and biomechanical characterisation. In tension, specimens underwent stress relaxation and strength testing. In compression, specimens underwent confined compression testing. The GAG-depleted group was found to have circa 86% reduction of GAG content compared to native and decellularised groups. There was no significant difference in GAG content between native (3.75 ± 0.58 µg/mg) and decellularised (3.40 ± 0.37 µg/mg) groups. Stress relaxation testing discovered the time-independent and time-dependent relaxation moduli of the decellularised group were reduced ≥50% compared to native and GAG-depleted groups. However, viscoelastic behaviour of native and GAG-depleted groups resulted similar. Strength testing discovered no differences between native and GAG-depleted group's properties, albeit a reduction ∼20% for decellularised specimens' linear modulus and tensile strength compared to native tissue. In compression testing, the aggregate modulus was found to be circa 74% lower in the GAG-depleted group than the native and decellularised groups, while the zero-strain permeability was significantly higher in the GAG-depleted group (0.86 ± 0.65 mm4/N) than the decellularised group (0.03 ± 0.04 mm4/N). The results indicate that GAGs may significantly contribute to the mechanical properties of pSFT in compression, but not in tension. Furthermore, the content and function of GAGs in pSFTs are unaffected by decellularisation and the mechanical properties of the tissue remain comparable to native tissue.

Evaluation of injectable nucleus augmentation materials for the treatment of intervertebral disc degeneration.

Back pain affects a person's health and mobility as well as being associated with large health and social costs. Lower back pain is frequently caused by degeneration of the intervertebral disc. Current operative and non-operative treatments are often ineffective and expensive. Nucleus augmentation is designed to be a minimally invasive method of restoring the disc to its native healthy state by restoring the disc height, and mechanical and/or biological properties. The majority of the candidate materials for nucleus augmentation are injectable hydrogels. In this review, we examine the materials that are currently under investigation for nucleus augmentation, and compare their ability to meet the design requirements for this application. Specifically, the delivery of the material into the disc, the mechanical properties of the material and the biological compatibility are examined. Recommendations for future testing are also made.

Decellularised human bone allograft from different anatomical sites as a basis for functionally stratified repair material for bone defects.

Tissue engineered bone solutions aim to overcome the limitations of autologous and allogeneic grafts. Decellularised tissues are produced by washing cellular components from human or animal tissue to produce an immunologically safe and biocompatible scaffold, capable of integration following implantation. A decellularisation procedure utilising low concentration sodium dodecyl sulphate (0.1% w/v) was applied to trabecular bone from human femoral heads (FH) and tibial plateaus (TP). Biological (histology, DNA quantification), biomechanical (compression testing) and structural (µCT) comparisons were made between decellularised and unprocessed cellular tissue. Total DNA levels of decellularised FH and TP bone were below 50 ng mg-1 dry tissue weight and nuclear material was removed. No differences were found between cellular and decellularised bone, from each anatomical region, for all the biomechanical and structural parameters investigated. Differences were found between cellular FH and TP and between decellularised FH and TP. Decellularised FH had a higher ultimate compressive stress, Young's modulus and 0.2% proof stress than decellularised TP (p = 0.001, 0.002, 0.001, Mann Whitney U test, MWU). The mineral density of cellular and decellularised TP bone was significantly greater than cellular and decellularised FH bone respectively (cellular: p = 0.001, decellularised: p < 0.001, MWU). The bone volume fraction and trabecular thickness of cellular and decellularised FH bone were significantly greater than cellular and decellularised TP bone respectively (cellular: p = 0.001, 0.005; decellularised: p < 0.001, <0.001, MWU). Characterisation of decellularised trabecular bone from different anatomical regions offers the possibility of product stratification, allowing selection of biomechanical properties to match particular anatomical regions undergoing bone graft procedures.

Reproducing the Biomechanical Environment of the Chondrocyte for Cartilage Tissue Engineering.

It is well known that the biomechanical and tribological performance of articular cartilage is inextricably linked to its extracellular matrix (ECM) structure and zonal heterogeneity. Furthermore, it is understood that the presence of native ECM components, such as collagen II and aggrecan, promote healthy homeostasis in the resident chondrocytes. What is less frequently discussed is how chondrocyte metabolism is related to the extracellular mechanical environment, at both the macro and microscale. The chondrocyte is in immediate contact with the pericellular matrix of the chondron, which acts as a mechanocoupler, transmitting external applied loads from the ECM to the chondrocyte. Therefore, components of the pericellular matrix also play essential roles in chondrocyte mechanotransduction and metabolism. Recreating the biomechanical environment through tuning material properties of a scaffold and/or the use of external cyclic loading can induce biosynthetic responses in chondrocytes. Decellularized scaffolds, which retain the native tissue macro- and microstructure also represent an effective means of recapitulating such an environment. The use of such techniques in tissue engineering applications can ensure the regeneration of skeletally mature articular cartilage with appropriate biomechanical and tribological properties to restore joint function. Despite the pivotal role in graft maturation and performance, biomechanical and tribological properties of such interventions is often underrepresented. This review outlines the role of biomechanics in relation to native cartilage performance and chondrocyte metabolism, and how application of this theory can enhance the future development and successful translation of biomechanically relevant tissue engineering interventions. Impact statement Physiological cartilage function is a key criterion in the success of a cartilage tissue engineering solution. The in situ performance is dependent on the initial scaffold design as well as extracellular matrix deposition by endogenous or exogenous cells. Both biological and biomechanical stimuli serve as key regulators of cartilage homeostasis and maturation of the resulting tissue-engineered graft. An improved understanding of the influence of biomechanics on cellular function and consideration of the final biomechanical and tribological performance will help in the successful development and translation of tissue-engineered grafts to restore natural joint function postcartilage trauma or osteoarthritic degeneration, delaying the requirement for prosthetic intervention.

An experimental simulation model to assess wear of the porcine patellofemoral joint.

A range of surgical techniques and osteochondral interventions have been developed for early stage chondral/osteochondral repair interventions in the knee however, methods for functional, pre-clinical assessment of these therapies are limited. In this study, a method for simulating physiological loading and motion in the porcine patellofemoral joint was developed using a 6-axis simulator. As an example of how the method can be used, the influence of surgical positioning of osteochondral allografts in the patella on cartilage wear, deformation and damage and graft stability was investigated in this porcine patellofemoral joint model. The functional performance of allografts implanted either optimally (flush with the cartilage surface) or 1 mm proud of the cartilage surface was compared to a positive control (stainless steel pin implanted 1 mm proud of the cartilage surface), a negative control (no intervention) and a defect model. Allografts implanted flush with the surrounding cartilage could restore the articulating surface of the patella resulting in low wear, damage and deformation of the opposing cartilage surface, similar to that of the negative control group. Implanting the graft proud of the patella surface resulted in cartilage lesions on the femoral trochlea (ICRS grade 2) and a cartilage volume difference of 2.0 ± 3.9 mm3; the positive controls resulted in more severe lesions, a higher volume difference (14.2 ± 7.4 mm3) which in some cases exposed subchondral bone (ICRS grade 4). Defects in the patella caused deformation of the opposing cartilage surface. All grafts implanted in the patella subsided over the duration of the study. This study demonstrated a method that can be used to evaluate osteochondral repair strategies in the patellofemoral joint applying physiological loading and motions.

Decellularized Intervertebral Discs: A Potential Replacement for Degenerate Human Discs.

Intervertebral disc (IVD) degeneration is a major cause of back pain. Current surgical interventions have limitations. An alternative approach is to replace degenerated IVDs with a natural biological scaffold. The removal of cellular components from human IVDs should render them nonimmunogenic upon implantation. The aim of this initial proof of technical feasibility study was to develop a decellularization protocol on bovine IVDs with endplates (EPs) and assess protocol performance before application of the protocol to human IVDs with attached EP and vertebral bone (VB). A decellularization protocol based on hypotonic low concentration sodium dodecyl sulfate (0.1% w/v) with proteinase inhibitors, freeze/thaw cycles, and nuclease and sonication treatments was applied to IVDs. Histological, biochemical, and biomechanical comparisons were made between cellular and decellularized tissue. Cell removal from bovine IVDs was demonstrated and total DNA levels of the decellularized inner annulus fibrosus (iAF), outer annulus fibrosus (oAF), and EP were 40.7 (±11.4), 25.9 (±3.8), and 29.3 (±3.1) ng.mg-1 dry tissue weight, respectively (n = 6, ±95% confidence level [CL]). These values were significantly lower than in cellular tissue. No significant difference in DNA levels between bovine cellular and decellularized nucleus pulposus (NP) was found. Glycosaminoglycans (GAGs) were largely retained in the NP, iAF, and oAF. Cyclic compression testing showed sufficient sensitivity to detect an increase in stiffness of bovine IVD postdecellularization (2957.2 ± 340.8 N.mm-1) (predecellularization: 2685.4 ± 263.1 N.mm-1; n = 5, 95% CL), but the difference was within natural tissue variation. Total DNA levels for all decellularized tissue regions of human IVDs (NP, iAF, oAF, EP, and VB) were below 50 ng.mg-1 dry tissue weight (range: 2 ng.mg-1, iAF to 29 ng.mg-1, VB) and the tissue retained high levels of GAGs. Further studies to assess the biocompatibility and regenerative potential of decellularized human IVDs in vitro and in vivo are now required; however, proof of technical feasibility has been demonstrated and the retention of bone in the IVD samples would allow incorporation of the tissue into the recipient spine. Impact statement Intervertebral disc (IVD) degeneration is a major cause of back pain. Current surgical treatments have limitations and relatively poor outcomes. An implantable cell-free biological scaffold, which will not invoke adverse immune responses, has the potential to preserve the natural mobility of the patient's spine and be regenerated with endogenous cells, preventing further degeneration and improving surgical outcomes. This study demonstrates, for the first time, that it is possible to create a cell-free human IVD biological scaffold with attached bone using decellularization technology, the first step toward the development of an implantable regenerative device for IVD replacement.

Development and characterisation of a decellularised bovine osteochondral biomaterial for cartilage repair.

It is proposed that an acellular natural osteochondral scaffold will provide a successful repair material for the early intervention treatment of cartilage lesions, to prevent or slow the progression of cartilage deterioration to osteoarthritis. Here, we investigated the efficacy of methods for the decellularisation of bovine osteochondral plugs. The plugs were subject to four freeze/thaw cycles followed by two cycles of washes in hypotonic solution and low concentration (0.1% w/v) sodium dodecyl sulphate with protease inhibitors. Plugs were treated with nuclease (DNase and RNase) treatment followed by sterilization in peracetic acid. Full tissue decellularisation was achieved as confirmed by histological analysis and DNA quantification, however the resultant acellular matrix had reduced glycosaminoglycan content which led to an increased percent deformation of cartilage. Furthermore, the acellular scaffold was not reproducibly biocompatible. Additional terminal washes were included in the process to improve biocompatibility, however, this led to visible structural damage to the cartilage. This damage was found to be minimised by reducing the cut edge to cartilage area ratio through decellularisation of larger cuts of osteochondral tissue.