Abstract. Objective. Clinical treatments to repair articular cartilage (AC) defects such as autologous cartilage implantation (mosaicplasty) often suffer from poor integration with host tissue, limiting their long-term efficacy. Thus to ensure the longevity of AC repair, understanding natural repair mechanisms that allow for successful integration between cartilaginous surfaces, as has been reported in juvenile tissue, may be key. Here, we evaluated cartilage integration over time in a pig explant model of natural tissue repair by assessing expression and localisation of major ECM proteins, enzymatic cross-linkers including the five isoforms of lysyl oxidase (LOX), small leucine-rich repeat proteoglycans (SLRP's), and proteases (e.g. ADAMTS4). Methods. AC was retrieved from the femoral condyles of 8-week-old pigs. Full thickness 6mmØ AC discs were prepared, defects were induced, and explants cultured for up to 28 days. After fixation, sections were stained using Safranin-O and antibodies against Collagen types I & II, LOX, and ADAMTS4. Gene expression analyses were performed using qPCR. We also cultured devitalized samples, either with or without enzymatic treatment to deplete proteoglycans, for 28 days and similarly assessed repair. Results. Safranin-O staining demonstrated successful integration of cartilage defects over a 28-day period. No significant regulation in the expression of Col1a1, Col2a1, LOX or SLPR genes was observed at any time point. Immunofluorescence staining revealed that only ADAMTS4 localized at the injury surface in integrated samples. Interestingly, we also observed successful spontaneous integration of proteoglycan-depleted devitalized tissue. Conclusion. Cartilage integration in our pig cartilage explant model did not appear to be mediated by upregulation of major cartilage ECM components, enzymatic cross-linkers, or SLRPs. However, spontaneous integration of devitalized, proteoglycan-depleted AC, and localised upregulation of ADAMTS4 at the injured surface in successfully integrated samples, suggest that ADAMTS4 may enhances normal repair in injured AC through local aggrecan depletion, therefore enabling spontaneous cross-linking of collagen fibrils. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Hydrogels are hydrated 3-dimensional (3D) polymer networks that can be chemically or physically crosslinked. Interest in the use of hydrogels for tissue engineering applications has been growing in the past few decades due to their excellent biocompatibility and biodegradability. One of the major drawbacks of the use of hydrogels in such applications is their lack of structural strength. To address this, in this work, we have combined two hydrogel types, namely gelatin and alginate. In this work, a 1 ml volume of gelatin alginate hydrogel was molded in each well of a 24 well-plate and crosslinked with different concentrations of calcium chloride (CaCl₂) (20, 40, 60, 80, and 100 mM) to investigate the influence of concentration on hydrogel properties and cell viability. The hydrogel was characterized using Fourier transform infrared (FTIR) spectrometry, environmental scanning electron microscopy (ESEM), and an Alamar blue assay to assess the chemical structure, the surface morphology, and the epithelial cell viability of the hydrogel, respectively. The FTIR analysis shows that network formation improved with increasing concentration; decreased ion-polymer interactions have been noted for concentrations ≤ 60 mM. This appears to be in agreement with ESEM images that show an evolution from a smooth, featureless surface to the appearance of surface pore structure for concentrations ≥ 80 mM. Perhaps as ion concentration increases and network formation improves, the effect is evidenced as surface porosity; low concentrations result in swelling and a smooth surface. In terms of cell viability, viability has been found to increase with increasing concentration. The cell viability is 90 % at 100 mM CaCl₂, in contrast to 50 % for a concentration of 20 mM after 9 days of incubation. It is possible that the reduced viability can be attributed to the high proportion of uncrosslinked polymer chains at low concentrations. Overall, these results provide useful information about the role of crosslinking concentration on hydrogel properties, knowledge that may be applied to 3D bioprinting.

Summary. Low energy irradiation of vitamin E blended UHMWPE is feasible to fabricate total joint implants with high wear resistance and impact strength. Introduction. Irradiated ultra-high molecular weight polyethylene (UHMWPE), used in the fabrication of joint implants, has increased wear resistance. But, increased crosslinking decreases the mechanical strength of the polymer, thus limiting the crosslinking to the surface is desirable. Here, we used electron beam irradiation with low energy electrons to limit the penetration of the radiation exposure and achieve surface cross-linking. Methods. Medical grade 0.1wt% vitamin E blended UHMWPE (GUR1050) was consolidated and irradiated using an electron beam at 0.8 and 3 MeV to 150 kGy. Fourier Transform Infrared Spectroscopy (FTIR) was used from the surface along the depth at an average of 32 scans and a resolution of 4 cm. −1. A transvinylene index (TVI) was calculated by normalizing the absorbance at 965 cm. −1. (950–980cm. −1. ) against 1895 cm. −1. (1850 – 1985 cm. −1. ). TVI in irradiated UHMWPE is linearly correlated with the radiation received [3]. Vitamin E indices were calculated as the ratio of the area under 1265 cm. −1. (1245–1275 cm. −1. ) normalized by the same. Pin-on-disc (POD) wear testing was conducted on cylindrical pins (9 mm dia., 13 mm length, n=3) as previously described at 2 Hz [4] for 1.2 million cycles (MC). Wear rate was measured as the linear regression of gravimetric weight change vs. number of cycles from 0.5 to 1.2 MC. Double notched IZOD impact testing was performed (63.5 × 12.7 × 6.35mm) in accordance with ASTM F648. Cubes (1 cm) from 0.1wt% blended and 150 kGy irradiated pucks (0.8 MeV) were soaked in vitamin E at 110°C for 1 hour followed by homogenization at 130°C for 48 hours. Results. The penetration of the electron beam for cross-linking was limited at low beam energy and cross-linking of the surface 2 mm was achieved. The wear rate of samples irradiated at 0.8 and 3 MeV was 1.12±0.15, and 0.98±0.11, respectively (p»0.5). In addition, the wear rate of the surface (0.8 MeV) irradiated UHMWPE was 0.33±0.02 mg/MC 1 mm below the surface. The impact strength of UHMWPE irradiated at 0.8 MeV was 73 kJ/m. 2. and 54.2 kJ/m. 2. for that irradiated at 3 MeV (p=0.001). Doping with vitamin E and homogenization increased the surface vitamin E concentration from undetectable levels to 0.11±0.01. Discussion. The wear rate of this surface cross-linked UHMWPE was comparable to uniformly cross-linked UHMWPEs irradiated at higher electron beam energies. Even lower wear rate subsurface suggested the feasibility of machining 1 mm from the surface in implant fabrication. Limiting cross-linking to the surface resulted in higher impact strength compared to a uniformly cross-linked UHMWPE. Vitamin E was optionally replenished by additional doping after cross-linking; an advantage of this method may be increased oxidation resistance

Abstract. Objectives. Ultra-High Molecular Weight Polyethylene (UHMWPE) can be made radiopaque through the diffusion of an oil-based contrast agent (Lipiodol Ultra-fluid). A similar process is used for Vitamin E incorporated polyethylene, which has a well-established clinical history. This study aimed to quantify the leaching of Lipiodol and compare to vitamin E polyethylene. Method. GUR 1050 polyethylene (4 mm thickness) was cut into squares, 10 mm. 2. Samples (n=5) were immersed in 25 ml Lipiodol (Guerbet, France), or 15 ml Vitamin E (L-atocopherol, Sigma-Aldrich, UK). To facilitate diffusion, samples were held at 105°C for 18 hours. After treatment, all samples were immersed in DMEM (Sigma-Aldrich, UK) with Penicillin Streptomycin (Sigma-Aldrich, Kent, UK) at 4%v/v and held at 37°C in an incubator. Untreated polyethylene samples were included as controls. Leaching was quantified gravimetrically at weeks 2, 4 and 8. The radiopacity of the Lipiodol-diffused samples was investigated from µCT images (162kV, resolution 0.2 mm, X Tec, XT H 225 ST, Nikon Metrology, UK). Results. The leaching of Lipiodol and Vitamin E followed the same trend and reached a steady state after week 2. At this point there was a 20% decrease in the Hounsfield Unit and droplets of radiopaque oil were visible in the DMEM solution; these were not evident in subsequent scans. Over 8 weeks of 20% Lipiodol leached out of the polyethylene, which was greater than of 10% Vitamin E. Conclusion. After 8 weeks the radiopaque polyethylene was still identifiable in CT scan images, even though 20% of leaching occurred. The leaching of Lipiodol may be mitigated through cross-linking, which has been shown to reduce leaching of Vitamin E; this will be investigated as future work. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Bone fractures are highly observed clinical situation in orthopaedic treatments. In some cases, there might be non-union problems. Therefore, recent studies have focused on tissue engineering applications as alternative methods to replace surgical procedures. Various biopolymer based scaffolds are produced using different fabrication techniques for bone tissue engineering applications. In this study, hydroxyapatite (HAp) and loofah containing carboxymethyl chitosan (CMC) scaffolds were prepared. In this regard, first 4 ml of CMC solution, 0.02 g of hydroxyapatite (HAP) and 0.06 g of poly (ethylene glycol) diglycidyl ether (PEGDE) were mixed in an ultrasonic bath until the HAp powders were suspended. Next, 0.04 g of loofah was added to the suspension and with the help of PEGDE as the cross-linking agent, then, the mixture was allowed to cross-link at 40. o. C overnight. Finally, the three-dimensional, porous and sponge-like scaffolds were obtained after lyophilization (TELSTAR - LyoQuest −85) at 0.1 mbar and −25°C for 2 days. Morphologies, chemical structures and thermal properties of the scaffolds were characterized by scanning electron microscopy (SEM), Fourier Transform infrared spectroscopy (FT-IR) and thermogravimetric differential thermal analysis (TGA/DTA), respectively. In addition, swelling behavior and mechanical properties of the scaffolds under compression loading were determined. In order to investigate biocompatibility of the scaffolds, WST-1 colorimetric assay at days 0, 1, 3, 5 and 7 was conducted by using human dermal fibroblast. Also, histological and morphological analysis were performed for cell attachment at day 7. In conclusion, the produced scaffolds showed no cytotoxic effect. Therefore, they can be considered as a candidate scaffold for bone tissue regeneration. Further studies will be performed by using bone marrow and periosteum derived mesenchymal stem cells with these scaffolds

Adherent cells are known to respond to physical characteristics of their surrounding microenvironment, adapting their cytoskeleton and initiating signaling cascades specific to the type of cue encountered. Scaffolds mimicking native biophysical cues have proven to differentiate stem cells towards tissue-specific lineages and to maintain the phenotype of somatic cells for longer periods of time in culture. Biomaterial-based tendon implants are designed to withstand high physiological loads but often lack the appropriate biochemical, biophysical and biological structure to drive tendon regeneration by populating cells. The objective of this study is to use tendon main component, collagen type I, to create scaffolds that reproduce tendon natural anisotropy and rigidity, in an effort to engineer functional tendon tissue with native organization and strength, able to maintain tenocyte phenotype and to differentiate stem cells towards the tenogenic lineage. Porcine collagen type I in solution was treated with one of the following cross-linkers: glutaraldehyde, genipin or 4-arm polyethylene glycol (4SP). The resulting mixture was poured on micro-grooved (2×2×2 um) or planar PDMS moulds and air-dried to obtain 5 mg/ml collagen films. Surface topography and elastic modulus were analyzed using SEM/AFM and rheometry, respectively. Human tendon cells were cultured on the micro-grooved/planar scaffolds for up to 10 days. Cell morphology, collagen III and tenascin C expression were analyzed by immunocytochemistry. Among the different cross-linkers used, only the treatment with 4SP resulted in scaffolds with a recognizable micro-grooved surface topography. Precise control over the micro-grooved topography and the rigidity of the scaffolds was achieved by cross-linking the collagen with varying concentrations of 4SP (0, 0.5, 1 and 1.5mM) at low pH and temperature. The elastic modulus of the scaffolds cross-linked with 4SP (0.5mM) matched the values previously reported to induce tenogenic differentiation in stem cells (50–90 kPa). Approximately eighty percent of the human tendon cells cultured on the micro-grooved collagen films aligned in the direction of the anisotropy for 10 days in culture, mimicking the alignment of tenocytes in the native tissue. Cell nuclei morphology, known to play a central role in the process of mechanotransduction, was significantly more elongated for the tenocytes cultured on the micro-grooved scaffolds after 4 days in culture for all the 4SP concentrations. Synthesis, deposition and alignment of collagen III and tenascin C, two important tenogenic markers, were up regulated selectively on the micro-grooved and rigid scaffolds after 10 days in culture, respectively. These results highlight the synergistic effect of matrix rigidity and cell alignment on tenogenic cell lineage commitment. Collectively, this study provides new insights into how collagen can be modulated to create scaffolds with precise imprinted topographies and controlled rigidities

Introduction. Exercise increases tendon collagen synthesis and cross-link formation. Exercise also increases the expression of TGF-β. 1. TGF-β. 1. may contribute to the upregulation of tendon collagen synthesis during exercise, but this relationship has not been established in vivo. The purpose of this study was to evaluate the effects of TGF-β. 1. receptor inhibition on tendon collagen. Materials and Methods. Male Wistar rats were divided into sedentary (SED, n = 9) or exercised (RUN, n=15) groups. Exercised animals completed four days of treadmill exercise (60 minutes/days). The peritendinous space of one Achilles tendon was injected with LY-364947 (ALK5 inhibitor; INHIB) while the opposite leg was injected with a vehicle (SHAM). Injections were given daily after each exercise bout. ERK and Smad 2/3 phosphorylation was evaluated by Western blotting. Collagen I and III gene expression were evaluated via qRT-PCR. Tendon hydroxyproline and hydroxylyslpyridinoline (HP) cross-linking were assayed via HPLC. A longitudinal section of tendon was stained with H&E for evaluation of cell numbers and fibril organization. Results. Phosphorylation of ERK increased by 2.5-fold in both legs given LY-364947 (p<0.05) but was not influenced by exercise (p>0.05). Smad 3 phosphorylation was reduced (p<0.05) in tendons treated with LY-364947. Neither type I nor type III collagen gene expression was affected by TGF-β. 1. receptor inhibition or exercise (p>0.05). Collagen content was not altered by either exercise or LY-364947 (p>0.05). HP cross-linking was 3-fold lower in the RUN-INHIB when compared to the RUN-SHAM tendon (p<0.05). No effect of inhibitor on HP was noted in the sedentary animals. Cell density was greater (p<0.05) in the Achilles tendon of exercised animals (SED: 7.5 cell/100 μm. 2. , RUN: 10.3 cell/100 μm. 2. ) but was not influenced by TGF-β. 1. receptor inhibition (p>0.05). Fiber structure scores were 45% lower (p<0.07) in SED animals treated with inhibitor but normal in RUN animals given inhibitor. Discussion. The changes in ERK and Smad phosphorylation suggest that LY-364947 was effective at altering TGF-β. 1. signaling. Our data suggest that neither acute exercise nor TGF-β. 1. receptor inhibition altered collagen gene expression. In contrast, TGF-β. 1. appears to be important for regulating Achilles tendon cross-link formation during exercise training and inhibition of TGF-β. 1. impacts fiber structure

Cellular therapies play an important role in tendon tissue engineering with tenocytes being described as the most prominent cell population if available in large numbers. In vitro expansion of tenocytes in standard culture leads to phenotypic drift and cellular senescence. Maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the tendon microenvironment. One approach used to modulate in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking through the activation of hypoxia-inducible factor 1-alpha (HIF1-α), we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance. SDS-PAGE and immunocytochemistry analysis demonstrated that human tenocytes treated with MMC at 2 % oxygen tension showed increased synthesis and deposition of collagen type I. Moreover, immunocytochemistry for the tendon-specific ECM proteins collagen type III, V, VI and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2 % oxygen tension. In addition, western blot analysis revealed increased expression of tendon-specific protein Scleraxis, while a detailed gene analysis illustrated upregulation of tendon-specific genes and downregulation of trans-differentiation genes again when cells cultured with MMC under hypoxic conditions. Collectively, results suggest that the synergistic effect of MMC and low oxygen tension can accelerate the formation of ECM-rich substitutes, which stimulates tenogenic phenotype maintenance

The enthesis is a specialised zonal tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones (tendon, fibrocartilage, mineralized fibrocartilage and bone). After injury, the native structure is often not re-established and a mechanically weaker fibrovascular scar is formed. Traditionally used monotherapies have failed to be effective, posing the need for multi-cargo localized delivery vehicles. We hypothesize that multilayer collagen-based scaffolds can serve as delivery vehicles for specific bioactive molecules with tenogenic, chondrogenic and osteogenic potential to enhance the functional regeneration of the enthesis. Three-layer scaffolds composed by a tendon-like layer of collagen type I, a cartilage-like layer of collagen type II and a bone-like layer of collagen type I and hydroxyapatite were fabricated by an iterative layering freeze-drying technique. The scaffolds were cross-linked with varying concentration of 4-arm polyethylene glycol (4s-PEG) and the biological and mechanical properties were assessed. Each layer was functionalized with platelet-derived growth factor, insulin growth factor, heparan sulfate or bone morphogenetic protein 7 and their tenogenic, chondrogenic and osteogenic potential on bone-marrow derived stem cells was investigated in vitro. Scaffolds cross-linked with 1 mM 4s-PEG showed 60% free amines reduction respect to non-cross-linked scaffolds, were stable in collagenase over 24 hours and had a compression modulus of 30 kPa. The bioactive molecules had a sustained release profile (approximately 50 ng/mL) over 5 days as a function of cross-linking. Preliminary in vitro studies confirmed the chondrogenic potential of heparin sulfate and insulin growth factor by the increase of proteoglycans

Cellular therapies play an important role in tendon tissue engineering and regenerative medicine with tenocytes being described as the most prominent cell population for these applications if available in large numbers. However, this is difficult to achieve, because in vitro expansion of tenocytes leads to phenotypic drift and loss of function. Recent work suggests that maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the native tendon microenvironment. One approach used to modulate in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). MMC is based on the addition of inert macromolecules to the culture media to mimic the dense extracellular matrix and accelerate the production of ECM-rich substitutes. In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking through the activation of hypoxia-inducible factor 1-alpha (HIF1-α), we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance by enhancing deposition of tissue-specific extracellular matrix. SDS-PAGE and immunocytochemistry analysis, demonstrated that human tenocytes treated with the optimal MMC concentration at 2% oxygen tension showed increased collagen type I synthesis and deposition after 7 days. Moreover, immunocytochemistry for collagen type III, type V, VI, elastin and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2% oxygen tension. In addition, it was shown that low oxygen tension and MMC did not affect the spindle-shape morphology, metabolic activity, proliferation and viability of human tenocytes Collectively, these results suggest that the synergistic effect of optimal macromolecular crowding concentration and low oxygen tension (2%) can accelerate the formation of ECM-rich substitutes, which may stimulate tenogenic phenotype maintenance. Further gene and protein analysis for tendon specific markers should be performed to validate our promising results

Introduction. Collagen is the predominant component of extracellular matrix in various connective tissues and makes up to 25% to 35% of the whole protein content in animal bodies. Type II collagen was first introduced from chicken sternal cartilage and presents supportive function in cartilaginous tissue. Since type II collagen is the major component of cartilage in joint, this study is aiming to determine an optimal type II collagen material for the development of medical devices for articular cartilage regeneration. In order to make more effective use of underutilized food waste, type II collagens from mammalian tissue sources (porcine tracheal cartilage; auricular cartilage; articular cartilage) and marine tissue sources (cuckoo ray, blonde ray, thorn back ray, lesser spotted dogfish) were isolated through acid-pepsin digestion under 4°C and characterized by various biological, biochemical and biophysical analysis. Pepsin cleaves the telopeptide region of the collagen molecule and pepsin treated collagen extraction ensures higher collagen yield. Telopeptide-free collagen reveals cytocompatibility, biodegradability and lower toxicity. The number and size of collagen chains were revealed by SDS-polyacrylamide gel electrophoresis. Intermolecular crosslinking density was quantified by Ninhydrin assay. Thermal stability was tested by differential scanning calorimetry (DSC) and enzymatic degradation was assessed by collagenase assay. Human chondrocytes were seeded on to collagen sponges at a density of 30,000 cells per sponge. Cell morphology (DAPI/ Rhodamine Phalloidin), viability(LIVE/DEAD®), proliferation(PicoGreen®) and metabolic activity (alamarBlue®) were analysed. Quantitative morphometric analysis was carried out using ImageJ software. Conclusion. Porcine articular cartilage and cartilaginous fishes yield high purity type II collagen. Type II collagen isolated from cartilaginous fishes exhibited similar crosslinking density and thermal stability. Among various porcine cartilaginous tissues, articular cartilage was the most resistant to enzymatic degradation and female trachea exhibited the highest cross-linking density. The biological, biochemical and thermal properties of type II collagen are dependent on the tissue and gender from which the collagen was extracted

The biomechanical evaluation of tendon repair with collagen-based scaffolds in rat model is a common method to determine the functional outcome of the tested material. We introduced a magnetic resonance imaging (MRI) approach to verify the biomechanical test data. In present study different collagen scaffolds for tendon repair were examined. Two collagen test materials: based on bovine stabilized collagen, chemically cross-linked with oriented collagenous fibres (material 1) and based on porcine dermal extracellular matrix, with no cross-linking (material 2) were compared. The animal study was approved by the local review board. Surgery was performed on male Sprague-Dawley rats with a body weight of 400 ± 19 g. Each rat underwent a 5 mm transection of the right Achilles tendon. The M. plantaris tendon was removed. The remaining tendon ends were re-joined with a 5 mm scaffold of either the material 1 or 2. Each scaffold material was sutured into place with two single stiches (Vicryl 4–0, Ethicon) each end. A total of 16 rats (n= 8 each group) were observed for 28 days follow up. The animals were sacrificed and hind limbs were transected proximal to the knee joint. MRI was performed using a 7 Tesla scanner (BioSpec 70/30, Bruker). T2-weighted TurboRARE sequences with an in-plane resolution of 0.12 mm and a slice thickness of 0.7 mm were analysed. All soft and hard tissues were removed from the Achilles tendon-calcaneus-foot complex before biomechanical testing. Subsequently, the specimens were fixed in a materials testing machine (Z1.0, Zwick, Ulm, Germany) for tensile testing. All tendons were preloaded with 1 N and subsequently stretched at a rate of 1 mm/s until complete failure was observed. Non-operated tendons were used as a control (n=4). After 28 postoperative days, MRI demonstrated that four scaffolds (material 1: n=2, material 2: n=2) were slightly dislocated in the proximal part of hind limb. In total five failures of reconstruction could be detected in the tendon repairs (material 1: n=3, material 2: n=2). Tendons augmented with the bovine material 1 showed a maximum tensile load of 57.9 ± 17.9 N and tendons with porcine scaffold material 2 of 63.1 ± 19.5 N. The native tendons demonstrated only slightly higher loads of 76.6 ± 11.6 N. Maximum failure load of the tendon-scaffold construct in both groups did not differ significantly (p < 0.05). Stiffness of the tendons treated with the bovine scaffold (9.9 ± 3.6 N/mm) and with the porcine scaffold (10.7 ± 2.7 N/mm) showed no differences. Stiffness of the native healthy tendon of the contralateral site was significantly higher (20.2 ± 6.6 N/mm, p < 0.05). No differences in the mechanical properties between samples of both scaffold groups could be detected, regardless of whether the repaired tendon defect has failed or the scaffold has been dislocated. The results show that MRI is important as an auxiliary tool to verify the biomechanical outcome of tendon repair in animal models

Summary. Coating of titanium implants with BMP-2-loaded polyelectrolyte multilayer films conferred the implant surface with osteoinductive properties which are fully preserved upon both air-dried storage and γ-sterilization. Although BMP-2 is recognised as an important molecule for bone regeneration, its supraphysiological doses currently used in clinical practice has raised serious concerns about cost-effectiveness and safety issues. Thus, there is a strong motivation to engineer new delivery systems or to provide already approved materials with new functionalities. Immobilizing the growth factor onto the surface of implants would reduce protein diffusion and increase residence time at the implantation site. To date, modifying the surfaces of metal materials, such as titanium or titanium alloys, at the nanometer scale for achieving dependable, consistent and long-term osseointegration remains a challenging approach. In this context, we have developed an osteoinductive coating of a porous titanium implant using biomimetic polyelectrolyte multilayer (PEM) films used as carriers of BMP-2. The PEM films were prepared by alternate deposition of 24 layer pairs of poly(L-lysine) (PLL) and hyaluronic acid (HA) layers (∼3.5 µm in thickness); such films were then cross-linked by means of a water-soluble carbodiimide (EDC) at different degrees. The amount of BMP-2 loaded in these films was tuned (ranging from 1.4 to 14.3 µg/cm. 2. ) depending on the cross-linking extent of the film and of the BMP-2 initial concentration. Because packaging, and storage of the devices are important issues that may limit a wide application of biologically functionalised materials, we assessed in the present study the osteoinductive performance of the BMP-2 loaded PEM coatings onto custom-made 3D porous scaffolds made of Ti-6Al-4V in vitro and in vivo pertinent to long-term storage in a dry state and to sterilization by gamma irradiation. Analysis of PEM films by infrared spectroscopy evidenced that the air-dried films were stable for at least one year of storage at 4°C and they resisted exposure to γ-irradiation at clinically approved doses. The preservation of the growth factor bioactivity was evaluated both in vitro (using C2C12 cell model) and in vivo (in a rat ectopic model). In vitro, BMP-2 loaded in dried PEM films exhibited shelf-life stability at 4°C over a one-year period. However, its bioactivity decreased from 50 to 80% after γ-irradiation at 25 and 50 kGy, respectively. Remarkably, the in vivo studies showed that the amount of new bone tissue formation induced by BMP-2 contained in PEM-coated Ti implants was not affected after air-drying of the implants and sterilization at 25 kGy indicating the full preservation of the growth factor bioactivity. Altogether, our results provided evidence of the remarkable property of PEM film coatings that both sequester BMP-2 and preserve its full in vivo osteoinductive potential upon both storage and γ-sterilization. The protective effects of PEM films on the growth factor bioactivity may be attributed to both the high water content in (PLL/HA) films (∼90%) and to their porosity, which may provide a “protein-friendly” environment similar to the natural extracellular matrix. This novel “off-the-shelf” technology of functionalised implants opens promising applications in prosthetic and tissue engineering fields

Summary Statement. In the present hip simulator studies, bearings with the newest generation of HXLPE, stabilised with vitamin E, did not show increased wear under severe conditions, such as accelerated ageing, component mal-orientation and third body wear. Introduction. Unfortunately, acetabular hip components cannot always be implanted in optimal condition. Therefore, we performed hip simulator studies with cups made from highly cross-linked, vitamin E stabilised UHMWPE in i) artificially aged condition, ii) with an inclination angle corresponding to 80 ° in vivo and iii) with third bodies coming from the Ti coating of the acetabular cup. Methods. For these hip simulator studies, seleXys cup inlays, size 28/EE, and RM Pressfit samples 50/28 (Mathys Ltd Bettlach, Switzerland) were used. Standard PE parts and vitamys® inlays (highly cross-linked, vitamin E stabilised UHMWPE) were tested in the same series. PE cups were machined out of sintered GUR 1020 slabs, packaged and gamma-sterilised in inert atmosphere at 30 kGy. The vitamys® material was made in-house by adding 0.1 wt.-% of vitamin E (Merck KGaA, Darmstadt/Germany) to GUR 1020 powder from Ticona GmbH, Kelsterbach/Germany. Cross-linking used 100 kGy gamma-irradiation and the final sterilisation was gas plasma. Artificial ageing was done under pressurised oxygen at 70 °C according to ASTM F2003 for 14 days (standard PE) and 60 days (vitamys®), respectively. The hip simulator test protocol of ISO 14242 was kept for the artificially aged cups, but the inclination angle altered to 80 ° for the test with the steep cup position. In the third test, the test fluid (diluted bovine serum stabilised with sodium azide and EDTA) was altered by adding about 10 Ti particles to the bearing for the first million cycles. This test condition imitates third body wear by particles shed from the coating of the RM cups. All testing was conducted at the RMS Foundation (Bettlach / Switzerland) on a servo-hydraulic six-station hip simulator (Endolab, Thansau/Rosenheim, Germany) at a temperature of 37±1°C. At lubricant change interval of 500’000 cycles, the inlays were measured gravimetrically with an accuracy of 0.01 mg. Results. The wear rate of the standard UHMWPE in the condition aged for 14 days reached 45 mg/Mcycle, corresponding to a 57 % increase over non-aged cups. For the vitamys® cups, the wear rate was virtually unchanged even after 60 days ageing (5.8 mg/Mcycle vs. 5.9 mg/Mcycle). For standard UHMWPE tested with an inclination of 80°, wear was 16% lower than those of the inlays with 45° inclination. Whereas for the vitamys® inlays, the wear rate was about the same for both inclination angles (5.4 mg/Mcycle vs. 5.9 mg/Mcycle,). The addition of Ti particles increased the wear rate of standard UHMWPE to 35 mg/Mcycle. However, vitamys® was hardly affected by the third bodies: the wear rate stood at 7.8 mg/Mcycle. Conclusions. Based on the present simulator study, it seems that hip bearings with the newest generation of HXLPE, stabilised with vitamin E, are exempt from increased wear rate when subjected to severe conditions, such as accelerated ageing, component mal-orientation and third body wear

Summary. Fifteen irradiated, vitamin E-diffused UHMWPE retrievals with up to three years in vivo service showed no appreciable oxidation, nor change in material properties from a never-implanted liner, and showed a 94% decrease in free radical content. Introduction. Radiation cross-linking, used to improve wear resistance of ultra-high molecular weight polyethylene (UHMWPE) bearings used in total joint arthroplasty, generates residual free radicals which are the precursors to oxidative embrittlement. First generation materials adopted thermal treatments to eliminate or reduce free radical content, but came with compromises in reduced mechanical properties or insufficient stabilization. A second generation alternative method infuses an antioxidant, vitamin E, into irradiated UHMWPE to stabilise free radicals while maintaining fatigue strength. In vitro studies predict excellent oxidation and wear resistance in vitamin E-stabilised bearings, but the long-term in vivo oxidation behavior, influenced by lipid absorption and cyclic loading, remains largely unknown. Our aim was to investigate in vivo changes in UHMWPE surgically-retrieved explants that were radiation cross-linked and stabilised by vitamin E. Patients & Methods. Fifteen surgically-retrieved irradiated, vitamin E-diffused and inert-gamma sterilised bearings (E1™, Biomet, Inc., Warsaw IN) with in vivo durations ranging from 3 days to 36.6 months were analyzed at unloaded rim/eminence and the articular surface along with one never-implanted component. Total lifetime of components was summed as shelf storage prior to implantation, in vivo duration and ex vivo duration in air. Fourier Transform Infrared Spectroscopy (FTIR) was used to measure carbonyl index (CI; per ASTM F2102-01ε1) both before and after 16 hour hexane extraction to. Extracted thin films were also reacted with nitric oxide to quantify hydroperoxides, an intermediate oxidation product associated with oxidation potential. Cross-link density was calculated from gravimetric swelling analysis per ASTM F2214. Crystallinity measurements were performed regionally using differential scanning calorimetry (DSC). Free radical content was measured by electron spin resonance (Memphis, TN). Results. Irradiated and vitamin E-diffused retrievals showed scratching at the articular surface, but retained machining marks up to three years in vivo, indicative of no measurable wear. Retrievals showed no significant oxidation at the time of surgical removal with maximum post-hexane carbonyl indices in the barely detectable range (MCI=0.029–0.154), located at the surface of retrievals. Ex vivo oxidation was not observed after 18 months of aging in air at room temperature. There was no increase in hydroperoxides (never-implanted HI=0.62±0.04; retrieval HI= 0.62±0.04), nor change in cross-link density (never-implanted: 0.275±0.015 mol/dm. 3. ; retrieval: 0.295±0.016 mol/dm. 3. ) or crystallinity (never-implanted: 58.3±1.4%; retrievals: 60.0±3.5%). Lipid penetration increased with time, showing a higher rate of diffusion in loaded regions. Free radical content was observed to decay with increasing in vivo duration (R. 2. =0.44; p<0.05), and by one order of magnitude (94%) by 36.6 months. A stronger negative correlation (R. 2. =0.65) was observed between the total lifetime of the liner and free radical content. Discussion/Conclusion. The free-radical scavenging activity of the vitamin E appears to successfully prevent both in vivo and ex vivo oxidation for short durations. Without an increase in hydroperoxides, the oxidation cascade initiated by radiation-induced and lipid-derived free radicals appears to have been halted. Retrievals also gave no indication of wear in this timeframe, similar to improved wear resistance seen in first generation materials. Continued monitoring will be necessary at longer implant durations

Improved understanding of the biomechanics and biology of rotator cuff tendons (RCT) may help reduce high re-rupture rates following repairs, particularly amongst larger tears. This study aims to use novel methods for quantitatively determining differences in the mechanical and thermal properties of intact healthy RCTs compared to torn ‘diseased’ tendons. A common problem in the mechanical testing of small tendon samples is that stress risers at the clamp-tendon interface can obscure measurements. As the shoulder is subject to shear, tension and compression, we developed a novel solution using Dynamic Shear Analysis (DSA), a form of rheology which studies material deformation. As collagen is the main component of RCT, the structure and mechanical properties may be affected by collagen conformational changes. Both dermis and rat tail tendon with increased collagen cross-linking exhibit stronger mechanical properties. Thermal changes detected by differential scanning calorimetry (DSC) can help to quantify collagen structural differences in torn RCT, and has been previously used to study muscle, cartilage and vertebral discs. There were 79 tears (mean age 65.2 years), which were classified according to the size of the tear as small, medium, large and massive. Two separate 3mm-sized biopsy samples were taken and subjected to DSA using oscillatory deformation under compression. The storage modulus (G') was calculated and used as an indicator of mechanical integrity. 18 control tendon specimens were obtained from patients aged between 22-89 years (mean age 58.8 years) during shoulder hemiarthroplasties and stabilisations. Additionally 7 normal, 7 small and 7 massive frozen specimens were thermally characterized. 3 samples per patient were heated between 20-80oC in hermetically sealed vessels. Useful thermal parameters were measured such as the melting temperature (TM) which apparently represents breaking of the amide-amide bonds and protein chains mobility, the denaturation temperature (TD) which supposedly corresponds to proteins falling out of solution and the denaturation enthalpy (ΔH) which reflects the relative amount of triple helical structure. Healthy tendons had a significantly higher modulus than torn tendons, indicating that torn tendons are mechanically weaker than normal tendons (p = 0.032). Normal tendons had significantly higher mean shear modulus than tendons with small and massive tears (p<0.01). Overall there was a negative correlation between moduli and severity of tendon tear (r = −0.698, p=0.189). The moduli did not significantly correlate with age, sex, hand dominance, or length of preservation in formalin. Massive RCT tears had significantly higher TM and TD when compared to normal RCT (p < 0.05), unlike small RCT tears. No significant difference was detected between the denaturation enthalpy of the different RCT groups. This case control study has demonstrated that normal RCTs have a significantly higher modulus than torn tendons, indicating that torn tendons have less mechanical integrity. Our study further demonstrated a trend between increasing tear size and decreasing mechanical integrity. This study has also demonstrated differences in some of the thermal properties of normal and torn RCTs. These are likely due to collagen structural changes. A decrease in the denaturation temperature of torn tendons, suggests that the material is intrinsically less stable. Torn tendons with reduced storage modulus and collagen integrity may be less able to withstand mechanical loads following repair. This pilot study provides some preliminary insight into the mechanisms that may contribute to, or represent adaptations to high rates of failure of RCT repairs

Objective

Excessive mechanical stress on synovial joints causes osteoarthritis (OA) and results in the production of prostaglandin E2 (PGE2), a key molecule in arthritis, by synovial fibroblasts. However, the relationship between arthritis-related molecules and mechanical stress is still unclear. The purpose of this study was to examine the synovial fibroblast response to cyclic mechanical stress using an in vitro osteoarthritis model.

We investigated the effect of mitomycin-C on the reduction of the formation of peritendinous fibrous adhesions after tendon repair. In 20 Wistar albino rats the tendo Achillis was cut and repaired using a modified Kessler technique. The rats were divided into two equal groups. In group 1, an injection of mitomycin-C was placed between the tendon and skin of the right leg. In group 2, an identical volume of sterile normal saline was injected on the left side in a similar fashion. All the rats received mitomycin-C or saline for four weeks starting from the day of operation. The animals were killed after 30 days. The formation of peritendinous fibrous tissue, the inflammatory reaction and tendon healing were evaluated. The tensile strength of the repaired tendons was measured biomechanically. Microscopic evidence of the formation of adhesions and inflammation was less in group 1. There was no significant difference in the tensile load required to rupture the repaired tendons in the two groups.

Mitomycin-C may therefore provide a simple and inexpensive means of preventing of post-operative adhesions.

Nanometre-sized particles of ultra-high molecular weight polyethylene have been identified in the lubricants retrieved from hip simulators. Tissue samples were taken from seven failed Charnley total hip replacements, digested using strong alkali and analysed using high-resolution field emission gun-scanning electron microscopy to determine whether nanometre-sized particles of polyethylene debris were generated in vivo. A randomised method of analysis was used to quantify and characterise all the polyethylene particles isolated.

We isolated nanometre-sized particles from the retrieved tissue samples. The smallest identified was 30 nm and the majority were in the 0.1 μm to 0.99 μm size range. Particles in the 1.0 μm to 9.99 μm size range represented the highest proportion of the wear volume of the tissue samples, with 35% to 98% of the total wear volume comprised of particles of this size. The number of nanometre-sized particles isolated from the tissues accounted for only a small proportion of the total wear volume. Further work is required to assess the biological response to nanometre-sized polyethylene particles.