To address the current challenge of anterior cruciate ligament (ACL) reconstruction, this study is the first to fabricate a braided collagen rope (BCR) which mimics native hamstring for ACL reconstruction. The study aims to evaluate the biological and biomechanical properties of BCR both in vivo and vitro. Rabbit ACL reconstruction model using collagen rope and autograft (hamstring tendon) was conducted. The histological and biomechanical evaluations were conducted at 6-, 12-, 18, 26-week post-operation. In vitro study included cell morphology analysis, cell function evaluation and RNA sequencing of the tenocytes cultured on BCR. A cadaver study was also conducted to verify the feasibility of BCR for ACL reconstruction. BCR displays satisfactory mechanical strength similar to hamstring graft for ACL reconstruction in rabbit. Histological assessment showed BCR restore ACL morphology at 26 weeks similar to native ACL. The superior dynamic ligamentization in BCR over autograft group was evidenced by assessment of cell and collagen morphology and orientation. The in vitro study showed that the natural collagen fibres within BCR enables to signal the morphology adaptation and orientation of human tenocytes in bioreactor. BCR enables to enhance cell proliferation and tenogenic expression of tenocytes as compared to hydrolysed collagen. We performed an RNA-Sequencing (RNA-seq) experiment where RNA was extracted from tenocyte seeded with BCR. Analysis of enriched pathways of the up-regulated genes revealed that the most enriched pathways were the Hypoxia-inducible factor 1-alpha (HIF1A) regulated networks, implicating the possible mechanism BCR induced ACL regeneration. The subsequent cadaver study was conducted to proof the feasibility of BCR for ACL reconstruction. This study demonstrated the proof-of-concept of bio-textile braided collagen rope for ACL reconstruction, and the mechanism by which BCR induces natural collagen fibres that positively regulate morphology and function of tenocytes

Jellyfish collagens exhibit auspicious perspectives for tissue engineering applications primarily due to their outstanding compatibility with a wide range of cell types, low immunogenicity and biodegradability. Furthermore, derived from a non-mammalian source, jellyfish collagens reduce the risk of disease transmission, minimising therefore the ethical and safety concerns. The current study aims to investigate the potential of 3-dimensional jellyfish collagen sponges (3D-JCS) in promoting bone tissue regeneration. Both qualitative and quantitative analyses were performed in order to assess adhesion and proliferation of MC3T3 cells on 3D-JCL, as well as cell migration and bone-like ECM production. Histological and fluorescent dyes were used to stain mineral deposits (i.e. Alizarin Red S (ARS), Von Kossa, Tetracycline hydrochloride) while images were acquired using optical and confocal microscopy. Qualitative data indicated successful adhesion and proliferation of MC3T3 cells on the 3D-JCS as well as cell migration along with ECM production both on the inner and outer surface of the scaffolds. Moreover, quantitative analyses indicated a four-fold increase of ARS uptake between 2- and 3-dimensional cultures (N=3) as well as an eighteen-fold increase of ARS uptake for the 3D-JCS (N=3) when cultured in osteogenic conditions compared to control. This suggests the augmented osteogenic potential of MC3T3 cells when cultured on 3D-JCS. Nevertheless, the cell-mediated mineral deposition appeared to alter the mechanical properties of the jellyfish collagen sponges that were previously reported to exhibit low mechanical properties (compressive modulus: 1-2 kPa before culture). The biocompatibility, high porosity and pore interconnectivity of jellyfish collagen sponges promoted adhesion and proliferation of MC3T3 cells as well as cell migration and bone-like ECM production. Their unique features recommend the jellyfish collagen sponges as superior biomaterial scaffolds for bone tissue regeneration. Further studies are required to quantify the change in mechanical properties of the cell-seeded scaffolds and confirm their suitability for bone tissue regeneration. We predict that the 3D-JCS will be useful for future studies in both bone and bone-tendon interface regeneration. Acknowledgments. This research has been supported by a Medical Research Scotland Studentship award (ref: -50177-2019) in collaboration with Jellagen Ltd

Mesenchymal stem cells (MSC) have potent immunomodulatory and regenerative effects via soluble factors. One approach to improve stem cell-based therapies is encapsulation of MSC in hydrogels based on natural proteins such as collagen and fibrin, which play critical roles in bone healing. In this work, we comparatively studied the influence of collagen and fibrin hydrogels of varying stiffness on the paracrine interactions established by MSC with macrophages and osteoblasts. Type I collagen and fibrin hydrogels in a similar stiffness range loaded with MSC from donants were prepared by modifying the protein concentration. Viability and morphology of MSC in hydrogels as well as cell migration rate from the matrices were determined. Paracrine actions of MSC in hydrogels were evaluated in co-cultures with human macrophages from healthy blood donors or with osteoblasts from bone explants of patients with osteonecrosis of the femoral head. Lower matrix stiffness resulted in higher MSC viability and migration. Cell migration rate from collagen hydrogels was higher than from fibrin matrices. The secretion of the immunomodulatory factors interleukin-6 (IL-6) and prostaglandin E. 2. (PGE. 2. ) by MSC in both collagen and fibrin hydrogels increased with increasing matrix stiffness. Tumor necrosis factor-α (TNF-α) secretion by macrophages cultured on collagen hydrogels was lower than on fibrin matrices. Interestingly, higher collagen matrix stiffness resulted in lower secreted TNF-α while the trend was opposite on fibrin hydrogels. In all cases, TNF-α levels were lower when macrophages were cultured on hydrogels containing MSC than on empty gels, an effect partially mediated by PGE. 2. Finally, mineralization capacity of osteoblasts co-cultured with MSC in hydrogels increased with increasing matrix stiffness, although this effect was more notably for collagen hydrogels. Paracrine interactions established by MSC in hydrogels with macrophages and osteoblasts are regulated by matrix composition and stiffness

Abstract. Objectives. Musculoskeletal injuries are the leading contributor to disability globally, yet current treatments do not offer complete restoration of the tissue. This has resulted in the exploration of novel interventions based on tissue engineering as a therapeutic solution. This study aimed to explore novel collagen sponges as scaffolds for bone tissue engineering as an initial step in the construction of tendon-bone co-culture constructs in vitro. Methods. Collagen sponges (Jellagen, UK), manufactured from Jellyfish collagen were seeded with 10,000 rat osteoblast cells (dROBs) and maintained in culture for 6 days (37°C, 5% CO. 2. ). Qualitative viability was assessed by a fluorescent Calcein-AM live cell stain and quantitively via the CYQUANT cell viability assay (Invitrogen, UK) on days 0, 1, 4 and 6 in culture (n=3 per time point). Digital imaging was also used to assess size and shape changes to the collagen sponge in culture. Results. The collagen sponge biomaterial supported dROB adhesion, viability and proliferation with an abundance of viable cells detected by fluorescent microscopy on day 6. Indeed, the quantitative assessment confirmed that cellular proliferation was evident with increases in fluorescence detected from 517 (± 88) RFU to 8730 ± (2228) RFU from day 0 to 6. In addition, the size of the collagen sponges appeared to decrease over time, indicating contraction of the collagen sponges in culture. Conclusions. This preliminary study has demonstrated that the novel collagen sponges support cellular attachment and proliferation of osteoblasts, and is an important first step in building a bone-tendon construct in vitro. Our future work is focussed on using the osteoblast-seeded sponges in combination with tendon cells, to build a co-culture to represent the bone-tendon interface in vitro. This work has the potential to advance the clinical translation of tissue-engineered tendons to the clinic. 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

Previous studies showed that telo-peptides degraded from type II collagen, a type of collagen fragments, could induce cartilage damage in bovine stifle joints. We aim to investigate the role of integrins (ITGs) and matrix metalloproteinases (MMPs) in collagen fragment-induced human cartilage damage that is usually observed in osteoarthritis (OA). We hypothesized that N-telopeptide (NT) derived from type II collagen could up-regulate the expression of β1 integrin (ITGB1) and then MMPs that may lead to osteoarthritic cartilage damage. Human chondrocytes were isolated from femoral head or tibial plateau of patients receiving arthroplasty (N = 24). Primary chondrocyte cultures were either treated with 30 µM NT, or 30 µM scrambled NT (SN), or PBS, or left untreated for 24 hrs. Total proteins and RNAs were extracted for examination of expression of ITGB1 and MMPs-3&13 with Western blotting and quantitative real-time PCR. Compared to untreated or PBS treated chondrocytes, NT-treated chondrocytes expressed significantly higher levels of ITGB1 and MMPs-3&-13. However, SN also up-regulated expression of ITGB1 and MMP-13. ITGB1 and MMPs-3&-13 might mediate the catalytic effect of NT, a type of collagen fragments, on human cartilage damage that is a hallmark of OA

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

Although autografts represent the gold standard for anterior cruciate ligament (ACL) reconstruction, tissue-engineered ACLs provide a prospect to minimize donor site morbidity and limited graft availability. This given study characterizes the ligamentogenesis in embroidered poly(L-lactide-co-ε-caprolactone) (P(LA-CL)) / polylactic acid (PLA) constructs using a dynamic nude mice xenograft model. (P(LA-CL))/PLA scaffolds remained either untreated (co) or were functionalized by gas fluorination (F), collagen foam cross-linked with hexamethylene diisocyanate (HMDI) (coll), or gas fluorination combined with the foam (F+coll). Cell free constructs or those seeded for 1 week with lapine ACL ligamentocytes were implanted into nude mice for 12 weeks. Following explantation, biomechanical properties, cell vitality and content, histopathology of scaffolds (including organs: liver, kidney, spleen), sulphated glycosaminoglycan (sGAG) contents and biomechanical properties were assessed. Implantation of the scaffolds did not negatively affect mice weight development and organs, indicating biocompatibility. All scaffolds maintained their size and shape for the duration of the implantation. A high cell viability was detected in the scaffolds prior to and following implantation. Coll or F+coll scaffolds seeded with cells yielded superior macroscopic properties when compared to the controls. Mild signs of inflammation (foreign-body giant cells, hyperemia) were limited to scaffolds without collagen. Microscopical score values and sGAG content did not differ significantly. Although remaining stable in vivo, elastic modulus, maximum force, tensile strength and strain at Fmax were significantly lower in the in vivo compared to the samples cultured 1 week in vitro, but did not differ between scaffold subtypes, except for a higher maximum force in F+coll compared with F samples (in vivo). Scaffold functionalization with fluorinated collagen foam provides a promising approach for ACL tissue engineering. (shared first authorship). Acknowledgement: The study was supported by DFG grants SCHU1979/9-1 and SCHU1979/14-1

Introduction and Objective. Alveolar bone resorption following tooth extraction or periodontal disease compromises the bone volume required to ensure the stability of an implant. Guided bone regeneration (GBR) is one of the most attractive technique for restoring oral bone defects, where an occlusive membrane is positioned over the bone graft material, providing space maintenance required to seclude soft tissue infiltration and to promote bone regeneration. However, bone regeneration is in many cases impeded by a lack of an adequate tissue vascularization and/or by bacterial contamination. Using simultaneous spray coating of interacting species (SSCIS) process, a bone inspired coating made of calcium phosphate-chitosan-hyaluronic acid was built on one side of a nanofibrous GBR collagen membrane in order to improve its biological properties. Materials and Methods. First, the physicochemical characterizations of the resulting hybrid coating were performed by scanning electron microscopy, X-ray photoelectron, infrared spectroscopies and high-resolution transmission electron microscopy. Then human mesenchymal stem cells (MSCs) and human monocytes were cultured on those membranes. Biocompatibility and bioactivity of the hybrid coated membrane were respectively evaluated through MSCs proliferation (WST-1 and DNA quantification) and visualization; and cytokine release by MSCs and monocytes (ELISA and endothelial cells recruitment). Antibacterial properties of the hybrid coating were then tested against S. aureus and P. aeruginosa, and through MSCs/bacteria interactions. Finally, a preclinical in vivo study was conducted on rat calvaria bone defect. The newly formed bone was characterized 8 weeks post implantation through μCT reconstructions, histological characterizations (Masson's Trichrome and Von Kossa stain), immunohistochemistry analysis and second harmonic generation. Biomechanical features of newly formed bone were determined. Results. The resulting hybrid coating of about 1 μm in thickness is composed of amorphous calcium phosphate and carbonated poorly crystalline hydroxyapatite, wrapped within chitosan/hyaluronic acid polysaccharide complex. Hybrid coated membrane possesses excellent bioactivity and capability of inducing an overwhelmingly positive response of MSCs and monocytes in favor of bone regeneration. Furthermore, the antibacterial experiments showed that the hybrid coating provides contact-killing properties by disturbing the cell wall integrity of Gram-positive and Gram-negative bacteria. Its combination with MSCs, able to release antibacterial agents and mediators of the innate immune response, constitutes an excellent strategy for fighting bacteria. A preclinical in vivo study was therefore conducted in rat calvaria bone defect. μCT reconstructions showed that hybrid coated membrane favored bone regeneration, as we observed a two-fold increase in bone volume / total volume ratios vs. uncoated membrane. The histological characterizations revealed the presence of mineralized collagen (Masson's Trichrome and Von Kossa stain), and immunohistochemistry analysis highlighted a bone vascularization at 8 weeks post-implantation. However, second harmonic generation analysis showed that the newly formed collagen was not fully organized. Despite a significant increase in the elastic modulus of the newly formed bone with hybrid coated membrane (vs. uncoated membrane), the obtained values were lower than those for native bone (approximately 3 times less). Conclusions. These significant data shed light on the regenerative potential of such bioinspired hybrid coating, providing a suitable environment for bone regeneration and vascularization, as well as an ideal strategy to prevent bone implant-associated infections

Abstract. Objectives. Human articular cartilage chondrocytes undergo changes to their morphology and clustering with cartilage degeneration as occurs in osteoarthritis. (1). The consequences of chondrocyte de-differentiation on mechanically-resilient extracellular matrix metabolism are, however, unclear. We have assessed whether there is a relationship between abnormal chondrocyte morphology, as demonstrated by the presence of cytoplasmic processes, and chondrocyte clustering, with cell-associated type-I collagen during cartilage degeneration. Methods. The femoral heads of 9 patients were obtained (with Ethical permission/consent) following hip replacement surgery and cartilage areas graded (Grade-0 non-degenerate; Grade-1 mildly degenerate). In situ chondrocyte morphology and cell-associated type-I collagen were labelled fluorescently with CMFDA Cell tracker green, and immuno-fluorescence respectively then visualised/quantified using confocal laser scanning microscopy and imaging software. Results. When comparing data from 9 femoral heads with Grade-0 [N(n)=6(72)] or Grade-1 cartilage [(N(n)=9(108)], the latter had a higher percentage of chondrocytes with cytoplasmic processes (length >5µm) (P=0.018) and clusters (≥5 cells within a lacuna) (P<0.001). The percentage of chondrocytes with processes and clusters displaying cell-associated type-I collagen, was also higher in degenerate cartilage (P<0.001 for both). However, some morphologically-normal chondrocytes exhibited cell-associated collagen type-I labelling while some clusters did not label with collagen type-I. Intriguingly, even in Grade-0 cartilage, some chondrocytes were morphologically abnormal and exhibited cell-associated type-I collagen. Conclusions. These results suggest a complex relationship between chondrocyte morphology/clusters and cell-associated collagen type-I. The presence of this collagen type implies chondrocyte de-differentiation to a fibroblastic phenotype even in non-degenerate cartilage. This cell type produces a fibro-cartilaginous ‘repair’ matrix which is considerably weaker than the collagen type-II matrix of healthy hyaline cartilage and may give rise to focal points of mechanical weakness. Funder. Chief Scientist's Office, Scotland (Grant TCS/18/01). 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

Objectives. Sustained intra-articular delivery of pharmacological agents is an attractive modality but requires use of a safe carrier that would not induce cartilage damage or fibrosis. Collagen scaffolds are widely available and could be used intra-articularly, but no investigation has looked at the safety of collagen scaffolds within synovial joints. The aim of this study was to determine the safety of collagen scaffold implantation in a validated in vivo animal model of knee arthrofibrosis. Materials and Methods. A total of 96 rabbits were randomly and equally assigned to four different groups: arthrotomy alone; arthrotomy and collagen scaffold placement; contracture surgery; and contracture surgery and collagen scaffold placement. Animals were killed in equal numbers at 72 hours, two weeks, eight weeks, and 24 weeks. Joint contracture was measured, and cartilage and synovial samples underwent histological analysis. Results. Animals that underwent arthrotomy had equivalent joint contractures regardless of scaffold implantation (-13.9° versus -10.9°, equivalence limit 15°). Animals that underwent surgery to induce contracture did not demonstrate equivalent joint contractures with (41.8°) or without (53.9°) collagen scaffold implantation. Chondral damage occurred in similar rates with (11 of 48) and without (nine of 48) scaffold implantation. No significant difference in synovitis was noted between groups. Absorption of the collagen scaffold occurred within eight weeks in all animals. Conclusion. Our data suggest that intra-articular implantation of a collagen sponge does not induce synovitis or cartilage damage. Implantation in a native joint does not seem to induce contracture. Implantation of the collagen sponge in a rabbit knee model of contracture may decrease the severity of the contracture. Cite this article: J. A. Walker, T. J. Ewald, E. Lewallen, A. Van Wijnen, A. D. Hanssen, B. F. Morrey, M. E. Morrey, M. P. Abdel, J. Sanchez-Sotelo. Intra-articular implantation of collagen scaffold carriers is safe in both native and arthrofibrotic rabbit knee joints. Bone Joint Res 2016;6:162–171. DOI: 10.1302/2046-3758.63.BJR-2016-0193

Porcine and fish by-products in particular are rich sources for collagen, which is the main component of the extracellular matrix (ECM). Although there are studies investigating different collagen derived from various tissue sources for the purpose of creating biomaterials, the comparison of biophysical, biochemical and biological properties of type II collagen isolated from cartilaginous tissues has yet to be assessed. In addition, it has been shown from previous studies that sex steroid hormones affect the collagen content in male and female animals, herein, type II collagens from male and female porcine cartilage were assessed in order to investigate gender effects on the property of collagen scaffolds. Moreover, type II collagen has a supportive role in articular cartilage in the knee joint. Therefore, the aim is to assess the properties of type II collagen scaffolds as a function of species, tissue and gender for cartilage regeneration. Type II collagen was extracted from male and female porcine trachea, auricular, articular cartilage and cartilaginous fish through acid-pepsin digestion at 4°C. SDS-PAGE was conducted to confirm the purity of extracted collagen. Collagen sponges were created via freeze-drying. Scaffold structure and pore size were evaluated by scanning electron microscopy (SEM). Thermal stability was assessed by differential scanning calorimetry (DSC). Sponges were seeded with human adipose derived stem cells to assess chondro-inductive potential of collagen sponges after 7, 14 and 21 days of culture. In conclusion, collagen sponges support the proliferation and differentiation of human adipose derived stem cells to different extents

Orthopaedic infection with bacteria leads to high societal cost and is detrimental to the life quality. Particularly, deep bone infection leading to osteomyelitis results in an inflammatory response whereby localized bone destruction occurs. Current treatments like antibiotic-containing polymethymethacrylate (PMMA) still has the high risk of bacterial resistance. Taking advantages of silver which has antibacterial and anti-inflammatory effect and bioactive collagen, we fabricated a silver nanoparticle (AgNP)-coated collagen membrane by sonication and sputtering. SEM showed good deposition of AgNPs on collagen membrane by both coating methods. The optimal coating concentration was finalized by assessing optimal antibacterial effect against cytotoxicity and finally collagen membrane coated with 1mg/mL AgNPs solution was selected. We also found that the coated collagen membrane demonstrating short-term cytotoxicity within 24 hours with damage to the cell membrane, which was evidenced by MTS and LDH release test, but had no significant influence (p > 0.05) thereafter. The amount of released AgNPs from coated collagen membrane had negligible cytotoxicity (p > 0.05). Confocal laser scanning microscope displayed similar cell morphology in both coated and uncoated collagen membrane. ELISA and qPCR presented the decreased secretion and expression (p < 0.001) of IL-6 and TNF-alpha. Upregulated expression (p < 0.001) of osteogenesis markers (RUNX2, ALP and OPN) could be found and this might be attributed to the modified collagen fibre surface coated by AgNPs. Collectively, the osteogenesis induced by AgNPs demonstrates a promising application in orthopaedic surgery for its use both as an antimicrobial agent, and to enhance bone regeneration

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

The hydrolysed collagen has a molecular weight of 3–6 KDa, is soluble in water, colourless and odourless. Hydrolysed collagen was obtained by proteolysis of the native ovine collagen. The enzymatic treatment was carried out with Heliozym under alkaline treatment (pH 8) for different periods of time (0 min, 10 min, 20 min, 30 min, 1 h, 2h, 3h y 4h) at 60°C. The hydroxyproline concentration increased significantly from time 0 min (11.44±2.81 mg/L) to the 4 h (24.47±1.60 mg/L); this change in concentration was observed in the FTIR spectra at a length of 1,037 cm-1 for OH group as well a change in the Amide I (1641 cm-1). The viscosity showed significant differences (P≤ 0.05) between the different hydrolysis times. This parameter was correlated with the molecular weight; when the viscosity was 0 cP the molecular weight showed the lowest value at 5.62 KDa. The antioxidant activity for ABTS radical scavenging showed significant differences (P≤ 0.05) between the times of hydrolysis, the greater the time, the higher the inhibition resulting with 67.61% at the end of the treatment. The DPPH radical scavenging resulted with 27.89 % at the beginning of the hydrolysis. However, the end of the hydrolysis (4h) showed inhibition at 52.75%. The antioxidant activity increased when molecular weight decreased, and this is related with the amino acids present in the peptides obtained for the hydrolysis of the collagen

Surgical repair of rotator cuff tears have high failure rates (20–70%), often due to a lack of biological healing. Augmenting repairs with extracellular matrix-based scaffolds is a common option for surgeons, although to date, no commercially available product has proven to be effective. In this study, a novel collagen scaffold was assessed for its efficacy in augmenting rotator cuff repair. The collagen scaffold was assessed in vitro for cytocompatability and retention of tenocyte phenotype using alamarBLUE assays, confocal imaging and real-time PCR. Immunogenicity was assessed in vitro by the activation of pre-macrophage cells. In vivo, using a modified rat rotator cuff defect model, supraspinatus tendon repairs were carried out in 46 animals. Overlay augmentation with the collagen scaffold was compared to unaugmented repairs. At 6- and 12-weeks post-op the repairs were tested biomechanically to evaluate repair strength, and histologically for quality of healing. The collagen scaffold supported human tenocyte growth in vitro, with cells appearing morphologically tenocytic and expressing higher tendon gene markers compared to plastic controls. No immunogenic responses were provoked compared to suture material control. In vivo, augmentation with the scaffold improved the histological scores at 12 weeks (8.37/15 vs. 6.43/15, p=0.0317). However, no significant difference was detected on mechanical testing. While the collagen scaffold improved the quality of healing of the tendon, a meaningful increase in biomechanical strength was not achieved. This is likely due to its inability to affect the bone-tendon junction. Future materials/orthobiologics must target both the repaired tendon and the regenerating bone-tendon junction

Introduction. Polymethylmethacrylate(PMMA) bone cement has been used in joint reconstruction surgery and recently introduced for treatment of osteoporotic vertebral compression fracture. However, the use of PMMA bone cement in vertebroplasty leads to extensive bone stiffening and high rate of adjacent vertebrae fracture. Aim. The purpose of this study was to investigate the properties of PMMA bone cement augmented with collagen and assess its characteristics and relevance for the reduction of complication rate associated with vertebroplasty. Methods. Bone cement was produced using 2 types of PMMA based bone cement. Augmented groups were prepared using 40g of bone cement with 1% of rat tail liquid collagen. Mixing was conducted in controlled laboratory environment and at room temperature. The working and setting time and the mechanical properties were determined in accordance to ASTM standards for acrylic cements. The effect of ageing in simulated body fluid(SBF) on mechanical properties of these cements and the microstructure were studied. Results. Addition of collagen to bone cement has shown no marked effect on the working and setting time and produces bone cement with good injectability. The compressive strength is not affected but the modulus shows the material is less brittle than PMMA. Conclusion. Addition of liquid collagen to PMMA based bone cement does not necessarily compromise the properties of the cements and produce cement with good injectability and less brittle than PMMA based bone cement alone. However, bone cement augmented with different concentration of collagen need to be studied further in order to assess its clinical relevance especially in vertebroplasty

Collagen scaffolds are generally characterized by their random fibre distribution and weak mechanical properties, which makes them unsuitable as substitutes for highly anisotropic tissues such as cornea or tendon. Recently, we developed a technique to create collagen type I scaffolds with well-defined anisotropic micro-patterns. Porcine collagen was mixed with PBS10X, NaOH and one of the following cross-linkers: glutaraldehyde (GTA), genipin and 4-arm polyethylene glycol (4SP). The resulting mixture was casted on micro-grooved (2×2×2 μm) polydimethylsiloxane (PDMS) moulds and allowed to dry in a laminar flow hood to obtain 5mg/ml collagen films. Different pH, temperatures (Tº), and cross-linker concentrations were tested in the process. Collagen gelation kinetics was analysed with rheometry and surface topography was assessed with scanning electron microscopy (SEM). Human bone marrow stem cells (HBMSCs) were seeded on the films and cell alignment was analysed by rhodamine/phalloidin staining and imaged with fluorescence microscopy. From all three cross-linkers tested, only 4SP cross-linked scaffolds showed a well-defined micro-grooved pattern. Increasing pH and Tº on 4SP-treated collagen decreased gelation time, which resulted in complete inhibition of the pattern, suggesting that an initial low viscous solution is required for a correct PDMS pattern infiltration. A wide range of 4SP concentrations (0.5, 1, 1.5 mM) maintained the well-defined topography on the films, opening the door to future fine-tuning of the stiffness sensed by cells. hBMSCs seeded on top of the scaffolds aligned along the pattern for 14 days in culture. Collectively, this data highlights the potential of these collagen scaffolds as tendon substitutes

Purpose. Collagen-rich structures of the knee are prone to damage through acute injury or chronic “wear and tear”. Collagen becomes more disorganised in degenerative tissue e.g. osteoarthritis. An alignment index (AI) used to analyse orientation distribution of collagen-rich structures is presented. Method. A healthy caprine knee was scanned in a Siemens Verio 3T Scanner. The caprine knee was rotated and scanned in nine directions to the main magnetic field B. 0. A 3D PD SPACE sequence with isotropic 1×1×1mm voxels (TR1300ms, TE13ms, FOV256mm,) was optimised to allow for a greater angle-sensitive contrast. For each collagen-rich voxel the orientation vector is computed using Szeverenyi and Bydder's method. Each orientation vector reflects the net effect of all the fibres comprised within a voxel. The assembly of all unit vectors represents the fibre orientation map. Alignment Index (AI) in any direction is defined as a ratio of the fraction of orientations within 20° (solid angle) centred in that direction to the same fraction in a random (flat) case. In addition, AI is normalised in such a way that AI=0 indicates isotropic collagen alignment. Increasing AI values indicate increasingly aligned structures: AI=1 indicates that all collagen fibres are orientated within the cone of 20° centred at the selected direction. AI = (nM - nRnd)/(nTotal - nRnd) if nM >= nRnd. AI = 0 if nM < nRnd. Where:. nM is a number of reconstructed orientations that are within a cone of 20° centred in selected direction. nRnd is a number of random orientations within a cone of 20° around selected direction. nTotal is a number of collagen reach voxels. By computing AI for a regular gridded orientation space we are able to visualise change of AI on a hemisphere facilitating understanding of the collagen fibre orientation distribution. Results. The patella tendon had an AI=0.6453. The Anterior Cruciate Ligament (ACL) had an AI=0.2732. The meniscus had an AI=0.1847. Discussion. The most aligned knee structure is the patella tendon where the collagen fibres align with the skeleton to transmit forces through bones and muscles. This structure had the AI closest to 1. The ACL had the second highest AI and is composed of two fibre bundles aligned diagonally across the knee. The meniscus acts as a shock absorber and is made up of vertical, radial and circumferential fibres which disperse forces more equally. The complexity of the meniscal structure resulted in the lowest AI. To date, this technique has only been performed with healthy tissue; the AI may become closer to zero if there is damage disrupting the collagen fibre alignment. The AI can further our understanding of collagen orientation distribution and could be used as a quantitative, non-invasive measure of structural health

Collagen is a key component of the extracellular matrix in a variety of tissues and hence is widely used in tissue engineering research, yet collagen has had limited uptake in the field of 3D printing. In this study we successfully adapted an existing electronic printing method, aerosol jet printing (AJP), to print high resolution 3D constructs of recombinant collagen type III (RHCIII). Circular samples with a diameter of 4.5mm and 288 layers thick, or a diameter of 6.5mm and 400 layers thick were printed on glass cover slips with print lines of 60µm. Attenuated Total Reflectance Fourier-Transorm Infa-red (ATR-FTIR) spectroscopy performed on the 4 of the printed samples and dried non-printed RHCIII samples showed that no denaturation had occurred due to the printing process. Printed samples were crosslinked using EDC [N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, Sigma Aldrich] to improve their stability and mechanical strength. Differential scanning calorimetry (DSC) performed showed a marked difference in the denaturation temperature between crosslinked printed samples and fibrillar non-printed samples and nano-indentation showed that the construct was relatively stiff. Previous results with similar samples have shown that mesenchymal stem cells (MSCs) align with and travel parallel to print direction. Results obtained from these samples show signs that they might be applied in other areas such as bone tissue engineering

Complex pathophysiologies involve different signalling mechanisms, with a multitude of often interconnected potential therapeutic targets. Therefore, there is a need for the development of multi-compartment delivery vehicles for combinatorial and synergistic therapeutic approaches. In this study it was hypothesized that multi-compartment crosslinked collagen type I systems can deliver multiple bioactive agents in a controlled manner in an in vitro model condition of skin fibrosis. Multi-compartment collagen-based systems were made using solutions of dialyzed type I collagen mixed with 10× PBS, after which they were neutralised and crosslinked with 1 and 2.0 mM 4 arm-succinimidyl glutarate ester PEG (4 arm-PEG-SG), respectively, followed by incubation at 37ºC. The systems were characterised through swelling assessment, collagenase degradation assay and compression tests. The release of encapsulated drugs from the hydrogels was studied by ELISA and the effect of the delivered bioactive agents was assessed through imaging and quantification for fibrotic markers in an in vitro model. A pilot study using FITC-dextran proved that the inner compartment was capable of promoting a sustained release over a long period of time (7 days), which was further confirmed with drug release assays using a TNF-α antagonist and recombinant decorin, fitting the intended therapeutic release profile. Protein expression studies showed a decrease of endogenous collagen type I and α-smooth muscle actin expression (p<0.05) indicating amelioration of fibrosis. In summary, this indicates that this system is suitable for dual delivery of multiple bioactive agents, resulting in a controlled release in vitro and illustrating its potential in therapy