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

Restoration a joint's articular surface following degenerative or traumatic pathology to the osteochondral unit pose a significant challenge. Recent advances have shown the utility of collagen-based scaffolds in the regeneration of osteochondral tissue. To provide these collagen scaffolds with the appropriate superstructure novel techniques in 3D printing have been investigated. This study investigates the use of polyɛ-caprolactone (PCL) collagen scaffolds in a porcine cadaveric model to establish the stability of the biomaterial once implanted. This study was performed in a porcine cadaveric knee model. 8mm defects were created in the medial femoral trochlea and repaired with a PCL collagen scaffold. Scaffolds were secured by one of three designs; Press Fit (PF), Press Fit with Rings (PFR), Press Fit with Fibrin Glue (PFFG). Mobilisation was simulated by mounting the pig legs on a continuous passive motion (CPM) machine for either 50 or 500 cycles. Biomechanical tensile testing was performed to examine the force required to displace the scaffold. 18 legs were used (6 PF, 6 PFR, 6 PFFG). Fixation remained intact in 17 of the cohort (94%). None of the PF or PFFG scaffolds displaced after CPM cycling. Mean peak forces required to displace the scaffold were highest in the PFFG group (3.173 Newtons, Standard deviation = 1.392N). The lowest peak forces were observed in the PFR group (0.871N, SD = 0.412N), while mean peak force observed in the PF group was 2.436N (SD = 0.768). There was a significant difference between PFFG and PFR (p = 0.005). There was no statistical significance in the relationship between the other groups. PCL reinforcement of collagen scaffolds provide an innovative solution for improving stiffness of the construct, allowing easier handling for the surgeon. Increasing the stiffness of the scaffold also allows press fit solutions for reliable fixation. Press fit PCL collagen scaffolds with and without fibrin glue provide dependable stability. Tensile testing provides an objective analysis of scaffold fixation. Further investigation of PCL collagen scaffolds in a live animal model to establish quality of osteochondral tissue regeneration are required

INTRODUCTION. Intervertebral disc (IVD) degeneration is not completely understood because of the lack of relevant models. In vivo models are inappropriate because animals are quadrupeds. IVD is composed of the Nucleus Pulposus (NP) and the Annulus Fibrosus (AF), an elastic tissue that surrounds NP. AF consists of concentric lamellae made of collagen I and glycosaminoglycans with fibroblast-like cells located between layers. In this study, we aimed to develop a novel 3D in vitro model of Annulus Fibrosus to study its degeneration. For this purpose, we reproduced the microenvironment of AF cells using 3D printing. METHOD. An ink consisting of dense collagen (30 mg.mL. -1. ) and tyramine-functionalized hyaluronic acid (THA) at 7.5 mg.mL. -1. was first designed by modulating pH and [NaCl] in order to inhibit the formation of polyionic complexes between collagen and THA. Then, composite inks were printed in different gelling baths to form collagen hydrogels. Last, THA photocrosslinking using eosin and green light was performed to strengthen hydrogels. Selected 3D printed constructs were then cellularized with fibroblasts. RESULTS. The physicochemical study revealed that collagen/THA solutions (4:1 ratio) used at pH 5 with 200 mM NaCl were homogenous. In addition, collagen fibrils were observed in these solutions. The dense composite collagen/THA inks printed in a 2X PBS bath rapidly gelled and the photo-crosslinking increased the mechanical properties by 2 to reach 25 kPa (Young's modulus). Then, 3D printing parameters were optimized (85 kPa, extrusion, 4.5 mm/s speed and 80% fill-in percentage) to generate flat and anisotropic lamellae observed by polarized light microscopy. For the in vitro study, several anisotropic layers were printed and fibroblasts seeded between them. Cells adhered to layers, spread, proliferate and aligned along the axis of printed layers. CONCLUSION. Taken together, these results show it is possible to reproduce in vitro the main AF's biochemical and physical properties

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

Aims. To test the hypothesis that reseeded anterior cruciate ligament (ACL)-derived cells have a better ability to survive and integrate into tendon extracellular matrix (ECM) and accelerate the ligamentization process, compared to adipose-derived mesenchymal stem cells (ADMSCs). Methods. Acellularized tibialis allograft tendons were used. Tendons were randomly reseeded with ACL-derived cells or ADMSCs. ACL-derived cells were harvested and isolated from remnants of ruptured ACLs during reconstruction surgery and cultured at passage three. Cell suspensions (200 µl) containing 2 × 10. 6. ACL-derived cells or ADMSCs were prepared for the purpose of reseeding. At days 1, 3, and 7 post-reseeding, graft composites were assessed for repopulation with histological and immunohistochemical analysis. Matrix protein contents and gene expression levels were analyzed. Results. In the graft reseeded with ACL-derived cells, a large number of elongated cells that integrated into the matrix were evident at day 3 and day 7. However, in the graft reseeded with ADMSCs, only a small number of elongated cells were found integrated into the matrix. Immunofluorescence for Ki-67 and type I collagen confirmed the pronounced production of type I collagen by Ki-67-positive ACL-derived cells integrated into the ECM. A messenger RNA (mRNA) expression assay demonstrated significantly higher gene expression levels of types I (p = 0.013) and III (p = 0.050) collagen in the composites reseeded with ACL-derived cells than ADMSCs. Conclusion. ACL-derived cells, when reseeded to acellularized tendon graft, demonstrated earlier better survival and integration in the tendon ECM and resulted in higher gene expression levels of collagen, which may be essential to the normal ligamentization process compared to ADMSCs. Cite this article: Bone Joint Res 2022;11(11):777–786

It is not known if the radiation sterilisation dose (RSD) of 25 kGy affects mechanical properties and biocompability of allograft bone by alteration of collagen triple helix or cross-links. Our aim was to investigate the mechanical and biological performance, cross-links and degraded collagen content of irradiated bone allografts. Human femoral shafts were sectioned into cortical bone beams (40 × 4 × 2 mm) and irradiated at 0, 5, 10, 15, 20, and 25 kGy for three-point bending tests. Corresponding cortical bone slices were used for in vitro determination of macrophage activation, osteoblast proliferation and attachment, and osteoclast formation and fusion. Subsequently, irradiated cortical bone samples were hydrolised for determination of pyridinoline (PYD), deoxypyridinoline (DPD), and pentosidine (PEN) by high performance liquid chromatography (HPLC) and collagen degradation by the alpha chymotrypsin (ï. j. CT) method. Irradiation up to 25 kGy did not affect the elastic properties of cortical bone, but the modulus of toughness was decreased from 87% to 74% of controls when the gamma dose increased from 15 to 25 kGy. Macrophages activation, the proliferation and attachment of osteoblasts on irradiated bone was not affected. Osteoclast formation and fusion were less than 40% of controls when cultured on bone irradiated at 25 kGy, and 80% at 15 kGy. Increasing radiation dose did not significantly alter the content of PYR, DPD or PEN but increased the content of denatured collagen. Cortical allografts fragility increases at doses above 15 kGy. Decreased osteoclast viability at these doses suggests a reduction in the capacity for bone remodelling. These changes were not correlated with alterations in collagen cross-links but in degradation to the collagen secondary structure as evidenced by increased content of denatured collagen

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

Aims. To explore the efficacy of extracorporeal shockwave therapy (ESWT) in the treatment of osteochondral defect (OCD), and its effects on the levels of transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, -3, -4, -5, and -7 in terms of cartilage and bone regeneration. Methods. The OCD lesion was created on the trochlear groove of left articular cartilage of femur per rat (40 rats in total). The experimental groups were Sham, OCD, and ESWT (0.25 mJ/mm. 2. , 800 impulses, 4 Hz). The animals were euthanized at 2, 4, 8, and 12 weeks post-treatment, and histopathological analysis, micro-CT scanning, and immunohistochemical staining were performed for the specimens. Results. In the histopathological analysis, the macro-morphological grading scale showed a significant increase, while the histological score and cartilage repair scale of ESWT exhibited a significant decrease compared to OCD at the 8- and 12-week timepoints. At the 12-week follow-up, ESWT exhibited a significant improvement in the volume of damaged bone compared to OCD. Furthermore, immunohistochemistry analysis revealed a significant decrease in type I collagen and a significant increase in type II collagen within the newly formed hyaline cartilage following ESWT, compared to OCD. Finally, SRY-box transcription factor 9 (SOX9), aggrecan, and TGF-β, BMP-2, -3, -4, -5, and -7 were significantly higher in ESWT than in OCD at 12 weeks. Conclusion. ESWT promoted the effect of TGF-β/BMPs, thereby modulating the production of extracellular matrix proteins and transcription factor involved in the regeneration of articular cartilage and subchondral bone in an OCD rat model. Cite this article: Bone Joint Res 2024;13(7):342–352

Aims. Mechanical stimulation is a key factor in the development and healing of tendon-bone insertion. Treadmill training is an important rehabilitation treatment. This study aims to investigate the benefits of treadmill training initiated on postoperative day 7 for tendon-bone insertion healing. Methods. A tendon-bone insertion injury healing model was established in 92 C57BL/6 male mice. All mice were divided into control and training groups by random digital table method. The control group mice had full free activity in the cage, and the training group mice started the treadmill training on postoperative day 7. The quality of tendon-bone insertion healing was evaluated by histology, immunohistochemistry, reverse transcription quantitative polymerase chain reaction, Western blotting, micro-CT, micro-MRI, open field tests, and CatWalk gait and biomechanical assessments. Results. Our results showed a significantly higher tendon-bone insertion histomorphological score in the training group, and the messenger RNA and protein expression levels of type II collagen (COL2A1), SOX9, and type X collagen (COL10A1) were significantly elevated. Additionally, tendon-bone insertion resulted in less scar hyperplasia after treadmill training, the bone mineral density (BMD) and bone volume/tissue volume (BV/TV) were significantly improved, and the force required to induce failure became stronger in the training group. Functionally, the motor ability, limb stride length, and stride frequency of mice with tendon-bone insertion injuries were significantly improved in the training group compared with the control group. Conclusion. Treadmill training initiated on postoperative day 7 is beneficial to tendon-bone insertion healing, promoting biomechanical strength and motor function. Our findings are expected to guide clinical rehabilitation training programmes. Cite this article: Bone Joint Res 2023;12(5):339–351

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

Abstract. INTRODUCTION. To preserve knee function and reduce degenerative, meniscal tears should be repaired where possible. Meniscal wrapping with collagen matrices has shown promising clinical outcome (AAOS meniscal algorithm), however there is limited basic science to support this. AIM. to model the contact pressures on the human tibial plateau beneath a (1) a repaired radial meniscal tear and (2) a wrapped and repaired radial meniscal tear. METHODOLOGY. Complete anterolateral radial tears were formed across 4 lateral human menisci, before repairing with ‘rip-stop’ H sutures using 2mm Arthrex Meniscal Suture tape. This was then repeated with the addition of a ChondroGide collagen matrix wrapping. From this experimental setup a finite element (FE) analysis model was construted. FE models of the two techniques (i) suture alone and (ii) suture and collagen-matrix wrap, were then modelled; bone was linear elastic, articular cartilage was a hyperelastic Yeoh model, and a linear elastic and transversely isotropic material model for the meniscus. The contact areas of the articulating surfaces, meniscus kinematics, and stress distribution around the repair were compared between the two systems. RESULTS. Meniscal suture-tape repair had higher local stresses and strains (σ_max=51 MPa ε_max=25%) around the repair compared to with Collagen wrapping (σ_max=36.6MPa ε_max=15%). Radial displacement and pressure on the meniscal contact surfaces were higher in the suture only repair. CONCLUSION. Collagen-matrix wrapping strengthens the repair, reducing local peak stresses and strains around the suture-tape. This could reduce the chance of suture-tape pull-out and subsequent repair failure

Aims. Autologous chondrocyte implantation (ACI) is a promising treatment for articular cartilage degeneration and injury; however, it requires a large number of human hyaline chondrocytes, which often undergo dedifferentiation during in vitro expansion. This study aimed to investigate the effect of suramin on chondrocyte differentiation and its underlying mechanism. Methods. Porcine chondrocytes were treated with vehicle or various doses of suramin. The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN); COL1A1; COL10A1; SRY-box transcription factor 9 (SOX9); nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX); interleukin (IL)-1β; tumour necrosis factor alpha (TNFα); IL-8; and matrix metallopeptidase 13 (MMP-13) in chondrocytes at both messenger RNA (mRNA) and protein levels was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot. In addition, the supplementation of suramin to redifferentiation medium for the culture of expanded chondrocytes in 3D pellets was evaluated. Glycosaminoglycan (GAG) and collagen production were evaluated by biochemical analyses and immunofluorescence, as well as by immunohistochemistry. The expression of reactive oxygen species (ROS) and NOX activity were assessed by luciferase reporter gene assay, immunofluorescence analysis, and flow cytometry. Mutagenesis analysis, Alcian blue staining, reverse transcriptase polymerase chain reaction (RT-PCR), and western blot assay were used to determine whether p67. phox. was involved in suramin-enhanced chondrocyte phenotype maintenance. Results. Suramin enhanced the COL2A1 and ACAN expression and lowered COL1A1 synthesis. Also, in 3D pellet culture GAG and COL2A1 production was significantly higher in pellets consisting of chondrocytes expanded with suramin compared to controls. Surprisingly, suramin also increased ROS generation, which is largely caused by enhanced NOX (p67. phox. ) activity and membrane translocation. Overexpression of p67. phox. but not p67. phox. AD (deleting amino acid (a.a) 199 to 212) mutant, which does not support ROS production in chondrocytes, significantly enhanced chondrocyte phenotype maintenance, SOX9 expression, and AKT (S473) phosphorylation. Knockdown of p67. phox. with its specific short hairpin (sh) RNA (shRNA) abolished the suramin-induced effects. Moreover, when these cells were treated with the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) inhibitor LY294002 or shRNA of AKT1, p67. phox. -induced COL2A1 and ACAN expression was significantly inhibited. Conclusion. Suramin could redifferentiate dedifferentiated chondrocytes dependent on p67. phox. activation, which is mediated by the PI3K/AKT/SOX9 signalling pathway. Cite this article: Bone Joint Res 2022;11(10):723–738

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

Aims. Proliferation, migration, and differentiation of anterior cruciate ligament (ACL) remnant and surrounding cells are fundamental processes for ACL reconstruction; however, the interaction between ACL remnant and surrounding cells is unclear. We hypothesized that ACL remnant cells preserve the capability to regulate the surrounding cells’ activity, collagen gene expression, and tenogenic differentiation. Moreover, extracorporeal shock wave (ESW) would not only promote activity of ACL remnant cells, but also enhance their paracrine regulation of surrounding cells. Methods. Cell viability, proliferation, migration, and expression levels of Collagen-I (COL-I) A1, transforming growth factor beta (TGF-β), and vascular endothelial growth factor (VEGF) were compared between ACL remnant cells untreated and treated with ESW (0.15 mJ/mm. 2. , 1,000 impulses, 4 Hz). To evaluate the subsequent effects on the surrounding cells, bone marrow stromal cells (BMSCs)’ viability, proliferation, migration, and levels of Type I Collagen, Type III Collagen, and tenogenic gene (Scx, TNC) expression were investigated using coculture system. Results. ESW-treated ACL remnant cells presented higher cell viability, proliferation, migration, and increased expression of COL-I A1, TGF-β, and VEGF. BMSC proliferation and migration rate significantly increased after coculture with ACL remnant cells with and without ESW stimulation compared to the BMSCs alone group. Furthermore, ESW significantly enhanced ACL remnant cells’ capability to upregulate the collagen gene expression and tenogenic differentiation of BMSCs, without affecting cell viability, TGF-β, and VEGF expression. Conclusion. ACL remnant cells modulated activity and differentiation of surrounding cells. The results indicated that ESW enhanced ACL remnant cells viability, proliferation, migration, and expression of collagen, TGF-β, VEGF, and paracrine regulation of BMSC proliferation, migration, collagen expression, and tenogenesis. Cite this article: Bone Joint Res 2020;9(8):457–467

Aims. The purpose of this study was to explore a simple and effective method of preparing human acellular amniotic membrane (HAAM) scaffolds, and explore the effect of HAAM scaffolds with juvenile cartilage fragments (JCFs) on osteochondral defects. Methods. HAAM scaffolds were constructed via trypsinization from fresh human amniotic membrane (HAM). The characteristics of the HAAM scaffolds were evaluated by haematoxylin and eosin (H&E) staining, picrosirius red staining, type II collagen immunostaining, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Human amniotic mesenchymal stem cells (hAMSCs) were isolated, and stemness was verified by multilineage differentiation. Then, third-generation (P3) hAMSCs were seeded on the HAAM scaffolds, and phalloidin staining and SEM were used to detect the growth of hAMSCs on the HAAM scaffolds. Osteochondral defects (diameter: 3.5 mm; depth: 3 mm) were created in the right patellar grooves of 20 New Zealand White rabbits. The rabbits were randomly divided into four groups: the control group (n = 5), the HAAM scaffolds group (n = 5), the JCFs group (n = 5), and the HAAM + JCFs group (n = 5). Macroscopic and histological assessments of the regenerated tissue were evaluated to validate the treatment results at 12 weeks. Results. In vitro, the HAAM scaffolds had a network structure and possessed abundant collagen. The HAAM scaffolds had good cytocompatibility, and hAMSCs grew well on the HAAM scaffolds. In vivo, the macroscopic scores of the HAAM + JCFs group were significantly higher than those of the other groups. In addition, histological assessments demonstrated that large amounts of hyaline-like cartilage formed in the osteochondral defects in the HAAM + JCFs group. Integration with surrounding normal cartilage and regeneration of subchondral bone in the HAAM + JCFs group were better than those in the other groups. Conclusion. HAAM scaffolds combined with JCFs promote the regenerative repair of osteochondral defects. Cite this article: Bone Joint Res 2022;11(6):349–361

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 culture time. Although the characteristic anisotropy of tendon tissue is commonly replicated in scaffolds, relevant physical cues such as tendon rigidity or mechanical loading are often neglected. The objective of this study is to use tendons' main extracellular matrix component, collagen type I, to create scaffolds with an anisotropic surface topography and controlled rigidity, in an effort to engineer functional tendon tissue equivalents, with native organization and strength. 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 μm) or planar polydimethylsiloxane (PDMS) molds and dried in a laminar flow hood to obtain 5 mg/ml collagen films. Surface topography and elastic modulus of the final scaffolds were analyzed using SEM/AFM and rheometry, respectively. Human tendon cells were isolated from adult tendon tissue and cultured on micro-grooved/planar scaffolds for 4, 7 and 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 at low pH and temperature. The elastic modulus of the scaffolds cross-linked with the highest concentration of 4SP matched the physiological values reported in developing tendons (∼15 kPa). Around eighty percent of the human tendon cells cultured on the cross-linked collagen films aligned in the direction of the anisotropy for 10 days in culture. At 4 days, tenoyctes cultured on micro-grooved substrates presented a significant higher nuclei aspect ratio than tenocytes cultured on planar substrates for all the 4SP concentrations. Synthesis, deposition and alignment of collagen III and tenascin C, two important tenogenic markers, were up regulated selectively in the rigid micro-grooved scaffolds after 7 days in culture. 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. Gene expression analysis and a replicate study with hBMSCs will be carried out to support the first results and to further identify the optimal biophysical conditions for tenogenic cell lineage commitment. This potentially leads to the design of smart implants that not only restore immediate tendon functionality but also provide microscopic cues that drive cellular synthesis of organized tissue-specific matrix

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