Aims. It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth. Methods. C57BL/6 mice rotator cuff (RC) repair model was established to explore the effects of mechanical stimulation on macrophage polarization, transforming growth factor (TGF)-β1 generation, and MSC chondrogenesis within T-B enthesis by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Macrophage depletion was performed by clodronate liposomes, and T-B healing quality was evaluated by histology and biomechanics. In vitro, bone marrow-derived macrophages (BMDMs) were stretched with CELLOAD-300 load system and macrophage polarization was identified by flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). MSC chondrogenic differentiation was measured by histochemical analysis and qRT-PCR. ELISA and qRT-PCR were performed to screen the candidate molecules that mediated the pro-chondrogenic function of mechanical stimulated BMDMs. Results. Mechanical stimulation promoted macrophage M2 polarization in vivo and in vitro. The conditioned media from mechanically stimulated BMDMs (MS-CM) enhanced MSC chondrogenic differentiation, and mechanically stimulated BMDMs generated more TGF-β1. Further, neutralizing TGF-β1 in MS-CM can attenuate its pro-chondrogenic effect. In vivo, mechanical stimulation promoted TGF-β1 generation, MSC chondrogenesis, and T-B healing, which were abolished following macrophage depletion. Conclusion. Macrophages subjected to appropriate mechanical stimulation could polarize toward the M2 phenotype and secrete TGF-β1 to promote MSC chondrogenesis, which subsequently augments T-B healing. Cite this article: Bone Joint Res 2023;12(3):219–230

The enthesis is a tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones, namely tendon, fibrocartilage, mineralized fibrocartilage and bone. Given the avascularity of the tendon and the gradual change in tissue architecture and cell phenotype, the enthesis original tissue is often not re-established after chronic injuries, resulting in scar formation. Conservative treatments and surgical approaches are still far from a functional regeneration, whilst tissue engineering based scaffolds have recently showed great potential. In this work, we hypothesised that collagen-based scaffolds that mimic the basic architecture of the enthesis, will be able to spatially direct stem cell differentiation, providing an in vitro platform to study enthesis regeneration. A three-layer sponge composed of a tendon-like layer (collagen type I), a fibrocartilage-like layer (collagen type II) and a bone-like layer (collagen type I and hydroxyapatite) was fabricated by an iterative layering freeze-drying technique. Scaffold pore size and structural continuity at the interfaces were assessed by SEM and μ-CT analysis. Bone-marrow derived stem cells (BMSCs) were seeded on the scaffold and cultured in basal and differentiation media (chondrogenic, tenogenic and osteogenic). At day 7 and 21 the scaffolds were stained with Alizarin Red and Alcian Blue; alkaline phosphatase activity (ALP) and calcium and glycosaminoglycans (GAGs) were quantified in order to evaluate BMSC differentiation towards osteogenic and chondrogenic lineage. The presence of collagen I, III, tenascin and decorin in the scaffolds was evaluated by immunofluorescence staining in order to evaluate tenogenic differentiation of BMSCs. Scaffolds with three distinct but interconnected layers of collagen type I, collagen type II and collagen type I + hydroxyapatite were fabricated, with pore sizes in the range of 100–200 μm. Increased ALP and calcium levels were detected in a localised manner within the bone-like layer when scaffolds were cultured in basal medium (p<0.025 vs the other 2 layers). Similarly, proteoglycans were detected specifically in the fibrocartilage-like layer when scaffolds were cultured in the chondrogenic differentiation medium (p<0.03 vs the other 2 layers). Increased expression of tenogenic markers was observed in the tendon-like layer of scaffolds cultured in tenogenic media (p<0.045 vs the other 2 layers). In conclusion, the different collagen composition of each layer was able to spatially direct BMSC differentiation in a localized manner within the scaffold. Ongoing work is evaluating the synergistic effect between growth factor functionalized within the fibrocartilage and tendon-like layers for improved BMSC differentiation. Overall, these scaffolds hold promising potential in developing novel and more efficient strategy towards enthesis regeneration

Despite extensive research aimed at improving surgical outcomes of enthesis injuries, re-tears remain a common problem, as the repairs often lead to fibrovascular scar as opposed to a zonal enthesis. Zonal enthesis formation involves anchoring collagen fibers, synthesizing proteoglycan-rich fibrocartilage, and mineralizing this fibrocartilage [1]. During development, the hedgehog signaling pathway promotes the formation and maturation of fibrocartilage within the zonal tendon-to-bone enthesis [1-4]. However, whether this pathway has a similar role in adult zonal tendon-to-bone repair is not known. Therefore, we developed a murine anterior cruciate ligament (ACL) reconstruction model [5] to better understand the zonal tendon-to-bone repair process and perturb key developmental regulators to determine the extent to which they can promote successful repair in the adult. In doing so, we activated the hedgehog signaling pathway both genetically using transgenic mice and pharmacologically via agonist injections. We demonstrated that both treatments improved the formation of zonal attachments and tunnel integration strength [6]. These improved outcomes were due in part to hedgehog signaling's positive role in proliferation of the bone marrow stromal cell (bMSC) progenitor pool and subsequent fibrocartilage production of bMSC progeny cells that form the attachments. These results suggest that, similar to growth and development, hedgehog signaling promotes the production and maturation of fibrocartilage during tendon-to-bone integration in adults. Lastly, we developed localized drug delivery systems to further improve the treatment of these debilitating injuries in future translational studies. Acknowledgements: This work was supported by NIH R01AR076381, R21AR078429, R00AR067283, F31AR079840, T32AR007132, and P30AR069619, in addition to the McCabe Fund Pilot Award at the University of Pennsylvania

Obesity is associated with poor outcomes and increased risk of failure after rotator cuff (RC) repair surgery. The effect of diet-induced obesity (DIO) on enthesis healing has not been well characterised and whether its effects can be reversed with dietary intervention is unknown. We hypothesised that DIO would result in inferior enthesis healing in a rat model of RC repair and that dietary intervention in the peri-operative period would improve enthesis healing. A total of 78 male Sprague-Dawley rats were divided into three weight-matched groups from weaning and fed either: control diet (CD), high-fat diet (HFD), or HFD until surgery, then CD thereafter (HF-CD). After 12 weeks the left supraspinatus tendon was detached, followed by immediate surgical repair. At 2 and 12 weeks post-surgery, animals were cullers and RCs harvested for biomechanical and histological evaluation. Body composition and metabolic markers were assessed via DEXA and plasma analyses, respectively. DIO was established in the HFD and HF-CD groups prior to surgery, and subsequently reversed in the HF-CD group after surgery. At 12 weeks post-surgery, plasma leptin concentrations were higher in the HFD group compared to the CD group (5.28 vs. 2.91ng/ml, P=0.003). Histologically, the appearance of the repaired entheses was poorer in both the HFD and HF-CD compared to the CD group at 12 weeks (overall histological score 6.20 (P=0.008), 4.98 (P=0.001) and 8.68 out of 15, respectively). The repaired entheses in the HF-CD group had significantly lower (26.4 N, P=0.028) load-at-failure 12 weeks post-surgery compared to the CD group (34.4 N); while the HFD group was low, but not significantly different (28.1 N, P=0.096). Body mass at the time of surgery, plasma leptin and body fat percentage were negatively correlated with histological scores and plasma leptin with load-at-failure 12 weeks post-surgery. DIO impaired enthesis healing in this rat RC repair model, with inferior biomechanical and histological outcomes. Restoring normal weight with dietary change after surgery did not improve healing outcomes. Exploring interventions that improve the metabolic state of obese patients and counselling patients appropriately about their modest expectations after repair should be considered

We hypothesised that diet-induced obesity (DIO) would result in inferior enthesis healing in a rat model of rotator cuff (RC) repair and that dietary intervention in the peri-operative period would improve enthesis healing. A total of 78 male Sprague-Dawley rats were divided into three weight-matched groups from weaning and fed either: control diet (CD), high-fat diet (HFD), or HFD until surgery, then CD thereafter (HF-CD). After 12 weeks, the left supraspinatus tendon was detached, followed by immediate surgical repair. At 2 and 12 weeks post-surgery, animals were culled, and RCs harvested for biomechanical and histological evaluation. Body composition and metabolic markers were assessed via DEXA and plasma analyses, respectively. DIO was established in the HFD and HF-CD groups before surgery and subsequently reversed in the HF-CD group after surgery. Histologically, the appearance of the repaired entheses was poorer in both the HFD and HF-CD groups compared with the CD group at 12 weeks after surgery, with semiquantitative scores of 6.2 (P<0.01), 4.98 (P<0.01), and 8.7 of 15, respectively. The repaired entheses in the HF-CD group had a significantly lower load to failure (P=0.03) at 12 weeks after surgery compared with the CD group, while the load to failure in the HFD group was low but not significantly different (P=0.10). Plasma leptin were negatively correlated with histology scores and load to failure at 12 weeks after surgery. DIO impaired enthesis healing in this rat RC repair model, with inferior biomechanical and histological outcomes. Restoring normal weight with dietary change after surgery did not improve healing outcomes. Circulating levels of leptin significantly correlated with poor healing outcomes. This pre-clinical rodent model demonstrates that obesity is a potentially modifiable factor that impairs RC healing and increases the risk of failure after RC surgery

The enthesis is a specialised zonal tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones, namely tendon, fibrocartilage, mineralized fibrocartilage and bone. Following injuries and surgical repair, the enthesis is often not reestablished and so far, traditionally used tissue substitutes have lacked to reproduce the complexity of the native tissue. In this work, we hypothesised that a collagen-based three-layer scaffold that mimic the composition of the enthesis, in combination with bioactive molecules, will enhance the functional regeneration of the enthesis. A three-layer sponge composed of a tendon-like layer (collagen I), a cartilage-like layer (collagen II) and a bone-like layer (collagen I and hydroxyapatite) was fabricated by an iterative layering freeze-drying technique. Scaffold porosity and structural continuity at the interfaces were assessed through SEM analysis. Bone-marrow derived stem cells (BMSCs) were seeded by syringe vacuum assisted technique on the scaffold. Scaffolds were cultured in basal media for 3 days before switching to differentiation media (chondrogenic, tenogenic and osteogenic). BMSCs metabolic activity, proliferation and viability were assessed by alamarBlue, PicoGreen and Live/Dead assays. At D21 the scaffolds were fixed, cryosectioned and Alizarin Red and Alcian Blue stainings were performed in order to evaluate BMSC differentiation towards osteogenic and chondrogenic lineage. The presence of collagen I and tenascin in the scaffolds was evaluated by immunofluorescence staining at D21 in order to assess tenogenic differentiation of BMSCs. Subsequently, the cartilage-like layer was functionalized with IGF-1, seeded with BMSCs and cultured in basal media up to D21. Structural continuity at the interfaces of the scaffolds was confirmed by SEM and scaffold porosity was assessed as >98%. The scaffolds supported cell proliferation and infiltration homogeneously throughout all the layers up to D21. Osteogenic differentiation of BMSC selectively in the bone-like layer was confirmed by Alizarin red staining in scaffolds cultured in basal and osteogenic media. Alcian blue staining revealed the presence of proteoglycans selectively in the cartilage-like layer in scaffolds cultured in chondrogenic media but not in basal media. Increased expression of the tenogenic markers collagen I and tenascin were observed in the tendon-like layer of scaffolds cultured in tenogenic but not in basal media for 21 days. The presence of IGF-1 increased osteogenic and chondrogenic differentiation of BMSCs, whereas no difference was observed for tenogenic differentiation. In conclusion, a 3-layer collagen sponge was successfully fabricated with distinct but integrated layers; the different collagen composition of the non-functionalized 3-layer sponge was able to regulate BMSC differentiation in a localized manner within the scaffold. The scaffold functionalization with IGF-1 accelerated chondrogenic and osteogenic BMSC differentiation. Overall, functionalization of the 3-layer scaffolds holds promising potential in enthesis regeneration

Tendons and tendon-to-bone entheses don't usually regenerate after injury, and the hierarchical organization of such tissues makes them challenging sites of study for tissue engineers. In this study, we have tried a novel approach using miRNA and a bioactive bioink to stimulate the regeneration of the enthesis. microRNAs (miRNAs) are short, non-coding sequences of RNA that act as post-transcriptional regulators of gene and protein expression [1]. Mimics or inhibitors of specific miRNAs can be used to restore lost functions at the cell level or improve healing at the tissue level [2,3]. We characterized the healing of a rat patellar enthesis and found that miRNA-16-5p was upregulated in the fibrotic portion of the injured tissue 10 days after the injury. Based on the reported interactions of miRNA-16-5p with the TGF-β pathway via targeting of SMAD3, we aimed to explore the effects of miRNA-16-5p mimics on the tenogenic differentiation of adipose-derived stem cells (ASCs) encapsulated in a bioactive bioink [4,5]. Bioinks with different properties are used for the 3D printing of biomimetic constructs. By integrating cells, materials, and bioactive molecules it is possible to tailor the regenerative capacity of the ink to meet the particular requirements of the tissue to engineer [5]. Here we have encapsulated ASCs in a gelatin-methacryloyl (GelMa) bioink that incorporates miR-16-5p mimics and magnetically responsive microfibers (MRFs). When the bioink is crosslinked in the presence of a magnetic field, the MRFs align unidirectionally to create an anisotropic construct with the ability to promote the tenogenic differentiation of the encapsulated ASCs. Additionally, the obtained GelMA hydrogels retained the encapsulated miRNA probes, which permitted the effective 3D transfection of the ASC and therefore, the regulation of gene expression, allowing to investigate the effects of the miR-16-5p mimics on the tenogenic differentiation of the ASCs in a biomimetic scenario

Objectives. Rotator cuff tears are among the most frequent upper extremity injuries. Current treatment strategies do not address the poor quality of the muscle and tendon following chronic rotator cuff tears. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that activates many genes that are important in skeletal muscle regeneration. HIF-1α is inhibited under normal physiological conditions by the HIF prolyl 4-hydroxylases (PHDs). In this study, we used a pharmacological PHD inhibitor, GSK1120360A, to enhance the activity of HIF-1α following the repair of a chronic cuff tear, and measured muscle fibre contractility, fibrosis, gene expression, and enthesis mechanics. Methods. Chronic supraspinatus tears were induced in adult rats, and repaired 28 days later. Rats received 0 mg/kg, 3 mg/kg, or 10 mg/kg GSK1120360A daily. Collagen content, contractility, fibre type distribution and size, the expression of genes involved in fibrosis, lipid accumulation, atrophy and inflammation, and the mechanical properties of the enthesis were then assessed two weeks following surgical repair. Results. At two weeks following repair, treatment groups showed increased muscle mass but there was a 15% decrease in force production in the 10 mg/kg group from controls, and no difference between the 0 mg/kg and the 3 mg/kg groups. There was a decrease in the expression of several gene transcripts related to matrix accumulation and fibrosis, and a 50% decrease in collagen content in both treated groups compared with controls. Additionally, the expression of inflammatory genes was reduced in the treated groups compared with controls. Finally, PHD inhibition improved the maximum stress and displacement to failure in repaired tendons. Conclusions. GSK1120360A resulted in improved enthesis mechanics with variable effects on muscle function. PHD inhibition may be beneficial for connective tissue injuries in which muscle atrophy has not occurred. Cite this article: J. P. Gumucio, M. D. Flood, A. Bedi, H. F. Kramer, A. J. Russell, C. L. Mendias. Inhibition of prolyl 4-hydroxylase decreases muscle fibrosis following chronic rotator cuff tear. Bone Joint Res 2017;6:57–65. DOI: 10.1302/2046-3758.61.BJR-2016-0232.R1

Re-rupture rates after rotator cuff repair remain high because of inadequate biological healing at the tendon-bone interface. Single-growth factor therapies to augment healing at the enthesis have so far yielded inconsistent results. An emerging approach is to combine multiple growth factors over a spatiotemporal distribution that mimics normal healing. We propose a novel combination treatment of insulin-like growth factor 1 (IGF-1), transforming growth factor β1 (TGF-β1) and parathyroid hormone (PTH) incorporated into a controlled-release tyraminated poly-vinyl-alcohol hydrogel to improve healing after rotator cuff repair. We aimed to evaluate this growth factor treatment in a rat chronic rotator cuff tear model. A total of 30 male Sprague-Dawley rats underwent unilateral supraspinatus tenotomy. Delayed rotator cuff repairs were then performed after 3 weeks, to allow tendon degeneration that resembles the human clinical scenario. Animals were randomly assigned to: [1] a control group with repair alone; or [2] a treatment group in which the hydrogel was applied at the repair site. All animals were euthanized 12 weeks after rotator cuff surgery and the explanted shoulders were analyzed for biomechanical strength and histological quality of healing at the repair site. In the treatment group had significantly higher stress at failure (73% improvement, P=0.003) and Young's modulus (56% improvement, P=0.028) compared to the control group. Histological assessment revealed improved healing with significantly higher overall histological scores (10.1 of 15 vs 6.55 of 15, P=0.032), and lower inflammation and vascularity. This novel combination growth factor treatment improved the quality of healing and strength of the repaired enthesis in a chronic rotator cuff tear model. Further optimization and tailoring of the growth factors hydrogel is required prior to consideration for clinical use in the treatment of rotator cuff tears. This novel treatment approach holds promise for improving biological healing of this clinically challenging problem

Successful anterior cruciate ligament (ACL) reconstructions strive a firm ligament-bone integration. Therefore, the aim of this study was to address in more detail the enthesis as the thriphasic bone attachment of the ACL using a tissue engineering approach. To establish a tissue-engineered enthesis-like construct, triphasic scaffolds embroidered from poly(L-lactide-co-caprolactone) and polylactic acid functionalized with collagen foam were colonized with osteogenically differentiated human mesenchymal stromal cells (hMSCs) and lapine (L) ACL fibroblasts. These triphasic scaffolds with a bone-, a fibrocartilage transition- and a ligament phase were seeded directly after spheroid assembly or with 14 days precultured LACL fibroblast spheroids and 14 days osteogenically differentiated hMSCs spheroids (=longer preculture) and cultured for further 14 days. Cell survival was tested. Collagen type I and vimentin were immunolabeled and the content of DNA and sulfated glycosaminoglycan (sGAG) was quantified. The relative gene expression of tenascin C, type I and X collagens, Mohawk and Runx2 was analyzed. Compared to the LACL spheroids the hMSC spheroids adhered better to the scaffold surface with faster cell outgrowth on the fibers. Collagen type I and vimentin were mainly detected in the hMSCs colonizing the bone zone. The DNA content was generally higher in the bone (hMSCs) than in the ligament zones and after short spheroid preculture higher than after longer preculture whereas the sGAG content was greater after longer preculture for both cell types. The longer precultivated hMSCs expressed more type I collagen in comparison to those only shortly precultured before scaffold seeding. Type I collagen and tenascin C were higher expressed in scaffolds directly colonized with LACL compared to those seeded after longer spheroid preculture. The gene expression of ECM components and transcription factors depended on cell type and preculturing condition. Zonal colonization of triphasic scaffolds using the spheroid method is possible offering a novel approach for enthesis tissue engineering

Abstract. Objectives. The scapholunate interosseous ligament (SLIL) has a unique C-shape following the arc of the scaphoid and lunate surfaces from distal dorsal around to distal volar. This ligament comprises of three subregions: dorsal, proximal and volar. The SLIL enthesis, a specialized region where this ligament attaches to the scaphoid and lunate, has not previously been studied despite its important mechanical function in the biomechanics of the wrist joint. This study therefore aims to compare the histomorphological differences between the SLIL subregions, including at their entheses. This study will examine the qualitative and quantitative differences between the three subregions, as well as between the scaphoid and lunate attachments. Methods. Twelve fresh-frozen human cadaveric wrists were dissected and the gross dimensions of each SLIL subregion measured. Subregions were then histologically processed for qualitative and quantitative morphological and compositional analyses, including quantification of enthesis calcified fibrocartilage (CF) area. Results. From the gross measurements taken, the dorsal subregion was the thickest. There were no significant differences in lengths and widths between the three subregions. Qualitatively, the dorsal and volar subregions had fibrocartilaginous entheses while the proximal subregion inserted into cortical bone via articular cartilage. Quantitatively, the dorsal subregion had significantly more CF than the volar subregion. There was no significant difference in the enthesis CF between scaphoid and lunate attachments in the three subregions. Conclusions. There are significant histomorphological differences between the SLIL subregions. The dorsal subregion has the largest amount of CF, which is consistent with the greater biomechanical force subjected to this subregion compared to the other subregions. This result confirms that the dorsal subregion is the strongest of the three subregions. The similar histomorphology of the ligament at the scaphoid and lunate entheses suggests that similar biomechanical forces are applied to both attachments. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

Abstract. Objectives. The enthesis is a specialised structure at the interface between bone and tendon with gradual integration to maintain functionality and integrity. In the process of fabricating an in-vitro model of this complex structure, this study aims to investigate growth and maturation of bone, tendon and BMSC spheroids followed by 3D mini-tissue production. Methods. Cell spheroids Spheroids of differentiated rat osteoblasts (dRObs), rat tendon fibroblasts (RTFs) and bone marrow stem cells (BMSC) were generated by culturing in 96 well U bottom cell repellent plates. With dROb spheroids previously analysed [1], RTF spheroids were examined over a duration of up to 28 days at different seeding densities 1×10. 4. , 5×10. 4. , 1×10. 5. , 2×10. 5. in different media conditions with and without FBS (N=3). Spheroid diameter was analysed by imageJ/Fiji; Cell proliferation and viability was assessed by trypan blue staining after dissociating with accutase + type II collagenase mix; necrotic core by H&E staining; and extracellular matrix by picro-sirius red (RTFs) staining to visualise collagen fibres under bright-field and polarised light microscope. 3D mini-tissue constructs. 15 day old mineralised dROb spheroids (∼1.5mm diameter) were deposited in pillar array supports using a customised spheroid deposition system to allow 3D mini-tissue formation via fusion (N=3). Similarly BMSC and RTF spheroids were deposited after determining the seeding density that produced spheroid size equivalent to 15 day old dROb spheroids. Gentle removal of spheroids from supports was performed on day 2, 4 and 6 to assess spheroid fusion. Histological staining was performed to observe cellular arrangement and extracellular matrix. Results. RTF spheroids diameter reduced over the course of 28 days regardless of the seeding density. A substantial decline in cell numbers over time was observed and suggests lack of cell proliferation due to tenogenic differentiation. Absence of a necrotic core in RTF spheroids, in all seeding densities, reveals their inherent capacity to maintain cell viability in avascular conditions. Picro-sirius red staining demonstrated the presence of collagen type I fibres predominantly in peripheral regions of spheroids maintaining its shape. Small amounts of collagen type III were also noticed. The dROb spheroids fused rapidly within 2 days resulting in the formation of a mini-tissue. 2×10. 5. RTFs and 3×10. 5. BMSCs produced spheroids of ∼1.5mm on day 3 and day 1 respectively. When these spheroids were deposited in pillar array supports, they did not undergo fusion even up to 6 days. This suggests inadequate aggregation of spheroids and insufficient ECM production at this early stage. Conclusions. This study has demonstrated the ability of RTFs to produce necrotic core-free spheroids with collagen fibres maintaining their structural integrity. For mini-tissue formation, we predict a longer initial culture time of RTF and BMSC spheroids will allow increased cellular interaction and ECM production before deposition, and will facilitate spheroid fusion. These findings will be applied in producing heterogenous mini-tissues, serving as a 3D in-vitro enthesis model. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

Abstract. The lateral ligaments of the ankle composed of the anterior talofibular (ATFL), calcaneofibular (CFL) and posterior talofibular ligaments (PTFL), are amongst the most commonly injured ligaments of the human body. Although treatment methods have been explored exhaustively, healing outcomes remain poor with high rates of re-injury, chronic ankle instability and pain persisting. The introduction and application of tissue engineering methods may target poor healing outcomes and eliminate long-term complications, improving the overall quality of life of affected individuals. For any surgical procedure or tissue-engineered replacement to be successful, a comprehensive understanding of the complete anatomy of the native structure is essential. Knowledge of the dimensions of ligament footprints is vitally important for surgeons as it guides the placement of bone tunnels during repair. It is also imperative in tissue-engineered design as the creation of a successful replacement relies on a thorough understanding of the native anatomy and microanatomical structure. Several studies explore techniques to describe ligament footprints around the body, with limited studies describing in-depth footprint dimensions of the ATFL, CFL and PTFL. Techniques currently used to measure ligament footprints are complex and require resources which may not be readily available, therefore a new methodology may prove beneficial. Objectives. This study explores the application of a novel technique to assess the footprint of ankle ligaments through a straightforward inking method. This method aims to enhance surgical technique and contribute to the development of a tissue-engineered analogue based on real anatomical morphometric data. Methods. Cadaveric dissection of the ATFL, CFL and PTFL was performed on 12 unpaired fresh frozen ankles adhering to regulations of the Human Tissue (Scotland) Act. The ankle complex with attaching ligaments was immersed in methylene blue. Dissection of the proximal and distal entheses of each ligament was carried out to reveal the unstained ligament footprint. Images of each ligament footprint were taken, and the area, length and width of each footprint were assessed digitally. Results. The collective area of the proximal entheses of the ATFL, CFL and PTFL measures 142.11 ± 12.41mm2. The mean areas of the superior (SB) and inferior band (IB) of the distal enthesis of the ATFL measured 41.72 ± 5.01mm2 and 26.66 ± 3.12mm2 respectively. The footprint of the distal enthesis of the CFL measured 146.07 ± 14.05mm2, while the footprint of the distal PTFL measured 126.26 ± 8.88mm2. The proximal footprint of the ATFL, CFL and PTFL measured 11.06 ± 0.69mm, 7.87 ± 0.43mm and 10.52 ± 0.63mm in length and 8.66 ± 0.50mm, 9.10 ± 0.92mm and 14.41 ± 1.30mm in width on average. The distal footprint of the ATFL (SB), ATFL (IB), CFL and PTFL measured 10.92 ± 0.81 mm, 8.46 ± 0.46mm, 13.98 ± 0.93mm and 11.25 ± 0.95mm in length and 7.76 ± 0.59mm, 7.51 ± 0.64mm, 18.98 ± 1.15mm and 24.80 ± 1.25mm in width on average. Conclusions. This methodology provides an effective approach in the identification of the footprint of the lateral ligaments of the ankle to enhance surgical precision and accuracy in tissue-engineered design. 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. After an injury, the biological reattachment of tendon to bone is a challenge because healing takes place between a soft (tendon) and a hard (bone) tissue. Even after healing, the transition zone in the enthesis is not completely regenerated, making it susceptible to re-injury. In this study, we aimed to regenerate Achilles tendon entheses (ATEs) in wounded rats using a combination of kartogenin (KGN) and platelet-rich plasma (PRP). Methods. Wounds created in rat ATEs were given three different treatments: kartogenin platelet-rich plasma (KGN-PRP); PRP; or saline (control), followed by histological and immunochemical analyses, and mechanical testing of the rat ATEs after three months of healing. Results. Histological analysis showed well organised arrangement of collagen fibres and proteoglycan formation in the wounded ATEs in the KGN-PRP group. Furthermore, immunohistochemical analysis revealed fibrocartilage formation in the KGN-PRP-treated ATEs, evidenced by the presence of both collagen I and II in the healed ATE. Larger positively stained collagen III areas were found in both PRP and saline groups than those in the KGN-PRP group. Chondrocyte-related genes, SOX9 and collagen II, and tenocyte-related genes, collagen I and scleraxis (SCX), were also upregulated by KGN-PRP. Moreover, mechanical testing results showed higher ultimate tensile strength in the KGN-PRP group than in the saline control group. In contrast, PRP treatment appeared to have healed the injured ATE but induced no apparent formation of fibrocartilage. The saline-treated group showed poor healing without fibrocartilage tissue formation in the ATEs. Conclusions. Our results show that injection of KGN-PRP induces fibrocartilage formation in the wounded rat ATEs. Hence, KGN-PRP may be a clinically relevant, biological approach to regenerate injured enthesis effectively. Cite this article: J. Zhang, T. Yuan, N. Zheng, Y. Zhou, M. V. Hogan, J. H-C. Wang. The combined use of kartogenin and platelet-rich plasma promotes fibrocartilage formation in the wounded rat Achilles tendon entheses. Bone Joint Res 2017;6:231–244. DOI: 10.1302/2046-3758.64.BJR-2017-0268.R1

The fibrocartilaginous enthesis displays a complex interface between two mechanically dissimilar tissues, namely tendon and bone. This graded transition zone consists of parallel collagen type I fibres arising from the tendon and inserting into bone across zones of fibrocartilage with aligned collagen type I and collagen type II fibres and mineralised fibrocartilage. Due the high stress concentrations arising at the interface, entheses are prone to traumatic and chronic overuse injuries such as rotator cuff and anterior cruciate ligament (ACL) tears. Treatment strategies range from surgical reattachment for complete tears and conservative treatments (physiotherapy, anti-inflammatory drugs) in chronic inflammatory conditions. Generally, the native tissue architecture is not re-established and mechanically inferior scar tissue is formed. Current interfacial tissue engineering approaches pose scaffold-associated drawbacks and limitations, such as foreign body response. Using a thermo-responsive electrospun scaffold that provides architectural signals similar to native tissues and can be removed prior to implantation, we aim to develop an ECM-rich, cell-based implant for tendon-enthesis regeneration. Alcian blue staining revealed highest sGAG deposition in cell (human adipose derived stem cells) sheets grown on random electrospun fibres and lowest sGAG deposition in collagen type I sponges. Cells did not show an equal distribution throughout the collagen type II scaffolds but tended to form localised aggregates. Thermo-responsive electrospun fibres with random and aligned fibre orientation provided an adequate three-dimensional environment for chondrogenic differentiation of multilayer hADSC-sheets shown by high ECM-production, especially high sGAG deposition. Chondrogenic cell sheets showed increased expression of SOX9, COL2A1, COL1A1, COMP and ACAN after 7 days of chondrogenic induction when compared to pellet culture. Anisotropic fibres enabled the generation of aligned chondrogenic cell sheets, shown by cell and collagen fibre alignment. Thermoresponsive electrospun fibres showed high chondro-inductivity due to their three-dimensionality and therefore pose a promising tool for the generation of scaffold-free multilayer constructs for tendon-enthesis repair within short culture periods. Aligned chondrogenic cell sheets mimic the zonal orientation of the native enthesis as the fibrocartilaginous zone exhibits high collagen alignment

After surgical tendon repair, the tendon-to-bone enthesis often doesn't regenerate, which leads to high numbers of rupture recurrences. To remedy this, tissue engineering techniques are being pursued to strengthen the interface and improve regeneration. In this study, we used biphasic 3D printed PLGA scaffolds with aligned pores at the tendon side and random pores at the bone side to mimic the native enthesis. We seeded these with mesenchymal stem cells and inserted them into dual-flow bioreactors, allowing us to employ tenogenic and chondrogenic differentiation medium in separate flows. MTS assay demonstrated metabolism in dual-flow bioreactors at levels similar to tissue culture plate and rotating bioreactors. After 7, 14 and 21 days, samples were collected and analyzed by histology, RT-PCR and GAG production. H&E staining confirmed a compact cell layer attached to fibers and between porous cavities of scaffolds that increased with time of culture. Interestingly, cultured constructs in dual-flow bioreactors biased towards a chondrogenic fate regardless of which flow they were exposed to, possibly due to high porosity of the scaffold allowing for fluid mixture. Sox9 was upregulated at all timepoints (up to 30× compared to control), and by day 21 Col2A1 was also highly upregulated. Additionally, GAG production in treated constructs (serum-free) was able to match constructs exposed to 10% FBS in controls, demonstrating the functional matrix forming capabilities of this system. Overall, we have validated this dual-flow system as a potential platform to form the enthesis, and future studies will further optimize parameters to achieve distinctly biphasic constructs

This study documents the gross and histologic structure of the infrapatellar plica, and fat pad, and adds to an earlier report to the COA. The important new findings are that the femoral attachment of the plica is an enthesis, and that the plica itself is. This study seeks to demonstrate that the structure of the fat pad (FP) and infrapatellar plica (IPP) is that of an enthesis organ. Twelve fresh frozen cadaver knees, each with an IPP, were dissected and the gross anatomic features recorded. The IPP and FP were harvested for study. Representative histologic sections were prepared on tissue fixed in 10% neutral buffered formalin, embedded in paraffin, cut at 4 microns on a rotatory microtome. Staining techniques included hematoxylin and eosin, Masson's trichrome, elastic stain and S100. Appropriate decalcification of sections of the femoral insertion of the IPP was performed. All sections were examined by light microscopy at low, medium and high power. IPP types included 8 separate, 1 split, 2 fenestrated, and one vertical septum. The origin of the IPP is a fibrous arc arising from the apex of the notch separate from the margin of the articular cartilage. This attachment site is the instant centreof rotation of the IPP and FP; they are thus not isometric. The central zone of the IPP consists of a mix of connective tissue types. Representative sections taken of the femoral attachment of the IPP display a transition zone between dense fibrillar collagen of the IPP, then fibrocartilage and cortical bone similar to a ligament attachment site or enthesis. The central plica histology is composed predominantly of dense regular connective tissue with variable clear space between the collagen bundles, and is thus ligamentous. There is abundant elastase staining throughout, as well as crimping of the collagen suggesting capacity for stretch. S100 staining demonstrates nerves around and in the substance of the IPP. The central body shows lobulated collections of mature adipose tissue admixed with loose connective tissue, containing abundant small peripheral nerves and vessels (all showing crimping and redundancy), merging with the dense fibrous tissue of the IPP. The FP is highly innervated, deformable, and fibro-fatty. Its histology shows lobules of fat, separated by connective tissue septa, which merge with the synovial areolar membrane surrounding the FP. The linked structures, IPP, central body, and FP occupy the anterior compartment, and function as an enthesis organ: the IPP tethers the FP via the central body and together they rotate around the femoral origin of the IPP. They are not isometric, and must stretch and relax with knee motion. The histology correlates with this requirement. The origin of the IPP is an enthesis, a new observation. Elastase staining, redundancy of vessels and nerves, crimping and redundancy of the dense connective tissue all reflect the requirement to deform. The fat pad merges with the central body, both highly innervated space fillers, tethered by the IPP, which is a non-isometric ligament, also containing nerves. The important clinical significance of these structures is that release of the IPP at the origin reuces or eliminates anterior knee pain in most

Background. Re-attachment of tendon to bone is challenging with surgical repair failing in up to 90% of cases. Poor biological healing is common and characterised by the formation of weak scar tissue. Previous work has demonstrated that decellularised allogenic demineralised bone matrix (DBM) regenerates a physiologic enthesis. Xenografts offer a more cost-effective option but concerns over their immunogenicity have been raised. We hypothesised that augmentation of a healing tendon-bone interface with DBM incorporated with autologous mesenchymal stem cells (MSCs) would result in improved function, and restoration of the native enthesis, with no difference between xenogenic and allogenic scaffolds. Methods. Using an ovine model of tendon-bone retraction the patellar tendon was detached and a complete distal tendon defect measuring 1 cm was created. Suture anchors were used to reattach the shortened tendon and xenogenic DBM + MSCs (n=5) and allogenic DBM + MSCs (n=5) were used to bridge the defect. Functional recovery was assessed every 3 weeks and DBM incorporation into the tendon and its effect on enthesis regeneration was measured using histomorphometry. Results. By 12 weeks, DBM augmentation resulted in significantly improved functional weight bearing with no failures in either group. Compared to xenogenic DBM, allogenic DBM was associated with significantly higher functional weight bearing at 6 (P=0.047), 9 (P=0.028) and 12 weeks (P=0.009). This was accompanied by a more direct type of enthesis characterised by significantly more fibrocartilage and mineralised fibrocartilage. Xenograft was also associated with an immunogenic reaction despite preoperative decellularisation. Conclusion. This study shows that DBM enhances tendon-bone healing and may reduce the high failure rates associated with surgery. An immunogenic reaction, and inferior biomechanical and histological results were also associated with the use of xenograft. Allogenic DBM with autologous MSCs may be a suitable scaffold for the enhancement of tendon-bone healing in the clinical setting. Disclosures. Funded by IKC PoC grant awarded by the University of Leeds. Ethical approval. Granted by the study institution (University College London)

Tendons mainly consist of collagen in order to withstand high tensile forces. Compared to other, high turnover tissues, cellularity and vascularity in tendons are low. Thus, the natural healing process of tendons takes long and can be problematic. In case of injury to the enthesis, the special transition from tendon over cartilage to bone is replaced by a fibrous scar tissue, which remains an unsolved problem in rotator cuff repair. To improve tendon healing, many different approaches have been described using scaffolds, stem cells, cytokines, blood products, gene therapy and others. Despite promising in vitro and in vivo results, translation to patient care is challenging. In clinics however, tendon auto- or allografts remain still first choice to augment tendon healing if needed. Therefore, it is important to understand natural tendon properties and natural tendon healing first. Like in other tissues, senescence of tenocytes seems to play an important role for tendon degeneration which is interestingly not age depended. Our in vivo healing studies have shown improved and accelerated healing by adding collagen type I, which is now used in clinics, for example for augmentation of rotator cuff repair. Certain cytokines, cells and scaffolds may further improve tendon healing but are not yet used routinely, mainly due to missing clinical data, regulatory issues and costs. In conclusion, the correct diagnosis and correct first line treatment of tendon injuries are important to avoid the necessity to biologically augment tendon healing. However, strategies to improve and accelerate tendon healing are still desirable. New treatment opportunities may arise with further advances in tendon engineering in the future

Rotator cuff tears are common, with failure rates of up to 94% for large and massive tears. 1. For such tears, reattachment of the musculotendinous unit back to bone is problematic, and any possible tendon-bone repair heals through scar tissue rather than the specially adapted native enthesis. We aim to develop and characterise a novel soft-hard tissue connector device, specific to repairing/bridging the tendon-bone injury in significant rotator cuff tears, employing decellularised animal bone partially demineralised at one end for soft tissue continuation. Optimisation samples of 15×10×5mm. 3. , trialled as separate cancellous and cortical bone samples, were cut from porcine femoral condyles and shafts, respectively. Samples underwent 1-week progressive stepwise decellularisation and a partial demineralisation process of half wax embedding and acid bathing. Characterisations were performed histologically for the presence/absence of cellular staining in both peripheral and central tissue areas (n=3 for each cortical/cancellous, test/PBS control and peripheral/central group), and with BioDent reference point indentation (RPI) for pre- and post-processing mechanical properties. Histology revealed absent cellular staining in peripheral and central cancellous samples, whilst reduced in cortical samples compared to controls. Cancellous samples decreased in wet mass after decellularisation by 45.3% (p<0.001). RPI measurements associated with toughness (total indentation depth, indentation depth increase) and elasticity (1st cycle unloading slope) showed no consistent changes after decellularisation. X-rays confirmed half wax embedding provided predictable control of the mineralised-demineralised interface position. Initial optimisation trials show proof-of-concept of a soft-hard hybrid scaffold as an immune compatible xenograft for irreparable rotator cuff tears. Decellularisation did not appreciably affect mechanical properties, and further biological, structural and chemical characterisations are underway to assess validity before in vivo animal trials and potential clinical translation

Introduction: The intricate biomechanical function of the alar ligaments in the craniocervical articular complex has received considerable scientific attention. While allowing the greatest range of axial rotation of the entire spine with 40° to each side, definitive restraint at the extremes of motion by the alar ligaments is of vital importance. Detailed knowledge of the function of these ligaments is essential for comprehending the factors leading to potentially devastating instability. Methods: Bilateral alar ligaments including the bony entheses were removed from six adult cadavers aged 65–89 years within 48 hours of death. All specimens were judged to be free of abnormalities with the exception of non-specific degenerative changes. Dimensions of the alar ligaments were measured. Schematic multipla-nar reconstruction of axial atlanto-axial rotation was done in the transverse and frontal planes for the neutral position and for rotation to 30° and 40° in the neutral plane to assess schematic fibre elongation during axial rotation and to determine the change in the angle of insertion at the odontoid and condylar entheses. This was repeated with a 1mm descending translation of the occipital condyles at 30° and 3mm descending translation of the occipital condyles at 40° rotation. Results: The average diameter of the odontoid process measured in the sagittal plane was 10.6 mm (SD 1.1). The longest fibre length was measured from the posterior border of the odontoid enthesis to the posterior border of the condylar enthesis with an average of 13.2 mm (SD 2.5) and the shortest between the lateral (anterior) border odontoid enthesis and the anterior condylar enthesis with an average of 8.2 mm (SD 2.2). Attachment areas of the enthesis revealed an average of 60 mm2 (SD 12.4) for the odontoid and 50,6 mm2 (SD12.6) for the condylar enthesis. Schematic fibre elongation reaches 27,1% for the longest fibres at 40° axial rotation. This is reducible to 7,8% elongation by 3mm caudal translation of the atlas. Conclusions: This theoretical model confirms that the bi-convex shape of atlanto-axial joint allows for rotation when modelled with oblique alar ligaments. This provides baseline for further research with functional MRI which will be useful for rheumatoid and post traumatic spine

Surgical reattachment of torn rotator cuff tendons can lead to satisfactory clinical outcome but failures remain common. Ortho-R product is a freeze-dried formulation of chitosan (CS) that is solubilized in platelet-rich plasma (PRP) to form injectable implants. The purpose of the current pilot study was to determine Ortho-R implant acute residency, test safety of different implant doses, and assess efficacy over standard of care in a sheep model. The infraspinatus tendon (ISP) was detached and immediately repaired in 22 skeletally mature ewes. Repair was done with four suture anchors in a suture bridge configuration (n = 6 controls). Freeze-dried formulations containing 1% w/v chitosan (number average molar mass 35 kDa and degree of deacetylation 83%) with 1% w/v trehalose (as lyoprotectant) and 42.2 mM calcium chloride (as clot activator) were solubilized with autologous leukocyte-rich PRP and injected at the tendon-bone interface and on top of the repaired site (n = 6 with a 1 mL dose and n = 6 with a 2 mL dose). Acute implant residency was assessed histologically at 1 day (n = 2 with a 1 mL dose and n = 2 with a 2 mL dose). Outcome measures included MRI assessment at baseline, 6 weeks and 12 weeks, histopathology at 12 weeks and clinical pathology. MRI images and histological slides were scored by 2 blinded readers (veterinarian and human radiologist, and veterinarian pathologist) and averaged. The Generalized Linear Model task (SAS Enterprise Guide 7.1 and SAS 9.4) was used to compare the different groups with post-hoc analysis to test for pairwise differences. Ortho-R implants were detected near the enthesis, near the top of the anchors holes and at the surface of ISP tendon and muscle at 1 day. Numerous polymorphonuclear cells were recruited to the implant in the case of ISP tendon and muscle. On MRI, all repair sites were hyperintense compared to normal tendon at 6 weeks and only 1 out 18 repair sites was isointense at 12 weeks. The tendon repair site gap seen on MRI, which is the length of the hyperintense region between the greater tuberosity and tendon with normal signal intensity, was decreased by treatment with the 2 mL dose when compared to control at 12 weeks (p = 0.01). Histologically, none of the repair sites were structurally normal. A trend of improved structural organization of the tendon (p = 0.06) and improved structural appearance of the enthesis (p = 0.1) with 2 mL dose treatment compared to control was seen at 12 weeks. There was no treatment-specific effect on all standard safety outcome measures, which suggests high safety. Ortho-R implants (2 mL dose) modulated the rotator cuff healing processes in this large animal model. The promising MRI and histological findings may translate into improved mechanical performance, which will be assessed in a future study with a larger number of animals. This study provides preliminary evidence on the safety and efficacy of Ortho-R implants in a large animal model that could potentially be translated to a clinical setting

Aim. To determine the influence of tendo achilles (TA) rupture gap distance and location on clinical outcome managed with accelerated functional rehabilitation. Methods. Twenty six patients with acute complete TA ruptures underwent ultrasound (US) within a week of injury. Measurements included the distance of the rupture from the enthesis and the gap distance between the tendon edges in three positions –. foot plantigrade,. maximum equinus and. maximum equinus with 90. o. knee flexion. All patients were managed non-operatively in functional weightbearing orthoses. Nineteen patients were followed up at a mean of 6.1 years (range 5.8–6.5). Outcomes included ultrasound confirmation of healing, Achilles Tendon Rupture Score (ATRS) and Modified Lepilahti score (MLS). Results. The mean distance of the rupture from the enthesis was 52mm (range: 40–76mm). The mean gap distance with the foot plantigrade was 11.4mm (95%CI: 9.9, 12.9) which reduced to 4.8mm (95%CI: 3.3, 6.4) in equinus and 1.5mm (95%CI: 0.8, 2.2) with 90° knee flexion. At follow up, no re-ruptures had occurred. US demonstrated continuity in all healed tendons. Mean ATRS was 86 (95%CI: 78.8, 93.9). There was a significant correlation between the distance of the rupture with the MLS (p=0.015) and the ATRS domains of strength (p=0.037) and fatigue (p=0.017). There was no significant correlation between the measured gap distance in the three positions with respect to the MTLS, ATRS or individual ATRS domain scores. There was no significant difference when comparing outcomes between left and right TAs or comparing gaps less than 1cm with those greater than 1 cm. Discussion. The distance of the gap from the enthesis may be more predictive of mid term clinical outcome in patients with TA rupture managed with accelerated functional rehabilitation compared with the magnitude of the gap and extent of closure with equinus and knee flexion

The structure and extracellular matrix composition of the interface are complex and allow for a gradual mechanical stress transfer between tendons and bone. In this study, biphasic silk fibroin scaffolds designed to mimic the gradient in collagen molecule alignment present at the interface. The scaffolds had two different pore alignments: anisotropic at the tendon side and isotropic at the bone side. Total porosity ranged from 50–80% and the majority of pores were <100–300 µm. Young's modulus varied from 689–1322 kPa. In addition, human adMSC were cultured on the scaffolds to evaluate the effect of pore morphology on cell proliferation and gene expression. Biphasic scaffolds supported cell attachment and influenced cytoskeleton organization depending on pore alignment. In addition, the gene expression of tendon, enthesis and cartilage markers significantly changed in each region of the scaffolds. We functionalized those scaffolds with heparin and explored their ability to deliver TGF-β2 and GDF5. TGF-β2 and pore anisotropy synergistically increased the expression of tendon/ligament markers and collagen I protein content. The combined delivery of TGF-β2 and GDF5 enhanced the expression of cartilage markers and collagen II protein content on substrates with isotropic porosity, whereas enthesis markers were enhanced in areas of mixed anisotropic/isotropic porosity

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

Purpose of the study: Irrespective of the technique used, the average rate of bone-tendon healing after rotator cuff repair is about 50% One of the reasons is the poor vitality of the tissues implicated in repair, particularly progressive destruction of the enthesis. Using the rat Achilles tendon, we destroyed the enthesis mechanically then repaired it with and without local injection of chondrocytes in order to study the effect of cell therapy on healing phenomena. Material and method: Sixty 3-month-old Wistar rats were operated on under general anaesthesia to detach the Achilles tendon and destroy the enthesis. In the first group (RI), the tendon was reinserted via a transosseous tunnel using a 4/0 non absorbable knitted thread. In the second group (RIC), joint chondrocytes, harvested from 4-day-old rats were injected locally during the same repair procedure. Animals were sacrificed every 15 days (n=15 per group) for a biomechanical and histology study. Results: In group RI, the non-healing rate was 50% versus 33% in group RIC; the difference was not significant (n=0.3). Tear resistance was increased significantly at 45 days in the RC group (p=0.04). The histology study showed a statistically significant development of a neoenthesis in the RIC group (p< 0.05), which was not observed in the RI group. Discussion: This animal model is valid for exploring rotator cuff healing with a spontaneous rate of healing to the order of 50%. Addition of chondrocytes during the surgical repair induces the production of an enthesis and increases the healing rate 50% and the value of the different biomechanical parameters at 30 days, with a statistically significant difference at 45 days

We used demineralised bone matrix (DBM) to augment re-attachment of tendon to a metal prosthesis in an in vivo ovine model of reconstruction of the extensor mechanism at the knee. We hypothesised that augmentation of the tendon-implant interface with DBM would enhance the functional and histological outcomes as compared with previously reported control reconstructions without DBM. Function was assessed at six and 12 weeks postoperatively, and histological examination was undertaken at 12 weeks. A significant increase of 23.5% was observed in functional weight-bearing at six weeks in the DBM-augmented group compared with non-augmented controls (p = 0.004). By 12 weeks augmentation with DBM resulted in regeneration of a more direct-type enthesis, with regions of fibrocartilage, mineralised fibrocartilage and bone. In the controls the interface was predominantly indirect, with the tendon attached to the bone graft-hydroxyapatite base plate by perforating collagen fibres

Summary Statement. Demineralised bone matrix augmented tendon-bone fixations in the animal model show less scar tissue and an enthesis morphology closer to the physiologic one which may lead to a more resistant repair construct. Introduction. Rotator cuff repair is one of the most common operative procedures in the shoulder. Yet despite its prevalence recurrent tear rates of up to 94% have been reported in the literature. High failure rates have been associated with tendon detachment from bone at the tendon – bone interface. Exogenous agents as biological strategies to augment tendon – bone healing in the shoulder represent a new area of focus to improve patient outcomes. Demineralised bone matrix (DBM) contains matrix bound proteins, exposed through acid demineralization step of DBM manufacture, and has long been recognised for its osteoinductive and osteoconductive properties. We hypothesised that DBM administered to the bone bed prior to the reattachment of the tendon, will upregulate healing and result in enhanced tissue morphology that more closely resembles that of a normal enthesis. An established ovine transosseous equivalent rotator cuff model was used. Methods. Following ethics approval, 10 adult wethers (18 months) were randomly allocated to control, n=4 (without DBM) or DBM, n=6 (DBM administered to bone bed) groups. The infraspinatus tendon was detached from its insertion and repaired in a transosseous equivalent fashion using PEEK suture anchors. In treatment animals 0.25cc of ovine DBM, previously prepared using a modified Urist protocol, was injected into two drill holes within the bony tendon footprint. Animals were culled at 4 weeks following surgery and processed for tissue histology and microcomputed tomography (μCT) endpoints. Results. No infection or tendon detachment following repair was noted in either group. 3D reconstructed images of μCT scans verified correct DBM and suture anchor placement. Histological images demonstrated distinct differences in tissue morphology between the two groups; however there was no evidence of the four – zoned structure characteristic of a healthy tendon bone insertion, in any specimens. In the control group specimens, the tendon midsubstance was highly disorganised with randomly arranged collagen fibres and diminutive areas of fibrocartilage. In the treatment group, large regions between tendon and bone were occupied by fibrocartilage. Within the fibrocartilage region, insertional collagen fibres appeared organised and chondrocytes were orientated in the direction of the insertional collagen fibres. Organised collagen fibre orientation within the tendon midsubstance was observed, though this was not consistent throughout all the specimens. DBM particles were resorbed and trabecular bone occupied the DBM holes. The PEEK anchors were all in direct contact with the ongrowing bone indicating good quality integration and fixation. Discussion. This study showed that DBM augmented tendon to bone repair leads to an upregulated cellular activity resulting in increased amounts of fibrocartilage between the repaired tendon and underlying bone. The upshot of this is an improved tissue organization which more closely resembles the morphology of the normal enthesis. Introduction of osteoinductive DBM at the tendon – bone interface during surgery may reduce failure rates associated with rotator cuff repair and improve clinical outcomes

We used an in vivo model to assess the use of an autogenous cancellous bone block and marrow graft for augmenting tendon reattachment to metallic implants. We hypothesised that augmentation of the tendon-implant interface with a bone block would enable retention of the graft on the implant surface, enhance biological integration, and result in more consistent functional outcomes compared with previously reported morcellised graft augmentation techniques. A significant improvement in functional weight-bearing was observed between six and 12 weeks. The significant increase in ground reaction force through the operated limb between six and 12 weeks was greater than that reported previously with morcellised graft augmented reconstructions. Histological appearance and collagen fibre orientation with bone block augmentation more closely resembled that of an intact enthesis compared with the morcellised grafting technique. Bone block augmentation of tendon-implant interfaces results in more reliable functional and histological outcomes, with a return to pre-operative levels of weight-bearing by 24 weeks

Cell sheets are manufactured from a high-density cell layer stabilized by its own freshly produced extracellular matrix (ECM). They could serve as versatile scaffolds for tissue repair. Unfortunately, their production often remains time-consuming requiring weeks of culturing. Ligament cell sheets are so far barely available. Regarding musculoskeletal tissues exposed to high repetitive biomechanical forces, the stability of cell sheets is insufficient. It could help to combine them with a biomechanical competent scaffold e.g. produced by an embroidering technique. Hence, we wanted to (1) develop a very rapid strategy to produce ACL ligamentocyte sheets within 24 h by using a thermoresponsive polymer surface, (2) use the sheets for scaffold seeding and (3) reflect the fibrocartilaginous transition zone of an ACL enthesis by combining sheets of ligamentocytes with chondrocytes or chondrogenic precursor cells as a strategy for directed seeding of two cell types on topologically different scaffold areas. Different cell numbers of lapine ACL ligamentocytes (L-ACLs), lapine articular chondrocytes (L-ACs) and human mesenchymal stromal cells (H-MSCs) were used for sheet formation. Experiments were performed with novel, self-assembled poly(glycidyl ether) (PGE) brushes based on random glycidyl methyl ether and ethyl glycidyl ether copolymers on polystyrene 12-well cell culture plates, which allow rapid sheet formation within 24 h. Uncoated plates served as controls. Temperature-triggered detachment was performed by 10 min incubation with PBS at ambient temperature before treatment with fresh warm PBS for 5 min at 37 degrees Celsius. Harvested cell sheets were transferred on polyglycolic acid (PGA) or embroidered poly-lactic acid / poly-co-caprolactone (PLA/P[LA-CL]) scaffolds, functionalized with collagen foam and fluorine gas treatment (prepared at the IPF in Dresden and the FILK in Freiberg). Cell distribution, growth, vitality and synthesis of ECM components were monitored up to 7 days. Cell numbers required for sheet preparation (3.9 cm2) depended strongly on the cell type (L-ACLs: 0.395 mio/cm2, L-AC: 0.342 mio/cm2, H-MSCs: 0.131 mio/cm2) and was highest for L-ACLs. The majority of cells survived sheet assembly, detachment, transfer onto the scaffolds and culturing. Cells migrated from the sheets into the scaffolds and spread through the scaffolds. L-ACLs and L-ACs produced ECM and maintained their phenotypes (type II collagen and sulfated glycosaminoglycans in L-AC sheets, decorin and tenascin C in L-ACL sheets). The presence and distribution of two cell types in scaffold cocultures (L-ACLs and H-MSCs) was proven by anti-human vimentin labeling. Hence, the PGE brush surface allows rapid formation (24 h) of cell sheets

Research in to tendon-bone healing techniques focus on increasing bone growth at the interface such as cell or growth factor (e.g. BMP-2) augmentation. Demineralised bone matrix (DBM) is osseoinductive and is in use clinically. Hypothesis: DBM augmentation of a healing tendon-bone interface will result in improved function at 3, 6, 9 and 12 weeks, and a morphology that more closely resembles that of a normal enthesis at 12 weeks. Materials and methods: An ovine patellar tendon model was used. 19 skeletally mature ewes were allocated to the control group or DBM group. In both groups the patellar tendon was detached, and following tibial tubercle osteotomy, was re-attached using 3 suture anchors. In the DBM group a piece of DBM was placed between the tendon and bone. 2 animals were sacrificed at 6 weeks and 6 animals at 12 weeks. Animals underwent force plate analysis at 3, 6, 9 and 12 weeks. The tendon-bone interface length which was fibrous or fibrocartilaginous, and the area of fibrocartilage, mineralised fibrocartilage and new bone was quantified. Results: 3 control group animals (33%) failed within 6 weeks. None failed in the DBM group. The DBM group was significantly better than the control at all time points (p< 0.05). DBM produced a significantly more fibrocartilaginous enthesis than the control group (p< 0.05). Controls were significantly more fibrous than the DBM group (p< 0.05). DBM produced significantly more fibrocartilage (p< 0.05), and mineralised fibrocartilage (p< 0.05). Discussion: 33% of the control group failed within 6 weeks, whilst no failures were observed in the DBM group. DBM animals mobilised earlier and had significantly better function at all time points. Histologically, the DBM group showed a more mature direct type enthesis at earlier time points. Conclusion: DBM augmentation of a healing tendon-bone interface enhances functional and morphological recovery at earlier time points

Due to unsatisfactory results and reported drawbacks of anterior cruciate ligament (ACL) reconstruction new regenerative approaches based on tissue-engineering strategies are currently under investigation. It was the purpose of this study to determine if a novel silk fiber-based ACL scaffold is able to initiate osteointegration in the femoral and tibial bone tunnels under in vivo conditions. Furthermore we tested if the osteointegration process will be improved by intraoperatively seeding the scaffolds with the autologous stromal vascular fraction, an adipose-derived, stem cell-rich isolate from knee fat pads. In this controlled laboratory study, 33 sheep underwent ACL resection and were then randomly assigned to 2 experimental groups: ACL reconstruction with a scaffold alone and ACL reconstruction with a cell-seeded scaffold. Half of the sheep in each group were randomly chosen and euthanized 6 months after surgery and the other half at 12 months. To analyze the integration of the silk-based scaffold in the femoral and tibial bone tunnels, hard tissue histology and micro-computed tomography measurements were performed. The histological workup showed that in all treatment groups, with or without the application of the autologous stromal vascular fraction, an interzone of collagen fibers had formed between bone and silk-based graft. This collagen-fiber continuity partly consisted of Sharpey fibers, comparable with tendon-bone healing known for autografts and allografts. Insertion sites were more broad based at 6 months and more concentrated on the slightly protruding, bony knoblike structures at 12 months. Histologically, no differences between the treatment groups were detectable. Analysis of micro-computed tomography measurements revealed a significantly higher tissue density for the cell-seeded scaffold group as compared with the scaffold-alone group in the tibial but not femoral bone tunnel after 12 months of implantation. The novel silk fiber-based scaffold for ACL regeneration demonstrated integration into the bone tunnels via the formation of a fibrous interzone similar to allografts and autografts. Histologically, additional cell seeding did not enhance osteointegration. No significant differences between 6 and 12 months could be detected. After 12 months, there was still a considerable amount of silk present, and a longer observation period is necessary to see if a true ligament-bone enthesis will be formed

Introduction: Functional outcome following proximal tibial replacement can be impaired by extensor mechanism inefficiency. Current methods used to re-attach the patellar tendon result in varied levels of extensor function. Successful attachment of the patellar tendon requires initial mechanical stability and long-term biological fixation. We have employed a prosthesis, to model patellar tendon re-attachment, to test the hypothesis that biological augmentation of an implant which can provide sufficient mechanical integrity will allow a tendon-implant interface to develop that is similar in function and morphology to a normal tendon-bone interface. Methods: The right patellar tendon in 24 Skeletally mature Friesland ewes was transfixed between the interlocking spikes of a hydroxyapatite-coated, customized tendon clamp to simulate patellar tendon re-attachment to a proximal tibial replacement. In 12 animals (Autograft group) the clamp attachment was augmented with autologous cancellous bone and marrow graft harvested from the ipsilateral iliac crest at the time of surgery, whilst the remaining animals (HA group) served as un-supplemented controls. Functional outcome was assessed using force plate measurements and two-dimensional optical kinematic gait analysis. Animals were euthanised at 6 and 12 weeks. The specimens were harvested, processed for histology and examined using light microscopy. Results: The clamp device provided sufficient mechanical fixation of the patellar tendon to allow immediate weight bearing. Gait analysis showed that the range of movement of the stifle (knee) joint was not compromised by the surgical intervention at 6 or 12 weeks post-operation. An extensor lag observed at 6 weeks in both the Autograft and HA group was seen to fully recover by 12 weeks post-operation. There was a significant increase in functional weight bearing through the operated limb of the Autograft group animals between 6 and 12 weeks, which was not observed in the HA group. The tendon-implant interface in the HA group animals showed a fibrous tissue encapsulation of the HA coated surface, with collagen fibrils running parallel to the implant surface. In the Autograft group at 6 weeks post-operation a soft tissue – bone – HA interface had developed, similar in morphology to that of an indirect-type enthesis. Perpendicular orientated Sharpey’s-like fibres were observed spanning the region between the tendon and the HA coated implant and the bone graft material was seen to be undergoing active remodelling. By 12 weeks post-operation the interface was layered with regions of fibrocartilage clearly visible, more closely resembling the morphology of a direct-type enthesis. Discussion: The clamp device provided sufficient mechanical fixation of the patellar tendon to allow immediate use the operated limb. The incorporation of a bio-active implant coating and biological augmentation encouraged a neo-enthesis to develop with near normal functional properties, and morphology similar to that of a normal patellar tendon-bone direct-type enthesis

We developed an in vivo model of the attachment of a patellar tendon to a metal implant to simulate the reconstruction of an extensor mechanism after replacement of the proximal tibia. In 24 ewes, the patellar tendon was attached to a hydroxyapatite (HA)-coated titanium prosthesis. In 12, the interface was augmented with autograft containing cancellous bone and marrow. In the remaining ewes, the interface was not grafted. Kinematic gait analysis showed nearly normal function of the joint by 12 weeks. Force-plate assessment showed a significant increase in functional weight-bearing in the grafted animals (p = 0.043). The tendon-implant interface showed that without graft, encapsulation of fibrous tissue occurred. With autograft, a developing tendon-bone-HA-implant interface was observed at six weeks and by 12 weeks a layered tendon-fibrocartilage-bone interface was seen which was similar to a direct-type enthesis. With stable mechanical fixation, an appropriate bioactive surface and biological augmentation the development of a functional tendon-implant interface can be achieved

This study aimed to explore the relationship between the geometry of the tuberosity located superior to the Achilles tendon enthesis and the thickness of its fibro-cartilaginous periosteum. The tuberosity acts as a pulley for the tendon during dorsiflexion of the foot and is thus compressed by the overlying tendon. This can result in pressure-related injuries which account for a significant number of Achilles-related problems among sportsmen or women. We postulated that variations in the contact area between the tendon and the tuberosity (and consequently the pressure exerted by the tendon) affects the periosteum thickness. Here, we report four methods of portraying the two dimensional geometry of the superior tuberosity. Material was obtained from 10 elderly dissecting room cadavers donated to the Cardiff University for anatomical examination and prepared for routine histology. Serial sagittal sections were collected at 1 mm intervals, and stained with Masson’s trichrome, toluidine blue and haematoxylin & eosin. In the first method, the area of the bursal cavity was measured between the deep surface of the tendon and the tuberosity within a 9mm radius of the proximal part of the attachment site. The second technique was similar, though used the long axis of the tendon as a reference, rather than its deep surface. The third technique measured the area of the tuberosity within 20 degrees of the tendon long axis. The final technique measured the cumulative gradient of the first 5 mm of the tuberosity, with reference to the tendon long axis. The periosteum thickness was measured at 500 μm intervals from the proximal part of the enthesis and mean values calculated. A good correlation was seen between all techniques, with the tuberosities having the most localised area of contact with the tendon, showing the thickest periosteum

Summary. Our study shows that a tendon rupture can be successfully augmented with Demineralised Cortical Bone (DCB) giving initial appropriate mechanical strength suitable for in vivo use providing the biological reactions to the graft are favourable. Introduction. Treatment of tendon and ligament injuries remains challenging; the aim is to find a biocompatible substance with mechanical and structural properties that replicate those of normal tendon and ligament. Because of its structural and mechanical properties, we proposed that DCB can be used in repair of tendon and ligament as well as regeneration of the enthesis. DCB is porous, biocompatible and has the potential to be remodelled by the host tissues. 2 studies were designed; in the first we examined the mechanical properties of DCB after gamma irradiation (GI) and freeze drying (FD). In the second we used different techniques for repairing bone-tendon-bone with DCB in order to measure the mechanical performance of the construct. Methods. In the first study we allocated the DCB specimens into 4 groups; group-A non-freeze dried non-gamma irradiated, group-B freeze dried non-gamma irradiated, group-C non-freeze dried gamma irradiated and group-D freeze dried and gamma irradiated. The 4 groups were tested for maximum tensile strength. In the 2nd study, patella - patellar tendon - tibia construct of mature ewes were harvested and the distal 1cm of the patellar tendon was excised, 4 models of repair were tested;. • Model-1, DCB was used to bridge the gap between the tendon and the tibial tuberosity. The DCB strip was stitched to the tendon using one bone anchor. • Model-2, similar to model-1 with the use of 2 bone anchors. • Model-3, similar to model-2, construct was offloaded by Fiberwire continuous thread looped twice through bony tunnels sited in the patella and in the tibial tuberosity. • Model-4, similar to model-3 with 3 hand braided fiberwire threads as offloading loop. All 4 models were tested until failure and force displacement curves used to investigate the structural properties of the reconstruction. Results. The Median of maximum tensile force for group-A was 218N [95%C.I.=147.9–284.7N], group-B was 306N [95%C.I.=154.1–488.6N], group-C was 263N [95%C.I.=227.8–315.6N], group-D was 676N [95%C.I.=127-1094.9N]. Group-D results were statistically higher (p=<0.05) compared to group-A and group-C, while there was no statistical significance compared to group-B. The median failure force for model-1 was 250N, (95%C.I.=235-287), model-2 was 290N (95%C.I.=197-396), model-3 was 767N (95%C.I.=730-812) and for model-4 was 934N (95%C.I.=867-975). There was no statistical significance between model-1 and model-2 (p=0.249), however statistical significance was found between other models (p=<0.006). Discussion. Demineralised Bone is widely used as a bone graft substitute and may be used to augment bone formation in load bearing applications. In this study we focus on the potential use of demineralised bone in ligament and tendon repair. A previous animal study by our group found that the use of demineralised bone can enhance healing of the enthesis. Other published studies suggested the possibility of using DCB as ligament substitute. We examined the effect of gamma radiation as the most common sterilisation technique in medical field and the freeze drying as a possible technique for long term storage on the tensile strength of the DCB

Tendon is a bradytrophic and hypovascular tissue, hence, healing remains a major challenge. The molecular key events involved in successful repair have to be unravelled to develop novel strategies that reduce the risk of unfavourable outcomes such as non-healing, adhesion formation, and scarring. This review will consider the diverse pathophysiological features of tendon-derived cells that lead to failed healing, including misrouted differentiation (e.g. de- or transdifferentiation) and premature cell senescence, as well as the loss of functional progenitors. Many of these features can be attributed to disturbed cell-extracellular matrix (ECM) or unbalanced soluble mediators involving not only resident tendon cells, but also the cross-talk with immigrating immune cell populations. Unrestrained post-traumatic inflammation could hinder successful healing. Pro-angiogenic mediators trigger hypervascularization and lead to persistence of an immature repair tissue, which does not provide sufficient mechano-competence. Tendon repair tissue needs to achieve an ECM composition, structure, strength, and stiffness that resembles the undamaged highly hierarchically ordered tendon ECM. Adequate mechano-sensation and -transduction by tendon cells orchestrate ECM synthesis, stabilization by cross-linking, and remodelling as a prerequisite for the adaptation to the increased mechanical challenges during healing. Lastly, this review will discuss, from the cell biological point of view, possible optimization strategies for augmenting Achilles tendon (AT) healing outcomes, including adapted mechanostimulation and novel approaches by restraining neoangiogenesis, modifying stem cell niche parameters, tissue engineering, the modulation of the inflammatory cells, and the application of stimulatory factors.

Cite this article: Bone Joint Res 2022;11(8):561–574.

Hypertrophy of lumbar articular facets and dorsal joint capsule are well documented in degenerative instability, the molecular changes occurring in the extracellular matrix (ECM) are however unknown. The L4/L5 posterior articular complex was removed from seven individuals undergoing fusion for degenerative instability. After methanol fixation and decalcification in EDTA, specimens were cryosectioned at 12 μm and immunolabelled with monoclonal antibodies for collagen types I, II, III, V and VI; chondroitin-4 and 6 sulphates; dermatan and keratan sulphate; versican, tenascin, aggrecan and link-protein. Antibody binding was detected using the Vectastain ABC ‘Elite’ kit. Labelling patterns were compared to corresponding healthy specimens examined previously. In comparison, the degenerative capsule was more dense and hypertrophied and the enthesis more fibrocartilaginous, with immunolabelling extensive for collagen type II, chondroitin–6-sulfate, chondroitin-4-sulfate, aggrecan and link-protein. The articular surface showed extensive evidence of degeneration, while the thickened capsular entheses encircled the articular facets dorsally. Bony spurs capped with regions of cartilaginous metaplasia were prominent in this region, the ECM labelling strongly for type II collagen and chondroitin-6-sulfate. The hypertrophy of lumbar facet joints subject to instability of the functional spinal unit therefore appears to be due to proliferation of the capsular enthesis rather than the actual articular facet. In view of the physiological function of the dorsal joint capsule as a wrap-around ligament in assisting the limitation of axial rotation, the molecular changes found in degenerative instability suggest rotational instability, such as results from degenerative disc disease, to be a decisive factor in the development of spondylarthropathy. It is furthermore probable, that the pronounced sagittal joint orientation in degenerative instability is the result of reactive joint changes rather than a predisposing factor of instability

Introduction. Kager's fat pad (KFP) is located in Kager's triangle between the Achilles tendon (AT), the superior cortex of the calcaneus and Flexor Hallucis Longus (FHL) muscle & tendon. Although the biomechanical functions of KFP are not yet fully understood, a number of studies suggested that KFP performs important biomechanical roles including assisting in the dynamic lubrication of the AT subtendinous area, protection of AT vascular supply, and load and stress distribution within the retrocalcaneal bursa space. Similar to the knee meniscus, KFP has become under increasing investigations since strong indications were found that it serves more than just a space filler. Both KFP and the knee meniscus are anchored to their surrounding tissues via fibrous attachments, they can be found in encapsulated (or ‘air tight’) regions, lined by synovial membranes, and they both slide within their motion ranges. The protruding wedge (PW) of KFP was observed to slide in and out of the retrocalcaneal bursal space during ankle plantarflexion and dorsiflexion, respectively. In-vitro studies of KFP suggest that it reduces the load by 39%, which is similar to that of the knee meniscus (30%-70% of the load applied on the knee joint). This study investigated the in-vivo load bearing functionality of KFP. Materials and Methods. The ankles of 5 volunteers (3 males, 2 females, Age 20-28, BMI 21-26) were scanned using a 0.2T MRI scanner at ankle plantarflexion and neutral positions. The ankles of 2 of those volunteers were later scanned using a 3T MRI scanner for higher accuracy. The areas and volumes of KFP were measured using Reconstruct¯ 3D modelling software. The hind foot of the volunteers were scanned using dynamic ultrasound to measure in-vivo real time shape changes of PW. Results. The cross sectional area of KFP in the AT midline saggital plane increased on average by 10% when ankles were changed from neutral to plantarflexion positions. The volume of KFP showed less variation than cross sectional areas (3.9% variation in volume). Previous studies showed the cross sectional area of the knee meniscus changes by 9.8% during loading, or flexing the knee by 90°, and its volume was reduced by 3.5%-5.9% (medial and lateral menisci respectively). Ultrasound imaging showed that PW's thickness decreased during dorsiflexion compared to plantarflexion by an average of 1mm and a hysteresis was found between the location of PW's tip and the insertion angle of AT, suggesting the fibrous tip of PW bears load during dorsiflexion. Discussions and conclusions. The similarities in results between the knee meniscus (literature review) and KFP supports hypotheses that KFP assists in reducing the load applied at the AT enthesis organ. Furthermore, histological studies showed a fibrosis is evident at the tip of PW, which is thought to be developed as a result of resisting external loading. This also supports speculations that KFP removal is likely to reduce lubrication, pressure distribution, load bearing, and consequently, increasing the tear and wear level within AT enthesis

Aims

Adenosine, lidocaine, and Mg²⁺ (ALM) therapy exerts differential immuno-inflammatory responses in males and females early after anterior cruciate ligament (ACL) reconstruction (ACLR). Our aim was to investigate sex-specific effects of ALM therapy on joint tissue repair and recovery 28 days after surgery.

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.

Tendon detachment from its bony insertion is one of the most frequent injuries occurring in the musculoskeletal interface, constituting an unmet challenge in orthopaedics. Tendon-to-bone integration occurs at the enthesis, which is characterized by a complex structure organized in a gradient of cells and microenvironments. Hence, the maintenance of a heterotypic cellular niche is critical for tissue functionality and homeostasis. Replicating this unique complexity constitutes a challenge when addressing tendon-to-bone regeneration and interfacial tissue engineering strategies. Currently, mechanisms presiding to tendon-to-bone interface healing are not yet fully understood, particularly the interactions between tendon and bone cells in the orchestration of interfacial repair versus regeneration. Therefore, this study focused on the hypothesis that interactions between human tendon-derived cells (hTDCs) and pre-osteoblasts (pre-OB) can initiate a cascade of events, potentially leading to interfacial regeneration. Thus, hTDCs and pre-OB (pre-differentiated human adipose-derived stem cells) were used. Herein, five different ratios between basal and osteogenic media (100:0,75:25,50:50,25:75,0:100) were assessed to estimate their influence on cell behaviour and identify the ideal parameters for simultaneously supporting tenogenic and osteogenic differentiation before establishing a co-culture. Tenogenic and osteogenic differentiation were assessed through the expression of tendon and bone markers, mineralization (alizarin red, AZ) and alkaline phosphatase (ALP) quantification. Results showed that hTDCs exhibited osteogenic differentiation potential when cultured in the presence of osteogenic media, as demonstrated by an increase in ALP activity and mineralization. Pre-OB expressed osteogenic markers (OCN, OPN) in all media conditions confirming osteogenic commitment, which was simultaneously confirmed by ALP levels and AZ staining. Thus, three different conditions (100:0, 50:50, 0:100) were chosen for further studies in a direct contact co-culture system. Similarly to single cultures, a significant proliferation was observed in all conditions and mineralization was increased as soon as 7 days of culture. Additionally, osteogenic, tenogenic and interface-relevant markers will be assessed to study the effect of co-culture on phenotype maintenance. In summary, the present work addresses major limitations to clinical translation of cell-based therapies aiming at promoting interfacial regeneration. Particularly, we explored the influence of culture media on the maintenance of tenogenic and osteogenic niches, taking a basic and critical step towards the establishment of more complex cell-based systems. Acknowledgements. Authors thank Fundação para a Ciência e Tecnologia in the framework of FCT-POPH-FSE, SFRH/BD/96593/2013 (RCA) and IF/00593/2015 (MEG); and to FCT/MCTES and the FSE/POCH, PD/59/2013 for PD/BD/128088/2016 (IC)

Subscapularis repair and integrity after a primary total shoulder arthroplasty is critical for successful outcomes. One should be familiar with the 3 basic takedown and repair techniques commonly utilised. Subscapularis repair after reverse shoulder arthroplasty is not as critical and in some cases may be detrimental to return of external rotation strength and motion. Subscapularis tenotomy: The tendon is incised approximately 1 cm from the lesser tuberosity and an oblique incision is created from proximal lateral to distal medial stopping at the sentinel vessels. A combination of tendon-to-tendon figure of 8 sutures. Lesser tuberosity osteotomy: This approach is helpful not only in obtaining a bone-to-bone healing, but also in the exposure. Osteotomies range from a fleck of bone in patients with minimal deformity, to a C-shaped osteotomy including part of the head which facilitates exposure of the posterior glenoid. Despite an ability to document radiographic healing of the lesser tuberosity fragment, this technique does not prevent fatty infiltration of the subscapularis. Subscapularis Peel: This repair requires tendon healing to bone and probably incomplete, in most cases, reconstitution of a normal enthesis. External rotation can be gained by recessing the subscapularis insertion medially with the arm in external rotation. While bone-to-tendon sutures are the gold standard, augmentation of the sutures using a prosthesis as the anchor has led to the development of prostheses with multiple holes. Dual row repair of the tendon, however, may lead to medial rupture

Injuries to the quadriceps muscle group are common in athletes performing high-speed running and kicking sports. The complex anatomy of the rectus femoris puts it at greatest risk of injury. There is variability in prognosis in the literature, with reinjury rates as high as 67% in the severe graded proximal tear. Studies have highlighted that athletes can reinjure after nonoperative management, and some benefit may be derived from surgical repair to restore function and return to sport (RTS). This injury is potentially career-threatening in the elite-level athlete, and we aim to highlight the key recent literature on interventions to restore strength and function to allow early RTS while reducing the risk of injury recurrence. This article reviews the optimal diagnostic strategies and classification of quadriceps injuries. We highlight the unique anatomy of each injury on MRI and the outcomes of both nonoperative and operative treatment, providing an evidence-based management framework for athletes.

Cite this article: Bone Joint J 2023;105-B(12):1244–1251.

In this histological study, the canine infraspinatus tendons were repaired to different bone surfaces: 1, a tendon end adjacent to the insertion: 2, a calcified fibro-cartilage layer; 3, a cancellous surface. Tendon repair to tendon ends restored the four-layered enthesis in the healing period, whereas tendon repair to the calcified fibrocartilage considerably delayed fiber development into bone. Fiber connection to cancellous surface developed according to the remodeling of trabecular bone by 12 to 16 postoperative weeks. Therefore, ruptured tendons should be attached to the remaining end or to a cancellous surface; they should not be attached to a calcified fibrocartilage layer

Introduction. Tendon injuries remain challenging, secondary healing and prolonged immobilisation result in suboptimal outcome. Previous study by our group showed that demineralised bone matrix (DBM) can result in faster healing of a tendon enthesis. The aim of this study is to test different ways augmenting tendon with DBM to enhance tendon repair and regeneration. Methods. DBM strips were prepared from tibias of mature ewes. Patella, patellar tendon and tibias were dissected and the distal 1 cm of the patellar tendon was excised. 4 models were designed;. Model-1, DBM strip was used to bridge the gap between the tendon and the tibial tuberosity. The DBM strip was stitched to the tendon using one bone anchor. Model-2, similar to model 1 with the use of 2 anchors. Model-3, similar to model 2, construct was off loaded by continuous thread looped twice through bony tunnels sited in the patella and in the tibial tuberosity. Model-4, similar to model 3 with 3 threads as off loading loop. All models were tested for pullout force and mode of failure. Results. The median failure force for model-1 (N=5) was 250N while for model-2 (N=5) was 290N. In model-3 and model-4 failure of the off loading loop was used as end point, 6 samples were tested in each model. Median failure force of model-3 was 767N and for model-4 was 934N. There was no statistical significance between model-1 and model-2 (p=0.249), however statistical significance was found between other models (p=< 0.006). Discussion. A study published in 1996 proved that cortical DBM can be used as ACL graft with evidence of ligamentisation. DBM provides a biologic scaffold with potential for use as ligament and tendon replacement. Our study shows that a tendon rupture can be augmented with DBM giving intial appropriate mechanical strength suitable for in-vivo use

Aim. To determine the effects of knee and ankle position on tendo Achilles (TA) gap distance in patients with acute rupture using ultrasound. Methods. Twenty seven patients with twenty-eight acute complete TA ruptures confirmed on ultrasound were recruited within a week of injury. The mean age at presentation was 42 years (range 23-80 years). Ultrasound measurements included location of the rupture and the gap distance between the superficial tendon edges with the ankle in neutral and knee extended. The gap distance was sequentially measured with the foot in maximum equinus and 0°, 30°, 60° and 90° of knee flexion. Results. The mean distance of the rupture from the enthesis was 52mm (range: 40-76mm). The mean gap distance with the ankle in neutral and knee extended was 11.4mm (95%CI: 9.9, 12.9). This reduced to 4.8mm (95%CI: 3.3, 6.4) when the foot was in maximum equinus, 3.8mm (95%CI: 2.5, 5.1) with 30° knee flexion, 2.6mm (95%CI: 1.7, 3.6) with 60° knee flexion and 1.5mm (95% CI: 0.8, 2.2) with 90° knee flexion. Expressing the reduction in gap distance with each successive position as a percentage of the gap distance with the knee extended and ankle in neutral revealed a mean reduction of 58.9%, 66.9%, 77.1% and 85.9% with maximum equinus and 0°, 30°, 60° and 90° of knee flexion respectively. The difference in gap distance between each of these positions was statistically significant (p<0.05). Discussion. Maximum equinus alone significantly reduces the gap distance in acute TA rupture. Increasing knee flexion demonstrates a lesser effect throughout the initial 90° arc suggesting apposition is encouraged by use of a below knee maximum equinus cast permitting full knee flexion

Introduction: In a previous study (. Hayes et al., 2007. )we reported that novel chondroitin sulphate (CS) sulphation motifs on cell-associated proteoglycans (PGs) may be putative biomarkers of progenitor/stem cell sub-populations resident within the superficial zone of articular cartilage (. Dowthwaite et al., 2005. ). In this study, using the same panel of antibodies, we examine the distribution of novel CS sulphation epitopes in a more clinically relevant model – the developing human knee joint. Methods: Twelve-14 week human foetal knee joint rudiments were processed into paraffin wax then de-waxed and immunoperoxidase-stained with mAbs 3B3(−), 7D4 and 4C3 using the Vector Universal Elite kit with Nova Red, Mayers Haematoxylin, mounted under coverslips and then photographed. Results: All three CS sulphation motif epitopes localised prominently at sites of incipient articular cartilage formation at a stage before there was any histological evidence of secondary ossification at the epiphysis. Interestingly, these CS epitopes were also detectable in very defined regions within the perichondrium; growth plate; the fibrocartilage of both meniscus and enthesis; vasculature; and at sites of capillary invasion, with subtle differences in their distribution; for example, 3B3(−) identified the cellular lining of cartilage canals within the epiphyses, whereas 7D4 labelled more their cellular contents. Discussion: The results of this study show that novel CS sulphation motifs on cell and matrix PGs play important and diverse roles in the development of a wide range of musculoskeletal connective tissues, including articular cartilage. We hypothesize that the unique sulphation sequences on CS-containing PGs are involved in regulating cell proliferation and differentiation events, through interaction with soluble signalling molecules (e.g. growth factors) in the extracellular milieu. These antibodies show considerable promise for uses in tissue engineering applications for identifying and sorting stem/progenitor cells for regeneration of musculoskeletal tissues

In the treatment of ligament injuries there has been much interest in the restoration of the actual ligament anatomy, and the extent to which the original enthesis may be re-established. This study therefore seeks to uncover new information on ligament microstructure and its insertion into bone. Five bovine medial collateral ligaments (MCL) and five ovine anterior cruciate ligaments (ACL) were used in this study. All ligaments were harvested with the femoral and tibial bony insertions still intact. The bone ends were clamped and the MCL stretched to about 10% strain while the ACL underwent a 90° twist. The entire ligament-bone system, under load, was fixed in 10% formalin solution for 12 hours, following which it was partially decalcified to facilitate microsectioning. Thin 30 ìm-thick sections of the ligament-bone interface and ligament midsubstance were obtained. Differential Interference Contrast (DIC) optical microscopy was used to image the ligament and bone microarchitecture in the prescribed states of strain. Fibre crimp patterns were examined for the prescribed loading condition and showed distinct sections of fibre recruitment. Transverse micro-imaging of the ligament showed a significant variation in the sub-bundle cross-sectional area, ranging from 100ìm to 800 ìm. Those bundles closer to the central long axis of the ligament were numerous and small, while moving towards the periphery, they were large and singular. Both classifications of entheses, direct and indirect, were observed in the MCL insertions into the femur and tibia respectively. Of interest was the indirect insertion where the macro-level view of the near parallel attachment of fibres to bone via the periosteum was revealed, at the microscale, to involve a gradually increasing orthogonal insertion of fibres. This unique transition occurred closer to the joint line. In the ACL the anterior-medial (AM) and posterior-lateral (PL) bundles were easily discernable. All insertions into bone for the ACL were of the direct type. Fibres were thus seen to transition through the four zones of gradual mineralization to bone. However the manner in which the AM and PL bundles insert into bone, and the lateral soft tissue transition between these two bundles, revealed a structural complexity that we believe is biomechanically significant. This ‘mechano-structural’ investigation, using novel imaging techniques, has provided new insights into the microstructure of the ligament bone system. The images presented from this study are aimed to aid new approaches for reconstruction, and provide a blue-print for the design of ligament-bone systems via tissue engineering

Aims

In the native hip, the hip capsular ligaments tighten at the limits of range of hip motion and may provide a passive stabilizing force to protect the hip against edge loading. In this study we quantified the stabilizing force vectors generated by capsular ligaments at extreme range of motion (ROM), and examined their ability to prevent edge loading.

Sutures from intraosseous anchors are used to secure soft tissue down onto bone during healing. Increasingly anchors are made from absorbable materials. Poly lactide carbonate (PLC - poly lactide with calcium carbonate) is an absorbable formulation with osteoconductive properties that should enhance both tissue healing and its own replacement by bone over time. An animal model of soft-tissue-to-bone healing was used to assess the efficacy of PLC Bioraptor™ anchors in comparison to anchors of non-osteoconductive poly lactide (PLLA). Forty-seven ewes were used in two groups of PLC or PLLA anchors, surviving to either four or 12 weeks. The patellar tendon was pared off the tibia, the footprint decorticated then the tendon re-attached. An external fixator protected the tendon from load bearing for three weeks. At post mortem the patella/patellar tendon/tibia complex was either prepared for histological examination or stored deep frozen for later measurement of peak load at failure. Non-operated specimens failed within the tendon mid substance; the failure site of healing specimens was dependent on their strength, with the weakest through interpositional granulation tissue, stronger specimens through fibres at various distances from the bone and the strongest, by partial bone avulsion. Active healing of the enthesis consisted of merging regions of. a) re-established cortical bone plate;. b) advancing mineralization of new, oriented collagen;. c) dense, cellular collagen parallel to the tendon axis. Thin new bone was present around both PLLA and PLC anchors. Healing tissues held by PLC suture anchors, were significantly stronger by 12 weeks than those held by PLLA anchors, possibly due to the calcium carbonate in the PLC anchor. However, the macroscopic and microscopic appearances of the healing tissues seemed little different between the two groups. This study indicates that PLC is a suitable replacement for PLLA in the fabrication of suture anchors. As well as its ability to produce stronger healing tissues, PLC has a shorter longevity in vivo and longer term is replaced by bone

Insertional Achilles tendonitis is an inflammatory disorder affecting mainly active young patients. The etiology is multifactorial and include the combination of anatomical and biomechanical characteristics. One fifth of the tendon injuries in athletes are insertional complaints which includes bursitis and insertion tendinitis.The complex of the insertion of the Achilles tendon includes three main components of fibrocartilage sesamoid, periosteum and enthesis. A conservative regime is recommended as the first line of treatment. In case of failure a surgical decompression of the posterior margin of the calcaneum is indicated. Nine cadaveric legs were used for the experiment. The leg was mounted on an MTS machine and was axially loaded 360 N. The foot was attached to a plate which enabled dorsal and plantar flexion. The Achilles was sutured twice in an Ethibond No. 5 using the Krakow technique in order to anchor the tendon to an actuator. A thin pressure sensor plate (Teckscan) was inserted into the retrocalcaneal bursa to measure the force, pressure and contact area of the Achilles to the calcaneus in various positions of the foot. The conditions included 90 degrees of the foot, 15 and 30 degrees of dorsiflexion while the tension that was applied on the Achilles was 0, 200 N and 300 N. After resection of the posterior surface of the calcaneus in a 20 degrees inclination. The mean peak force, pressure and area did not change in Achilles tensioning while the foot was in 90 degrees and were close to zero. In 15 degrees of dorsiflexion there was increase in the mean peak force, pressure and area when the Achilles was tensed to 200 and 300 Newton. Larger increase in these parameters was achieved by further dorsiflexion of the foot to 30 degrees. After resection of the posterior margin of the calcaneus in an angle of 20 degrees the mean peak force, pressure and area dropped close to zero and remained almost unchanged during the various conditions of the experiment. Dorsiflexion and tension of the Achilles tendon increases the mean peak force, pressure and area in the Achilles retrocalcaneal bursa. These data may explain the mechanism for insertional Achilles tendinosis. Resection of the posterior surface of the calcaneus in 20 degrees efficiently decompresses the retrocalcaneal bursa in various angles of the foot and in various tensions of the Achilles

Aims

To evaluate graft healing of decellularized porcine superflexor tendon (pSFT) xenograft in an ovine anterior cruciate ligament (ACL) reconstruction model using two femoral fixation devices. Also, to determine if pSFT allows functional recovery of gait as compared with the preoperative measurements.

Objectives

The long head of the biceps (LHB) is often resected in shoulder surgery and could therefore serve as a cell source for tissue engineering approaches in the shoulder. However, whether it represents a suitable cell source for regenerative approaches, both in the inflamed and non-inflamed states, remains unclear. In the present study, inflamed and native human LHBs were comparatively characterized for features of regeneration.

Aims

The success of anterior cruciate ligament reconstruction (ACLR) depends on osseointegration at the graft-tunnel interface and intra-articular ligamentization. Our aim was to conduct a systematic review of clinical and preclinical studies that evaluated biological augmentation of graft healing in ACLR.

Aims

Although we often encounter patients with an aortic aneurysm who also have diffuse idiopathic skeletal hyperostosis (DISH), there are no reports to date of an association between these two conditions and the pathogenesis of DISH remains unknown. This study therefore evaluated the prevalence of DISH in patients with a thoracic aortic aneurysm (AA).

Objectives

To compare the effect of femoral bone tunnel configuration on tendon-bone healing in an anterior cruciate ligament (ACL) reconstruction animal model.

Objectives

The aim of this study was to investigate the effect of hyperglycaemia on oxidative stress markers and inflammatory and matrix gene expression within tendons of normal and diabetic rats and to give insights into the processes involved in tendinopathy.

Objectives

Emerging evidence indicates that tendon disease is an active process with inflammation that is critical to disease onset and progression. However, the key cytokines responsible for driving and sustaining inflammation have not been identified.

We examined the mechanical properties of Vicryl (polyglactin 910) mesh in vitro and assessed its use in vivo as a novel biomaterial to attach tendon to a hydroxyapatite-coated metal implant, the interface of which was augmented with autogenous bone and marrow graft. This was compared with tendon re-attachment using a compressive clamp device in an identical animal model. Two- and four-ply sleeves of Vicryl mesh tested to failure under tension reached 5.13% and 28.35% of the normal ovine patellar tendon, respectively. Four-ply sleeves supported gait in an ovine model with 67.05% weight-bearing through the operated limb at 12 weeks, without evidence of mechanical failure.

Mesh fibres were visible at six weeks but had been completely resorbed by 12 weeks, with no evidence of chronic inflammation. The tendon-implant neoenthesis was predominantly an indirect type, with tendon attached to the bone-hydroxyapatite surface by perforating collagen fibres.

Objectives

Although many clinical and experimental investigations have shed light on muscle atrophy and intramuscular accumulation of fat after rotator cuff disruption, none have reported on their onset in the absence of muscle retraction.

Objectives

All-suture anchors are increasingly used in rotator cuff repair procedures. Potential benefits include decreased bone damage. However, there is limited published evidence for the relative strength of fixation for all-suture anchors compared with traditional anchors.

Objectives

The effects of disease progression and common tendinopathy treatments on the tissue characteristics of human rotator cuff tendons have not previously been evaluated in detail owing to a lack of suitable sampling techniques. This study evaluated the structural characteristics of torn human supraspinatus tendons across the full disease spectrum, and the short-term effects of subacromial corticosteroid injections (SCIs) and subacromial decompression (SAD) surgery on these structural characteristics.

Lateral epicondylitis, or ’tennis elbow’, is a common condition that usually affects patients between 35 and 55 years of age. It is generally self-limiting, but in some patients it may continue to cause persistent symptoms, which can be refractory to treatment. This review discusses the mechanism of disease, symptoms and signs, investigations, current management protocols and potential new treatments.

Cite this article: Bone Joint J 2013;95-B:1158–64.

Introduction

The pathogenesis of rotator cuff disease (RCD) is complex and not fully understood. This systematic review set out to summarise the histological and molecular changes that occur throughout the spectrum of RCD.

We compared time-dependent changes in the biomechanical properties of single-and double-row repair of a simulated acute tear of the rotator cuff in rabbits to determine the effect of the fixation techniques on the healing process.

A tear of the supraspinatus tendon was created in 80 rabbits which were separated into two equal groups. A single-row repair with two suture anchors was conducted in group 1 and a double-row repair with four suture anchors in group 2. A total of ten intact contralateral shoulder joints was used as a control group. Biomechanical testing was performed immediately post-operatively and at four and eight weeks, and histological analysis at four and eight weeks.

The mean load to failure in group 2 animals was greater than in group 1, but both groups remained lower than the control group at all intervals. Histological analysis showed similar healing properties at four and eight weeks in both groups, but a significantly larger number of healed tendon-bone interfaces were identified in group 2 than in group 1 at eight weeks (p < 0.012).

The ultimate load to failure increased with the number of suture anchors used immediately post-operatively, and at four and eight weeks. The increased load to failure at eight weeks seemed to be related to the increase in the surface area of healed tendon-to-bone in the double-row repair group.