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

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.

Aims. Studies on long-term patient-reported outcomes after open surgery for triangular fibrocartilage complex (TFCC) are scarce. Surgeons and patients would benefit from self-reported outcome data on pain, function, complications, and satisfaction after this surgery to enhance shared decision-making. The aim of this study is to determine the long-term outcome of adults who had open surgery for the TFCC. Methods. A prospective cohort study that included patients with open surgery for the TFCC between December 2011 and September 2015. In September 2020, we sent these patients an additional follow-up questionnaire, including the Patient-Rated Wrist Evaluation (PRWE), to score satisfaction, complications, pain, and function. Results. A total of 113 patients were included in the analysis. At ≥ 60 months after an open TFCC reinsertion, we found a mean PRWE total score of 19 (SD 21), a mean PRWE pain score of 11 (SD 11), and a PRWE function score of 9 (SD 10). The percentage of patients obtaining minimum clinically important difference rose from 77% at 12 months to 83% at more than 60 months (p < 0.001). Patients reported fewer complications than surgeons, and overall complication rate was low. Conclusion. Outcomes of patient-reported pain, function scores, and satisfaction are improved five years after open surgery for the TFCC. Cite this article: Bone Jt Open 2021;2(11):981–987

Bone tendon junction (BTJ) healing after injury is often slow, without restoration of fibrocartilage transition zone. Fibrocartilage formation has been observed near articular cartilage. It was hypothesised that articular cartilage interposition could stimulate fibrocartilage transition zone regeneration and improve BTJ healing. Partial patellectomy repair was performed in goat. Articular cartilage harvested from excised patella segment was interposed between the patella and patellar tendon during repair. No cartilage interposition was used in control group. Samples were harvested at six, 12, and 24 weeks for histological examination (n=6 each). The histological images were digitised and analyzed using an image analysis system. Healing progress was assessed by the amount of new bone formation and fibrocartilage transition zone regeneration. Quantitative data were analyzed using SPSS version 14.0. Statistic al significance level was set at p < 0.05. There was progressive increase in maximum new bone length and area of new bone formed with time (p< 0.05, Kruskal-Wallis test). No difference was observed between treatment groups. Articular cartilage interposition resulted in more fibrocartilage regeneration and higher proteoglycan uptake at all time points. At 24 weeks, length of fibrocartilage formed measured 7760 ± 629 μm with articular cartilage interposition, compared with 787± 274 μm in control (p = 0.002, Mann-Whitney test). Safranin O length measured 3301 ± 1236 μm with articular cartilage interposition, compared with 277 ± 187 μm in control (p = 0.03, Mann-Whitney test). Autologous articular cartilage interposition stimulates fibrocartilage transition zone regeneration in BTJ repair without affecting bone formation

Summary Statement. The Dkk3-derived cells represent a branch of the periosteal mesenchymal lineage that produces fibrocartilage as well as regenerating the periosteal structures. Introduction. Mesenchymal progenitor cells are capable of generating a wide variety of mature cells that constitute the connective tissue system. Our Laboratory has been developing SMAA GFP reporter mice to prove to be an effective tool for identifying these cells prior to the expression of markers of differentiation characteristic of bone, fat, muscular blood vessels or fibrocartilage. Dkk3 was chosen as a candidate reporter because microarray of SMAA-sorted cells culture indicated high expression of this non-canonical anti-Wnt factor, which was not anticipated in a culture with strong osteogenic potential. Material and Methods. Fracture healing process was evaluated in 12 week old male mice at 3, 5, 7, 14, 21 and 28days post fracture. A 3 color reporter mouse was generated by crossing SMAA-GFPcherry × Col3.6GFPcyan × Dkk3-eGFP and subjected to tibial fracture. A closed transverse fracture was performed by Einhorn device under isoflurane anesthesia after insertion of intramedullary pinning. Longitudinal 5 mm non-calcified cryosections were stabilised with Cryofilm tape. Results. Three days post fracture, the proliferating SMAA-red cells were also beginning to express either Dkk3 or Col3.6. By day 5 the two populations had diverged with the Dkk3 cells being on the outer surface of the developing callus while the Col3.6 cells were forming bone at the base of the callus. By day 7 when the callus is filled with cartilage, Dkk3 is active in cells that are in transition from elongated cells on the external surface of the callus to fibrocartilagenous cells that now express low levels of Col3.6. The zone of cells that express Dkk3 appear to block the passage of the surrounding vasculature into the underlying cartilage and does not deposit fibronectin. By day 14–21 when the cartilage core is resorbed, the only remaining Dkk3 is located in the newly formed periosteum external to the active endocortical bone forming activity associated with the inward remodeling of the outer cortical shell. Discussion. We interpret these findings that Dkk3 marks a non-osteogenic limb of the SMAA progenitor population that within the fracture partitions the osteogenic signals away from the surrounding skeletal muscle and the underlying differentiating fibrocartilage. It is a progenitor to cells that form fibrocartilage in the fracture zone as well as the tenascin C positive cells that populate the fibrous zone of the periosteum, and it resides in the cambial zone of the periosteum. Knowing the biological and molecular function of these cells should lead to a fuller appreciation of the pro- and anti-osteogenic factors that regulate skeletal repair

Abstract. Background. We know that tears of the Triangular fibrocartilage complex (TFCC) can cause DRUJ instability and ulnar sided wrist pain. This study shows the clinical result of patients who had arthroscopic transosseous repair of the TFCC tear with DRUJ instability. Arthroscopic repair of TFCC tear is a promising, minimally invasive surgical technique especially in patients with DRUJ instability. Materials and methods. Fifteen patients who underwent TFCC one tunnel repair form 2018–2021 were reviewed retrospectively in hospital. The proximal component of TFCC was repaired through arthroscopic one- tunnel transosseous suture technique. VAS score for pain, wrist range of motion, grip strength and post operative complications were evaluated and each patient was rated according to the DASH score. Results. The patients had a TFCC tear confirmed on MRI and was confirmed on arthroscopy by doing a hook test. The patients were followed up for 6 months. Twelve patients had normal stability of DRUJ and three patients showed mild laxity compared with the contralateral side. The mean VAS score reduced from 4.7 to 0.8 (P=0.001) and grip strength increased significantly. The quick DASH score (P=0.001)also showed significant functional improvement. No surgical related complications occurred. Conclusions. Arthroscopic one tunnel transosseous TFCC foveal repair can be an excellent and safe method for repair of TFCC tear with DRUJ instability. Its a good treatment option in terms of reliable pain relief, functional improvement and reestablishment of DRUJ stability

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

There is a difference of opinion regarding the usefulness of MR Imaging as a diagnostic tool for triangular fibrocartilage complex (TFCC) tears in the wrist. Our aim was to determine the accuracy of direct magnetic resonance arthrography (MRA) in the diagnosis of triangular fibrocartilage complex (TFCC) tears of the wrist in a district general hospital setting. In a retrospective review of 21 patients who presented with complains of wrist pain and following a clinical examination, all had direct MR arthrography of the wrist in our hospital in a 1.5Tesla scanner. All had a diagnostic arthroscopy within 2-4 months of the MR scan. All patients had chronic ulnar sided wrist pain, although only two had a definite history of trauma. The findings of each diagnostic method were compared, with arthroscopy considered the gold standard. Twenty-one patients were studied (10 male: 11 female), mean age 42 years (range 27-71) years). Seventeen TFCC tears were diagnosed on arthroscopy. For the diagnosis of TFCC tears MRA had a sensitivity, specificity and accuracy of 67%. Our results echoed the opinion of some of the previous investigators with an unacceptable sensitivity or specificity for a diagnostic tool. MR arthrography needs to be further refined as a technique before it can be considered to be accurate enough to replace wrist arthroscopy for the diagnosis of TFCC tears. Other centres have reported better accuracy, using more advanced MRI technology. Until this iswidely available at all levels of healthcare the results of MRI for the diagnosis of TFCC tears should be interpreted with caution

Purpose: The purpose of this study was to evaluate the long-term results of arthroscopic treatment in patients affected by triangular fibrocartilage complex (TFCC) type 1b lesions associated with distal radio ulnar joint (DRUJ) instability. Method: 138 patients affected by TFCC type 1b lesions: Group A (117 patients, 27±7 yrs) were treated using an out-in arthroscopic technique and Group B (21 patients, 24±4 yrs) with an associated total DRUJ instability, were treated using an out-in arthroscopic technique in addition to an anchor placement. Inclusion criteria were: TFCC tears, type 1b lesions and no previous wrist fractures. SF-36, DASH, VAS, and ROM were accessed preoperatively and at four years follow-up. Results: All the patients have a significant improvement in terms of SF-36 (p0.05). Conclusion: Arthroscopy is a tool of paramount importance in both diagnosis and treatment of TFCC injuries even associated with DRUJ. Furthermore, type 1b lesions associated with total DRUJ instability should be managed combining an out-in arthroscopic technique with the use of an anchor to completely relieve pain and restore wrist function

Summary. When a TFCC tear is diagnosed, practitioners should maintain a high level of suspicion for the presence of a concomitant SL or LT ligament tear. Introduction. Disruption of the scapholunate (SL) or lunotriquetral (LT) ligament leads to dorsal and volar intercalated segment instability, respectively, while triangular fibrocartilage complex (TFCC) tears result in distal radioulnar joint (DRUJ) instability. Viegas et al. (1993) demonstrated that 56% of grossly visualised cadaveric wrists had one or more tears of a ligament or of the TFCC. The purpose of this investigation is to quantify the incidence, distribution, and correlation of SL, LT, and TFCC tears in a large group of cadaver wrists using magnetic resonance imaging (MRI). Additionally, statistical analysis was performed to predict. Methods. Spin density weighted, fat suppressed, and STIR MRI scans of the wrist were obtained in 48 fresh frozen cadaver arms using a 3 Tesla MRI scanner. The scans were scrutinised by one of us (PC) – a board certified musculoskeletal radiologist. The dorsal, volar, and membranous portions of the SL and LT ligaments were examined sequentially for the presence of a tear. Similarly, the central disk and radioulnar attachments of the TFCC were inspected for tears. Results. A ligament or the TFCC was labeled as torn if there was a complete tear, partial tear, or perforation of one or more of its components, but not if sole degenerative changes, thinning, or fraying of the fibers was observed. Four of the 48 images could not be interpreted due to unsatisfactory scans. The most prevalent injury was a TFCC tear, which was present in 28 (64%) of the 44 wrists examined. SL ligament tears were discovered in 20 (45%) of the wrists, and LT tears were present in 14 (32%) of the wrists. Moreover, 45% of the wrists examined had a TFCC tear and either a SL or LT ligament tear. Specifically, 50% of the 28 wrists with a TFCC tear had a concomitant LT tear, and 46% had a concomitant SL tear. Discussion. SL, LT, and TFCC tears were found in a substantial portion of the wrists examined. Moreover, the majority of wrists with a TFCC tear also had a SL or LT ligament tear. Viegas et al. found that 70% of wrists with a TFCC perforation also had a LT ligament tear. In our series, 71% had a TFCC tear, and 50% of those had a concomitant LT tear

Introduction. We reports the accuracy of direct Magnetic Resonance Arthrography (MRA) in detecting Triangular Fibrocartilage Complex (TFCC), Scapho-Lunate Ligament (SLL) and Luno-Triquetral Ligament (LTL) tears using wrist arthroscopy as the gold standard. Methods. We reviewed the records of all patients who underwent direct wrist MRA and subsequent arthroscopy over a 4-year period between June 2007 and March 2011. Demographic details, MRA findings, arthroscopy findings and the time interval between MRA and arthroscopy were recorded. The scans were performed using a 1.5T scanner and a high resolution wrist coil. All scans were reported by a musculoskeletal radiologist. Sensitivity, specificity, positive and negative predictive values (PPV & NPV) were calculated. Results. Two hundred and thirty four (234) MRA were performed over the study period. Fifty patients (50), who subsequently underwent 51 wrist arthroscopies (one bilateral), were included. The mean age was 35 years (range 16–64 years). The average delay between MRA and arthroscopy was 4.8 months (median 4 months, range 17 days–18 months). All patients were symptomatic with wrist pain. At arthroscopy, 26 TFCC tears, 7 SLL tears and 3 LTL tears were found. For TFCC, sensitivity was 96%, specificity 88%, PPV 89% and NPV 96%. For SLL, the values were 57%, 66%, 21% and 91% respectively. For LTL, 67%, 79%, 17% and 97%, respectively. Receiver Operating Characteristic (ROC) curve analysis showed that MRA only reliably differentiates between patients with and without TFCC tears (Area Under Curve AUC = 0.92, p < 0.0001) but not SLL (AUC = 0.62, p=0.28) or LTL (AUC = 0.73, p=0.17) tears. Conclusion. MRA is a sensitive and specific imaging modality for diagnosing TFCC tears. However, the diagnostic accuracy for SLL and LTL tears was not satisfactory. Wrist arthroscopy remains the gold standard if there is a clinical suspicion of inter-carpal ligament tears

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

For chondral damage in younger patients, surgical best practice is microfracture, which involves drilling into the bone to liberate the bone marrow. This leads to a mechanically inferior fibrocartilage formed over the defect as opposed to the desired hyaline cartilage that properly withstands joint loading. While some devices have been developed to aid microfracture and enable its use in larger defects, fibrocartilage is still produced and there is no clear clinical improvement over microfracture alone in the long term. Our goal is to develop 3D printed devices, which surgeons can implant with a minimally invasive technique. The scaffolds should match the functional properties of cartilage and expose endogenous marrow cells to suitable mechanobiological stimuli in-situ, in order to promote healing of articular cartilage lesions before they progress to osteoarthritis, and rapidly restore joint health and mobility. Importantly, scaffolds should direct a physiological host reaction, instead of a foreign body reaction, associated with chronic inflammation and fibrous capsule formation, negatively influencing the regenerative outcome. Our novel silica/polytetrahydrofuran/polycaprolactone hybrids were prepared by sol-gel synthesis and scaffolds were 3D printed by direct ink writing. 3D printed hybrid scaffolds with pore channels of ~250 µm mimic the compressive behaviour of cartilage. Our results show that these scaffolds support human bone marrow stem/stromal cell (hMSC) differentiation towards chondrogenesis in vitro under hypoxic conditions to produce markers integral to articular cartilage-like matrix evaluated by immunostaining and gene expression analysis. Macroscopic and microscopic evaluation of subcutaneously implanted scaffolds in mice showed that scaffolds caused a minimal resolving inflammatory response. Our findings show that 3D printed hybrid scaffolds have the potential to support cartilage regeneration. Acknowledgements: Authors acknowledge funding provided by EPSRC grant EP/N025059/1

The anterior cruciate ligament (ACL) is the connective tissue located at the end of long bones providing stability to the knee joint. After tear or rupture clinical reconstruction of the tissue remains a challenge due to the particular mechanical properties required for proper functioning of the tissue. The outstanding mechanical properties of the ACL are characterized by a viscoelastic behavior responsible of the dissipation of the loads that are transmitted to the bone. These mechanical properties are the result of a very specialized graded extracellular matrix that transitions smoothly between the heterotypic cells, stiffness and composition of the ACL and the adjacent bone. Thus, mimicking the zonal biochemical composition, cellular phenotype and organization are key to reset the proper functioning of the ACL. We have previously shown how the biochemical composition presented to cells in electrospun scaffolds results in haptokinesis, reverting contact-guidance effects. [1]. Here, we demonstrate that contact guidance can also be disrupted by structural parameters in aligned wavy scaffolds. The presentation of a wavy fiber arrangement affected the cell organization and the deposition of a specific ECM characteristic of fibrocartilage. Cells cultured in wavy scaffolds grew in aggregates, deposited an abundant ECM rich in fibronectin and collagen II, and expressed higher amounts of collagen II, X and tenomodulin as compared to aligned scaffolds. In-vivo implantation in rabbits of triphasic scaffolds accounting for aligned-wavy-aligned zones showed a high cellular infiltration and the formation of an oriented ECM, as compared to traditional aligned scaffolds. [2]

The triangular fibrocartilage complex (TFCC) is a known stabiliser of the distal radioulnar joint (DRUJ). An injury to these structures can result in significant disability including pain, weakness and joint stiffness. The contribution each of its components makes to the stability of the TFCC is not well understood. This study was undertaken to investigate the role of the individual ligaments of the TFCC and their contribution to joint stability. The study was undertaken in two parts. 30 cadaveric forearms were studied in each group. The ligaments of the TFCC were progressively sectioned and the resulting effect on the stability of the DRUJ was measured. A custom jig was created to apply a 20N force through the distal radius, with the ulna fixed. Experiment one measured the effect on DRUJ translation after TFCC sectioning. Experiment two added the measurement of rotational instability. Part one of the study showed that complete sectioning of the TFCC caused a mean increase in translation of 6.09(±3) mm. Sectioning the palmar radioulnar ligament of the TFCC caused the most translation. Part two demonstrated a change in rotation with a mean of 18 (± 6) degrees following sectioning of the TFCC. There was a progressive increase in rotational instability until the palmar radioulnar ligament was also sectioned. Linear translation consistently increased after sectioning all of the TFCC ligaments, confirming its importance for DRUJ stability. Sectioning of the palmar radioulnar ligament most commonly caused the greatest degree of translation. This suggests injury to this ligament would more likely result in a greater degree of translational instability. The increase in rotation also suggests that this type of instability would be symptomatic in a TFCC injury

Cartilage lacks the ability to self-repair when damaged, which can lead to the development of degenerative joint disease. Despite intensive research in the field of cartilage tissue engineering, there is still no regenerative treatment that consistently promotes the development of hyaline cartilage. Extracellular matrix (ECM) derived hydrogels have shown to support cell adhesion, growth and differentiation [1,2]. In this study, porcine articular cartilage was decellularized, solubilised and subsequently modified into a photo-crosslinkable methacrylated cartilage ECM hydrogel. Bone marrow derived mesenchymal stem/stromal cells (MSCs) were encapsulated into both methacrylated ECM hydrogels (ECM-MA) and gelatin methacryloyl (GelMA) as control hydrogel, and their chondrogenic potential was assessed using biochemical assays and histological analysis. We found that successful decellularization of the cartilage tissue could be achieved while preserving key ECM components, including collagen and glycosaminoglycans. A live-dead assay demonstrated good viability of MSCs withing both GelMA and ECM-MA hydrogels on day 7. Large increases in sGAG accumulation was observed after 21 days of culture in chondrogenic media in both groups. Histological analysis revealed the presence of a more fibrocartilage tissue in the GelMA group, while cells embedded within the ECM-MA showed a round and chondrocytic-like morphology. Both groups stained positively for proteoglycans and collagen, with limited evidence of calcium deposition following Alizarin Red staining. These results show that ECM-MA hydrogels support a hyaline cartilage phenotype and robust cartilaginous matrix production. Future studies will focus on the printability of ECM-MA hydrogels to enable their use as bioinks for the biofabrication of functional tissues

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

Lesions in the joint surface are commonly treated with osteoarticular autograft transfer system (OATS), autologous cell implantation (ACI/MACI), or microfracture. Tissue formed buy the latter commonly results in mechanically inferior fibrocartilage that fails to integrate with the surrounding native cartilage, rather than durable hyaline cartilage. Fractional laser treatment to make sub-millimeter (<500 µm) channels has been employed for tissue regeneration in the skin to facilitate rejuvenation without typical scarring. Additionally, we have pioneered a means to generate articular cartilage matrix from chondrocytes—dynamic Self-Regenerating Cartilage (dSRC). Combining these two approaches by performing fractional laser treatment of the joint cartilage and treating with dSRC is a new paradigm for joint surface restoration. This approach was refined in a series of in vitro experiments and tested in swine knee defects during a 6-month study in 12 swine. dSRC are generated by placing 10. 7. swine knee chondrocytes into sealed 15-mL polypropylene tubes and cultured on a rocker at 40 cycles per minute for 14 days at 37°C. The chondrocytes aggregate and generate new extracellular matrix to form a pellet of dSRC. Channels of approximately 300-500 µm diameter were created by infrared laser ablation in swine cartilage (in vitro) and swine knees (in vivo). The diameter and depth of the ablated channel in the cartilage was controlled by the light delivery parameters (power, spot size, pulse duration) from a fractional 2.94 µm Erbium laser. The specimens were evaluated with histology (H&E, safranin O, toluidine blue) and polarized-sensitive optical coherence tomography for collagen orientation. We can consistently create laser-ablated channels in the swine knee and successfully implant new cartilage from dSRC to generate typical hyaline cartilage in terms of morphology and biochemical properties. The neocartilage integrates with host cartilage in vivo. These findings demonstrate our novel combinatorial approach for articular cartilage rejuvenation

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

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