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.

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 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.

Abstract

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.

Abstract

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.

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.

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).

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;

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.

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.

Shoulder girdles of 20 cadavers (68–94yrs) were harvested. The anterior (ACHA) and posterior circumflex humeral arteries (PCHA) were injected with ink and the extra and intraosseous courses of the dyed vasculature dissected through the soft tissues and bone to the osteotendinous junctions of the rotator cuff. The ink injection and bone dissection method was newly developed for the study. Rates of cross-over at the osteotendinous juntion were 75% in the supraspinatus, 67% in subscapularis, 33% in infraspinatus and 20% in teres minor. The supraspinatus and subscapularis insertions were vascularised by the arcuate artery, a branch of the ACHA. The insertions of the infraspinatus and teres minor were supplied by an unnamed terminal branch of the PCHA. The insertions of the rotator cuff receive an arterial supply across their OTJ's in 50% of cases. This may explain observed rates of AVN in comminuted proximal humeral fractures. The terminal branch of the PCHA supplying the infraspinatus and teres minor insertions was named the “Posterolateral Artery”. Finally, the new method employed for this study which allowed for direct visualisation of intraosseous vasculature, will enhance our understanding of skeletal vascular anatomy and have clinical applications in orthopaedic and reconstructive surgery

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.

We have used Fourier transform infrared spectroscopy (FTIR) to characterise the chemical and structural composition of the tendons of the rotator cuff and to identify structural differences among anatomically distinct tears. Such information may help to identify biomarkers of tears and to provide insight into the rates of healing of different sizes of tear. The infrared spectra of 81 partial, small, medium, large and massive tears were measured using FTIR and compared with 11 uninjured control tendons. All the spectra were classified using standard techniques of multivariate analysis.

FTIR readily differentiates between normal and torn tendons, and different sizes of tear. We identified the key discriminating molecules and spectra altered in torn tendons to be carbohydrates/phospholipids (1030 cm⁻¹ to 1200 cm⁻¹), collagen (1300 cm⁻¹ to 1700 cm⁻¹ and 3000 cm⁻¹ to 3350 cm⁻¹) and lipids (2800 cm⁻¹ to 3000 cm⁻¹).

Our study has shown that FTIR spectroscopy can identify tears of the rotator cuff of varying size based upon distinguishable chemical and structural features. The onset of a tear is mainly associated with altered structural arrangements of collagen, with changes in lipids and carbohydrates. The approach described is rapid and has the potential to be used peri-operatively to determine the quality of the tendon and the extent of the disease, thus guiding surgical repair.