Histological and ultrastructural degenerative findings in the gluteus medius tendon after hip arthroplasty.

BACKGROUND: Despite gluteus medius (GMED) tendinosis being relatively common, its presence in association with hip osteoarthritis (OA) or total hip arthroplasty (THA) is not well studied. It was hypothesized that more tendon degeneration would be found in patients with OA of the hip and in those that had undergone THA than that in a control group. METHODS: One hundred patients were included between 2016 and 2019 and were included into 4 groups; the patients were undergoing revision surgery in two groups and primary THA in the other two groups; 22 patients had previously undergone primary THA through a direct lateral approach (involving sectioning of the GMED tendon), 24 patients had previously undergone primary THA through a posterior approach (leaving the GMED tendon intact), 29 patients had primary hip OA, and 25 patients who suffered a femoral neck fracture served as controls. Biopsies from the GMED tendon were obtained at the time of the primary THA or the hip revision surgery. The tendon biopsies were examined ultrastructurally and histologically. RESULTS: Ultrastructurally, the direct lateral and posterior revision groups had statistically significantly more collagen fibrils with smaller diameters compared with the fracture and primary THA groups. Moreover, the direct lateral revision group had more collagen fibrils with smaller diameters compared with the posterior revision group. Histologically, the direct lateral revision group had a higher total degeneration score (TDS) compared with the primary hip OA group. CONCLUSIONS: The GMED tendon shows more ultrastructural degeneration in patients who undergo hip revision arthroplasty than in patients with primary OA of the hip and control patients, who had suffered a femoral neck fracture. Furthermore, patients who had previously undergone primary THA through a direct lateral approach revealed more histological GMED tendon degeneration than patients who suffer primary hip OA.

Microstructural and Mechanical Properties of Grafts Commonly Used for Cruciate Ligament Reconstruction.

BACKGROUND: Injuries to the anterior cruciate ligament and posterior cruciate ligament are common, and often are treated with reconstruction. Limited quantitative data are available describing material properties of grafts used for reconstructions such as the bone-patellar tendon-bone (BPTB), hamstring tendon (HS), and quadriceps tendon (QT). The purpose of this study was to quantify and compare microstructural and mechanical properties of BPTB, HS, and QT grafts. METHODS: Forty specimens (13 BPTB, 13 HS, and 14 QT grafts) from 24 donors were used. Specimens were subjected to preconditioning, stress relaxation, and ramp to failure. Mechanical parameters were calculated for each sample, and polarization imaging was used to evaluate the direction and strength of collagen fiber alignment during testing. RESULTS: QT had the largest modulus values, and HS had the smallest. BPTB exhibited the least disperse collagen organization, while HS were the least strongly aligned. Microstructural properties showed more strongly aligned collagen with increasing load for all grafts. All tissues showed stress relaxation and subtle microstructural changes during the hold period. CONCLUSIONS: The mechanical and microstructural properties differed significantly among BPTB, HS, and QT grafts. QT exhibited the largest moduli and greatest strength of collagen alignment, while HS had the smallest moduli and least strongly aligned collagen. CLINICAL RELEVANCE: This study identified mechanical and microstructural differences among common grafts and between these grafts and the cruciate ligaments they replace. Further research is needed to properly interpret the clinical relevance of these differences.

Flexor tendon grafts for pulley reconstruction - Morphological aspects.

BACKGROUND: Pulleys are crucial to convert flexor tendon excursion into angular motion at the metacarpophalangeal and interphalangeal joints. Loss of pulley function can lead to significant impairment of hand function and may require surgical reconstruction. This reconstruction can be achieved using different flexor tendons grafts, such as the intrasynovial flexor digitorum superficialis (FDS) or the extrasynovial palmaris longus (PL). However, there is limited knowledge on the micromorphology of human pulleys and the suitability of flexor tendon grafts for their reconstruction remains elusive. METHODS: In the present cadaver study A2 and A4 pulleys were compared with FDS and PL tendons by means of scanning electron microscopy (SEM), histology and immunohistochemistry. Surface morphology, core structure and vascularization of the specimens were analyzed. RESULTS: SEM imaging of the gliding surfaces revealed morphological differences between tendons and pulleys. Moreover, the core structure of FDS samples was characterized by bundles of individual collagen fibrils whereas PL tendons exhibited a less hierarchical microstructure. In contrast, pulleys consisted of lamellar sheets of densely packed collagen fibrils. Finally, immunohistochemical analyses revealed that the flexor tendons and pulleys contain similar numbers of CD31+ microvessels, indicating a comparable tissue vascularization. CONCLUSION: This study provides novel SEM and immunohistochemical insights into the micromorphology of human pulleys and flexor tendon grafts. Intrasynovial flexor tendons may be particularly suitable for pulley reconstruction and preserving the paratenon may be crucial for graft revascularization.

Optical Microscopy and Electron Microscopy for the Morphological Evaluation of Tendons: A Mini Review.

The morphological characteristics of tendons have been thoroughly evaluated via microscopy. Optical microscopy and electron microscopy are the most commonly used techniques for tendon tissue observation. According to the principles of both microscopy types, preparation and evaluation methods vary. Simple optical microscopy is commonly used in the observation of cells and extracellular matrix, and many stains, including hematoxylin-eosin, Van Gieson, Prussian blue, Alcian blue, and toluidine blue, are used for evaluating cells, collagen fiber arrangement, and noncollagenous proteins. Histological scoring systems have been used in many studies for semi-quantification. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are the most commonly used electron microscopy types, and special consideration is needed for the fixation and embedding protocols. Glutaraldehyde followed by osmium is most commonly used in the chemical fixation of tendon tissue, followed by epoxy resin embedment. Longitudinal sections captured in SEM images show the arrangement of collagen fibrils and the cells and lipid drops among them, while cross sections captured in TEM images show the diameter and distribution of collagen fibrils. SEM and TEM are used together for comprehensive evaluations. This mini review is focused on the preparation methodology and related evaluation indexes for the morphological evaluation of tendons.

Tendon Extracellular Matrix Remodeling and Defective Cell Polarization in the Presence of Collagen VI Mutations.

Mutations in collagen VI genes cause two major clinical myopathies, Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), and the rarer myosclerosis myopathy. In addition to congenital muscle weakness, patients affected by collagen VI-related myopathies show axial and proximal joint contractures, and distal joint hypermobility, which suggest the involvement of tendon function. To gain further insight into the role of collagen VI in human tendon structure and function, we performed ultrastructural, biochemical, and RT-PCR analysis on tendon biopsies and on cell cultures derived from two patients affected with BM and UCMD. In vitro studies revealed striking alterations in the collagen VI network, associated with disruption of the collagen VI-NG2 (Collagen VI-neural/glial antigen 2) axis and defects in cell polarization and migration. The organization of extracellular matrix (ECM) components, as regards collagens I and XII, was also affected, along with an increase in the active form of metalloproteinase 2 (MMP2). In agreement with the in vitro alterations, tendon biopsies from collagen VI-related myopathy patients displayed striking changes in collagen fibril morphology and cell death. These data point to a critical role of collagen VI in tendon matrix organization and cell behavior. The remodeling of the tendon matrix may contribute to the muscle dysfunction observed in BM and UCMD patients.

Independent, Controllable Stretch-Perfusion Bioreactor Chambers to Functionalize Cell-Seeded Decellularized Tendons.

Tissue-engineered decellularized matrices can progress clinical replacement of full-thickness ruptures or tendon defects. This study develops and validates a custom-made automated bioreactor, called oscillating stretch-perfusion bioreactor (OSPB), consisting of multiple, independent culture chambers able to combine a bidirectional perfusion with a programmable, uniaxial strain to functionalize cell-seeded decellularized tendons. Decellularized tendon matrices were seeded on their surfaces and within the tendon fibers with mesenchymal stem cells. Then, they were subjected to a bidirectional perfusion and programmed stretching cycles of 15-30-60 min on-off two times per day for 7 days of culture. In vitro analyses showed viable cells, homogenously distributed on the surface of the constructs. More importantly, cell-seeded decellularized tendon grafts undergoing cyclic load in our bioreactor had a superior production and organization of newly formed collagen matrix compared to static cultured constructs. The coherency and local alignment of the new collagen deposition within the inner injected channels quantitatively supported histological findings. The designed OSPB could be considered a unique, cost-effective system able to involve multiple independently controlled chambers in terms of biological and mechanical protocols. This system allows parallel processing of several customized tendon constructs to be used as grafts to enhance the surgical repair of large tendon defects.

Development of a novel multiphysical approach for the characterization of mechanical properties of musculotendinous tissues.

At present, there is a lack of well-validated protocols that allow for the analysis of the mechanical properties of muscle and tendon tissues. Further, there are no reports regarding characterization of mouse skeletal muscle and tendon mechanical properties in vivo using elastography thereby limiting the ability to monitor changes in these tissues during disease progression or response to therapy. Therefore, we sought to develop novel protocols for the characterization of mechanical properties in musculotendinous tissues using atomic force microscopy (AFM) and ultrasound elastography. Given that TIEG1 knockout (KO) mice exhibit well characterized defects in the mechanical properties of skeletal muscle and tendon tissue, we have chosen to use this model system in the present study. Using TIEG1 knockout and wild-type mice, we have devised an AFM protocol that does not rely on the use of glue or chemical agents for muscle and tendon fiber immobilization during acquisition of transversal cartographies of elasticity and topography. Additionally, since AFM cannot be employed on live animals, we have also developed an ultrasound elastography protocol using a new linear transducer, SLH20-6 (resolution: 38 µm, footprint: 2.38 cm), to characterize the musculotendinous system in vivo. This protocol allows for the identification of changes in muscle and tendon elasticities. Such innovative technological approaches have no equivalent to date, promise to accelerate our understanding of musculotendinous mechanical properties and have numerous research and clinical applications.

Integration of mesenchymal stem cell sheet and bFGF-loaded fibrin gel in knitted PLGA scaffolds favorable for tendon repair.

Tendon injuries are common and require a long time to heal, and are particularly associated with some adverse problems such as adhesion and rupture. Herein, we aim to develop new bioactive scaffolds endowed with stem cell sheets and growth factors to enable cell migration and proliferation favorable for tendon regeneration in situ. An exogenous basic fibroblast growth factor (bFGF)-loaded fibrin gel was firstly incorporated into the porous network of knitted poly(lactide-co-glycolide) (PLGA) scaffolds and then sheets of mesenchymal stem cells (MSCs) were also integrated into the scaffolds. It was shown that the pores in the knitted PLGA scaffold were readily filled with a complex network of fibrin fiber gel and the fibrin fibers were beneficial for the controlled release of bFGF over a long time period. After transplantation in a critical-size Achilles tendon defect model (7 mm) in the rat right hindlimb, gross observation revealed no immunologic incompatibility or rejection derived from the scaffold systems. It was observed that the MSC sheets contributed directly to tendon regeneration, and exerted an environment-modifying effect on the injuries in situ, consistent with the beneficial effect of bFGF. It was interesting that the knitted PLGA-fibrin gel scaffolds loaded with MSC sheets and bFGF showed the highest expression of tendon-related gene markers and outstanding repair efficacy, including appreciable biomechanical strength and native-like histological microstructures. Therefore, the integration of MSC sheets and bFGF into PLGA/bFGF-fibrin gel scaffolds may stimulate the proliferation and tenogenic differentiation of MSCs in situ and synergistically enhance the injured tendon reconstruction.

Growth Factor-Mediated Tenogenic Induction of Multipotent Mesenchymal Stromal Cells Is Altered by the Microenvironment of Tendon Matrix.

Age-related degenerative changes in tendon tissue represent a common cause for acute tendon pathologies. Although the regenerative potential of multipotent mesenchymal stromal cells (MSC) was reported to restore functionality in injured tendon tissue, cellular mechanisms of action remain partly unclear. Potential tenogenic differentiation of applied MSC is affected by various intrinsic and extrinsic factors. The current study presents an in vitro model to evaluate the combined extrinsic effects of decellularized equine tendon matrix, transforming growth factor beta 3 (TGFß3) and bone morphogenetic protein 12 (BMP12) on the tenogenic fate of equine adipose tissue-derived MSC. Monolayer MSC cultures supplemented with TGFß3 and BMP12 as well as MSC cultured on tendon matrix scaffolds preloaded with the growth factors were incubated for 3 and 5 days. Histological evaluation and real time reverse transcription polymerase chain reaction (RT-PCR) revealed that growth factor-mediated tenogenic induction of MSC was modified by the conditions of the surrounding microenvironment. While the gene expression pattern in monolayer cultures supplemented with TGFß3 or TGFß3 and BMP12 revealed an upregulation for collagen 1A2, collagen 3A1, tenascin c, scleraxis and mohawk ( p < 0.05 ), the presence of tendon matrix led to an upregulation of decorin and osteopontin as well as to a downregulation of smad8 ( p < 0.05). Preloading of scaffolds with either TGFß3, or with TGFß3 and BMP12 promoted a tenocyte-like phenotype and improved cell alignment. Furthermore, gene expression in scaffold culture was modulated by TGFß3 and/or BMP12, with downregulation of collagen 1A2, collagen 3A1, decorin, scleraxis, smad8 and osteopontin, whereas gene expression of tenascin c was increased. This study shows that growth factor-induced tenogenic differentiation of equine MSC is markedly altered by topographical constraints of decellularized tendon tissue in vitro. While TGFß3 represents an effective mediator for tenogenic induction, the role of BMP12 in tenogenesis may be of modulatory character and needs further evaluation.

More histologic and ultrastructural degenerative signs in the subscapularis tendon and the joint capsule in male patients with shoulder impingement.

PURPOSE: The purpose of the present study was to analyze biopsy samples from the subscapularis tendon and from the joint capsule from male patients with shoulder impingement syndrome (SAIS) and compare them with samples from male patients with post-traumatic recurrent shoulder instability. The hypothesis of the study was that patients with SAIS would have more histologic and ultrastructural degenerative changes in their subscapularis tendon and joint capsule than patients with post-traumatic recurrent shoulder instability. METHODS: Male patients scheduled for surgery, with either subacromial decompression or Bankart reconstruction, were included. Four biopsies from each patient were obtained from the capsule and four from the subscapularis tendon during arthroscopic surgery. The histologic characteristics and the presence of glycosaminoglycans were assessed using the light microscope, and the ultrastructure was assessed using a transmission electron microscope. RESULTS: Eight patients, median age 53 (45-74) years (p < 0.0001), were included in the impingement group, and 12 patients, median age 27 (22-48) years, were included in the instability group. The histologic assessment revealed significantly higher cellularity and total degeneration score in the capsule (p = 0.016 and p = 0.014 respectively) in patients with subacromial impingement compared with the instability patients. The corresponding finding was not made for the subscapularis tendon. The ultrastructural evaluation revealed that the instability patients had more fibrils with a large diameter (indicating less degeneration) in both the subscapularis tendon and the capsule compared with the impingement patients (p < 0.0001). CONCLUSION: Male patients with subacromial impingement have more histologic and ultrastructural degenerative changes in their shoulder compared with patients with post-traumatic recurrent shoulder instability. CLINICAL RELEVANCE: It appears that in patients with subacromial impingement, the whole shoulder joint is affected and not only the subacromial space. It is the opinion of the authors that intra-articular therapeutic injections could be tried more often in these patients. LEVEL OF EVIDENCE: III.

Optimizing a 3D model system for molecular manipulation of tenogenesis.

PURPOSE: Tendon injuries are clinically challenging due to poor healing. A better understanding of the molecular events that regulate tendon differentiation would improve current strategies for repair. The mouse model system has been instrumental to tendon studies and several key molecules were initially established in mouse. However, the study of gene function has been limited by the absence of a standard in vitro tendon system for efficiently testing multiple mutations, physical manipulations, and mis-expression. The purpose of this study is therefore to establish such a system. METHODS: We adapted an existing design for generating three-dimensional (3D) tendon constructs for use with mouse progenitor cells harboring the ScxGFP tendon reporter and the Rosa26-TdTomato Cre reporter. Using these cells, we optimized the parameters for construct formation, inducing tenogenesis via transforming growth factor-ß2 (TGFß2), and genetic recombination via an adenovirus encoding Cre recombinase. Finally, for proof of concept, we used Smad4 floxed cells and tested the robustness of the system for gene knockdown. RESULTS: We found that TGFß2 treatment induced a tenogenic phenotype depending on the timing of initiation. Addition of TGFß2 after 3D "tensioning" enhanced tendon differentiation. Interestingly, while TGFß2-induced proliferation depended on Smad4, tenogenic parameters such as ScxGFP expression and fibril diameter were independent of Smad4. CONCLUSIONS: Our results demonstrate the feasibility of this optimized system for harnessing the power of mouse genetics for in vitro applications.

A single stranded fluorescent peptide probe for targeting collagen in connective tissues.

We herein report the construction of a novel single stranded fluorescent collagen mimetic peptide by introducing a bulky FAM dye in the central region rather than the N terminus. Without the need for any prior thermal or ultraviolet treatment, the peptide probe can be conveniently applied to specifically target collagen in connective tissues for fluorescence imaging.

Directing the Differentiation of Parthenogenetic Stem Cells into Tenocytes for Tissue-Engineered Tendon Regeneration.

Uniparental parthenogenesis yields pluripotent stem cells without the political and ethical concerns surrounding the use of embryonic stem cells (ESCs) for biomedical applications. In the current study, we hypothesized that parthenogenetic stem cells (pSCs) could be directed to differentiate into tenocytes and applied for tissue-engineered tendon. We showed that pSCs displayed fundamental properties similar to those of ESCs, including pluripotency, clonogenicity, and self-renewal capacity. pSCs spontaneously differentiated into parthenogenetic mesenchymal stem cells (pMSCs), which were positive for mesenchymal stem cell surface markers and possessed osteogenic, chondrogenic, and adipogenic potential. Then, mechanical stretch was applied to improve the tenogenic differentiation of pMSCs, as indicated by the expression of tenogenic-specific markers and an increasing COL1A1:3A1 ratio. The pSC-derived tenocytes could proliferate and secrete extracellular matrix on the surface of poly(lactic-co-glycolic) acid scaffolds. Finally, engineered tendon-like tissue was successfully generated after in vivo heterotopic implantation of a tenocyte-scaffold composite. In conclusion, our experiment introduced an effective and practical strategy for applying pSCs for tendon regeneration. Stem Cells Translational Medicine 2017;6:196-208.

Pleiotropic roles of the matricellular protein Sparc in tendon maturation and ageing.

Acute and chronic tendinopathies remain clinically challenging and tendons are predisposed to degeneration or injury with age. Despite the high prevalence of tendon disease in the elderly, our current understanding of the mechanisms underlying the age-dependent deterioration of tendon function remains very limited. Here, we show that Secreted protein acidic and rich in cysteine (Sparc) expression significantly decreases in healthy-aged mouse Achilles tendons. Loss of Sparc results in tendon collagen fibrillogenesis defects and Sparc-/- tendons are less able to withstand force in comparison with their respective wild type counterparts. On the cellular level, Sparc-null and healthy-aged tendon-derived cells exhibited a more contracted phenotype and an altered actin cytoskeleton. Additionally, an elevated expression of the adipogenic marker genes PPARγ and Cebpα with a concomitant increase in lipid deposits in aged and Sparc-/- tendons was observed. In summary, we propose that Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties favors lipid accretion in tendons.

Flexor Tendon Sheath Engineering Using Decellularized Porcine Pericardium.

BACKGROUND: The flexor tendon sheath is an ideal target for tissue engineering because it is difficult to reconstruct by conventional surgical methods. The authors hypothesized that decellularized porcine pericardium can be used as a scaffold for engineering a biologically active tendon sheath. METHODS: The authors' protocol removed cellular material from the pericardium and preserved the structural architecture in addition to the collagen and glycosaminoglycan content. The scaffold was successfully reseeded with human sheath synoviocytes and human adipose-derived stem cells. Cells were evaluated for 8 weeks after reseeding. RESULTS: The reseeded construct demonstrated continuous production of hyaluronic acid, the main component of synovial fluid. After being seeded on the membrane, adipose-derived stem cells demonstrated down-regulation of collagen I and III and up-regulation of hyaluronan synthase 2. CONCLUSION: The results indicate that decellularized porcine pericardium may be a potential scaffold for engineering a biologically active human tendon sheath.

Effects of immobilization and whole-body vibration on rat serum Type I collagen turnover.

OBJECTIVE: The aim of this study was to investigate the effects of short-term, high-magnitude whole-body vibration (WBV) on serum type I collagen turnover in immobilized rats. MATERIALS AND METHODS: Thirty Wistar albino rats were randomly divided into the following 5 groups: immobilization (IS), immobilization + remobilization (IR), immobilization + WBV (IV), control (C), and WBV control (CV). Immobilization was achieved by casting from the crista iliaca anterior superior to the lower part of the foot for 2 weeks. The applied WBV protocol involved a frequency of 45 Hz and amplitude of 3 mm for 7 days starting a day after the end of the immobilization period. Serum type I collagen turnover markers were measured by using ELISA kits. RESULTS: Serum NH2-terminal propeptide of type I collagen (PINP) levels were significantly lower in the immobilization groups (p < 0.02) compared with the control groups. Although WBV improved PINP levels in the control groups, there were no differences in PINP levels among the immobilization groups. Similarly, serum COOH-terminal telopeptide of type I collagen (CTX) levels were higher in the WBV controls than their own controls (p < 0,05). Immobilization led to deterioration of tendon tissue, as observed by histopathological analysis with a transmission electron microscope. CONCLUSION: Although 1 week of WBV had a positive effect on type I collagen turnover in controls, it is not an efficient method for repairing tissue damage in the early stage following immobilization.

The development of confocal arthroscopy as optical histology for rotator cuff tendinopathy.

MRI, ultrasound and video arthroscopy are traditional imaging technologies for noninvasive or minimal invasive assessment of the rotator cuff tendon pathology. However, these imaging modalities do not have sufficient resolution to demonstrate the pathology of rotator cuff tendons at a microstructural level. Therefore, they are insensitive to low-level tendon diseases. Although traditional histology can be used to analyze the physiology of rotator cuff tendons, it requires biopsy that traumatizes the rotator cuff, thus, potentially comprising the mechanical properties of tendons. Besides, it cannot offer real-time histological information. Confocal endoscopy offers a way to assess the microstructural disorder in tissues without biopsy. However, the application of this useful technique for detecting low-level tendon diseases has been restricted by using clinical grade fluorescent contrast agent to acquire high-resolution microstructural images of tendons. In this study, using a clinical grade sodium fluorescein contrast agent, we have reported the development of confocal arthroscopy for optical histological assessment without biopsy. The confocal arthroscopic technique was able to demonstrate rotator cuff tendinopathy in human cadavers, which appeared macroscopically normal under video arthroscopic examinations. The tendinopathy status of the rotator cuff tendons was confirmed by corresponding traditional histology. The development of confocal arthroscopy may provide a minimally invasive imaging technique for real-time histology of rotator cuff without the need for tissue biopsy. This technique has the potential for surgeons to gain in real time the histological information of rotator cuff tendons, which may assist planning repair strategies and potentially improve intervention outcomes.

Macrophage-like U937 cells recognize collagen fibrils with strain-induced discrete plasticity damage.

At its essence, biomechanical injury to soft tissues or tissue products means damage to collagen fibrils. To restore function, damaged collagen must be identified, then repaired or replaced. It is unclear at present what the kernel features of fibrillar damage are, how phagocytic or synthetic cells identify that damage, and how they respond. We recently identified a nanostructural motif characteristic of overloaded collagen fibrils that we have termed discrete plasticity. In this study, we have demonstrated that U937 macrophage-like cells respond specifically to overload-damaged collagen fibrils. Tendons from steer tails were bisected, one half undergoing 15 cycles of subrupture mechanical overload and the other serving as an unloaded control. Both halves were decellularized, producing sterile collagen scaffolds that contained either undamaged collagen fibrils, or fibrils with discrete plasticity damage. Matched-pairs were cultured with U937 cells differentiated to a macrophage-like form directly on the substrate. Morphological responses of the U937 cells to the two substrates-and evidence of collagenolysis by the cells-were assessed using scanning electron microscopy. Enzyme release into medium was quantified for prototypic matrix metalloproteinase-1 (MMP-1) collagenase, and MMP-9 gelatinase. When adherent to damaged collagen fibrils, the cells clustered less, showed ruffled membranes, and frequently spread: increasing their contact area with the damaged substrate. There was clear structural evidence of pericellular enzymolysis of damaged collagen-but not of control collagen. Cells on damaged collagen also released significantly less MMP-9. These results show that U937 macrophage-like cells recognize strain-induced discrete plasticity damage in collagen fibrils: an ability that may be important to their removal or repair.

Characterization of deposits in patients with calcific tendinopathy of the supraspinatus. Role of phytate and osteopontin.

Calcific tendinopathy of the tendons of the rotator cuff is common in adults. These calcifications tend to be reabsorbed after a period of acute pain. This study evaluated the morphologic characteristics of calcific deposits and the participation of phytate and osteopontin (OPN) in their development. Calcific deposits were removed from 21 patients with calcific tendinopathy by ultrasound-guided needle puncture under local anesthesia. The removed deposits were evaluated by scanning electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. The amounts of calcium and phosphorus in the deposits were semi-quantitatively determined by energy dispersive X-ray analysis. Phytate was determined in 2 h urine samples, and OPN was extracted from a pool of deposits. The calcific deposits consisted of amorphous and poorly crystalline carbonated hydroxyapatite containing molecular water and organic matter. OPN was associated with the hydroxyapatite deposits. Phytate concentrations were significantly lower in the urine of patients with calcific tendinopathy than in healthy controls. The deficit in crystallization inhibitors such as phytate, and the presence of regulators such as OPN, may play important roles in the development of calcific tendinopathy.

Scleraxis-overexpressed human embryonic stem cell-derived mesenchymal stem cells for tendon tissue engineering with knitted silk-collagen scaffold.

AIM: Despite our previous study that demonstrates that human embryonic stem cells (hESCs) can be used as seed cells for tendon tissue engineering after stepwise induction, suboptimal tendon regeneration implies that a new strategy needs to be developed for tendon repair. We investigated whether overexpression of the tendon-specific transcription factor scleraxis (SCX) in hESC-derived mesenchymal stem cells (hESC-MSCs) together with knitted silk-collagen sponge scaffold could promote tendon regeneration. METHODS AND RESULTS: hESCs were initially differentiated into MSCs and then engineered with scleraxis (SCX+hESC-MSCs). Engineered tendons were constructed with SCX+hESC-MSCs and a knitted silk-collagen sponge scaffold and then mechanical stress was applied. SCX elevated tendon gene expression in hESC-MSCs and concomitantly attenuated their adipogenic and chondrogenic potential. Mechanical stress further augmented the expression of tendon-specific genes in SCX+hESC-MSC-engineered tendon. Moreover, in vivo mechanical stimulation promoted the alignment of cells and increased the diameter of collagen fibers after ectopic transplantation. In the in vivo tendon repair model, the SCX+hESC-MSC-engineered tendon enhanced the regeneration process as shown by histological scores and superior mechanical performance compared with control cells, especially at early stages. CONCLUSION: Our study offers new evidence concerning the roles of SCX in tendon differentiation and regeneration. We demonstrated a novel strategy of combining hESCs, genetic engineering, and tissue-engineering principles for tendon regeneration, which are important for the future application of hESCs and silk scaffolds for tendon repair.