Fibrocartilage Tissue Research Articles

The role of laminins in cartilaginous tissues: from development to regeneration.

As a key molecule of the extracellular matrix, laminin provides a delicate microenvironment for cell functions. Recent findings suggest that laminins expressed by cartilage-forming cells (chondrocytes, progenitor cells and stem cells) could promote chondrogenesis. However, few papers outline the effect of laminins on providing a favorable matrix microenvironment for cartilage regeneration. In this review, we delineated the expression of laminins in hyaline cartilage, fibrocartilage and cartilage-like tissue (nucleus pulposus) throughout several developmental stages. We also examined the effect of laminins on the biological activities of chondrocytes, including adhesion, migration and survival. Furthermore, we scrutinized the potential influence of various laminin isoforms on cartilage-forming cells' proliferation and chondrogenic differentiation. With this information, we hope to facilitate the understanding of the spatial and temporal interactions between cartilage-forming cells and laminin microenvironment to eventually advance cell-based cartilage engineering and regeneration.

Endogenous PRRX1+ progenitors are present after full thickness cartilage injury but do not directly contribute to fibrocartilage or subchondral bone tissues during the repair process

Endogenous PRRX1+ progenitors are present after full thickness cartilage injury but do not directly contribute to fibrocartilage or subchondral bone tissues during the repair process

The combined use of kartogenin and platelet-rich plasma promotes fibrocartilage formation in the wounded rat Achilles tendon entheses.

ObjectivesAfter 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).MethodsWounds 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.ResultsHistological 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.ConclusionsOur 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.

Plantar Plate Pathology: A Review Article

The plantar plate is a thick fibrocartilage tissue located at the ball of the foot. Pathology of the plantar plate is frequently missed or misdiagnosed and additionally, can be challenging to treat. Current surgical techniques include direct and indirect repairs. The purpose of this article is to help better understand and review the evaluation and treatment of plantar plate tears.

A comparison of different bioinks for 3D bioprinting of fibrocartilage and hyaline cartilage

Cartilage is a dense connective tissue with limited self-repair capabilities. Mesenchymal stem cell (MSC) laden hydrogels are commonly used for fibrocartilage and articular cartilage tissue engineering, however they typically lack the mechanical integrity for implantation into high load bearing environments. This has led to increased interested in 3D bioprinting of cell laden hydrogel bioinks reinforced with stiffer polymer fibres. The objective of this study was to compare a range of commonly used hydrogel bioinks (agarose, alginate, GelMA and BioINK™) for their printing properties and capacity to support the development of either hyaline cartilage or fibrocartilage in vitro. Each hydrogel was seeded with MSCs, cultured for 28 days in the presence of TGF-β3 and then analysed for markers indicative of differentiation towards either a fibrocartilaginous or hyaline cartilage-like phenotype. Alginate and agarose hydrogels best supported the development of hyaline-like cartilage, as evident by the development of a tissue staining predominantly for type II collagen. In contrast, GelMA and BioINK™ (a PEGMA based hydrogel) supported the development of a more fibrocartilage-like tissue, as evident by the development of a tissue containing both type I and type II collagen. GelMA demonstrated superior printability, generating structures with greater fidelity, followed by the alginate and agarose bioinks. High levels of MSC viability were observed in all bioinks post-printing (∼80%). Finally we demonstrate that it is possible to engineer mechanically reinforced hydrogels with high cell viability by co-depositing a hydrogel bioink with polycaprolactone filaments, generating composites with bulk compressive moduli comparable to articular cartilage. This study demonstrates the importance of the choice of bioink when bioprinting different cartilaginous tissues for musculoskeletal applications.

GENERAL FEATURES OF CALCIUM PYROPHOSPHATE DEPOSITION DISEASE

Background. Calcium pyrophosphate deposition (CPPD) disease is due to deposition of calcium pyrophosphate to hyaline cartilage, fibrocartilage and soft tissues. Clinical presentation may be as acute arthritis, chronic arthritis and associated with osteoarthritis. CPPD can be idiopathic or secondary, due to different metabolic diseases. Objectives. The aim of this study was to evaluate the clinical characteristics, secondary causes and risk factors of symptomatic CPPD. Material and methods. Patients diagnosed with CPPD upon presentation for joint pain and/or limitation of motion in Cluj-Napoca Rheumatology Department, were prospectively included in the study. Demographic data, clinical features, laboratory parameters for secondary causes of CPPD and risk factors were recorded. Results. The study included 40 patients with CPPD. A percentage of 70% patients presented with acute arthritis. The knee was the most affected joint. During the assessment for secondary CPPD, three patients with hypomagnesaemia and one patient with hypophosphatasia were found. Screening for alkaptonuria was negative in all patients tested (30 patients). Regarding risk factors, 65% of patients were over 60 years old, osteoarthritis was associated in 80% of cases, joint injury in 2%, meniscectomy in 6%, diuretics use in 18%, family history of CPPD 2%. Conclusions. Acute arthritis of the knee is the most frequent form of CPPD presentation. Ageing and osteoarthritis are common risk factors for CPPD. Although secondary causes of CPPD are rare, screening is very important.

Kartogenin Enhances Tendon Graft And Bone Tunnel Healing In A Rat Model

Category: Basic Sciences/Biologics Introduction/Purpose: The normal tendon-bone junction (TBJ) is a strong structure protected by the fibrocartilage transition zone. This allows a gradual transition of mechanical forces between tendon and bone, thus decreasing stress-concentration effects. Healing of the TBJ interface after an injury is slow and even after healing the junction often lacks the transition zone. A study on human patients showed that even years after ACL reconstruction, patients had no fibrocartilage zone regeneration. This and other studies show that surgical repair alone does not restore the unique protective fibrocartilage transition zone. We have shown that KGN injection into injured rat Achilles tendon-bone junctions enhanced wound healing with restored fibrocartilage transition zone. Here, we examined the effects of KGN treatment, along with platelet-rich plasma (PRP), on tendon-bone tunnel healing in rats. Methods: KGN stock was prepared in DMSO and diluted to 100 μM with PRP. PRP was obtained from the blood of Sprague– Dawley rats and the platelet concentration in PRP was adjusted to 3 times over the baseline platelet concentration in whole blood. Thrombin (1 kU/mL), served as the PRP activator. 27 female rats (234˜268g) were used. A 1.5 mm tunnel was drilled at the distal end of tibia. Achilles tendon was resected and sutured in the tunnel. Rats were randomly divided into 1 of 3 treatment groups: Group A: 50µl KGN + PRP; Group B: 50µl PRP solution; Group C: control. Rats were sacrificed at 4, 8, and 12 weeks for histological analysis. Whole tibia with the tendon insertion were harvested, fixed in 10% formalin and decalcified in 10% EDTA. Tissue were sectioned and stained with Safranin O + Fast green, and immunostained for collagen types 1 and 2. Results: All animals were in good condition after surgery and complications were not present. Safranin O staining was higher in the KGN+PRP group than the other groups indicating more cartilage-like tissues regeneration in this group. Formation of the cartilage-like transitional zone was time dependent; i.e., it increased with increase in time (Fig. 1A-C). In contrast, both PRP and control groups had no cartilage-like tissues (Fig. 1D-I); in fact, some gaps in the control group were found in the tendon-bone interface after 4 and 8 weeks (Fig. 1G-I). Finally, the cartilage-like tissues in the KGN+PRP group also stained positive for both Col-1 and Col-2 indicating that these were fibrocartilage tissues (Fig. 2). Conclusion: Here we demonstrated that KGN promotes the formation of fibrocartilage-like interface between the tendon graft and bone tunnel. This result suggests that the delivery of KGN into the tendon-bone interface could be a promising, cell-free approach to augment the tendon-bone interface healing. PRP in this study, while not effective in promoting fibrocartilage formation of the interface in its own right, it functions as an effective carrier that supplies scaffolds and growth factors necessary for the enhancement of wound healing. Future research is required to determine the optimal KGN dosage regimens and the optimal delivery method (e.g. injection vs implantation).

Kartogenin

Category: Basic Sciences/Biologics Introduction/Purpose: Tendon-bone junction (TBJ) injuries are very common and optimal treatment modalities are lacking. TBJ injuries often heal without formation of the fibrocartilage transition zone increasing the risk of rerupture. Current treatments only attach tendons to bones but do not promote healing or regeneration of the fibrocartilage zone. Therefore, we developed a novel approach to heal wounded TBJ using a small molecule called kartogenin (KGN). KGN was previously shown to induce chondrogenesis of tendon stem/progenitor cells (TSCs) in vitro and enhance wound healing in injured rat TBJs in vivo after short- term treatment (2 weeks). It is not known whether these effects can be maintained longterm. Therefore, we studied the long term (3 months) effects of KGN on achilles-tendon-bone junction healing. Methods: KGN stock was prepared in DMSO and diluted to 100 μM with PRP solution. PRP was obtained from the blood of Sprague–Dawley rats and the platelet concentration in PRP was adjusted to 3 times higher than that in whole blood. Thrombin (10 kU/ml) served as the PRP activator. 42 female rats (2.5 – 3 months old) were used. 1 mm diameter defects were created at the Achilles tendon-bone junction area in each hind leg of all rats. Rats were divided into 3 treatment groups: Group 1 (KGN+PRP): KGN (100 μM) +25 μl PRP + 5 μl thrombin; Group 2 (PRP): 25 μl PRP + 5 μl thrombin; and Group 3 (Control): 30 μl saline. All the rats were sacrificed at 3 months, and hind legs harvested for analyses. N= 6 hind legs were used for histological and qPCR analysis. N = 16 hind legs were used for mechanical testing. Results: Single treatment of KGN+PRP demonstrates improved TBJ healing (Fig. 1A) compared to PRP only and control groups (Fig. 1B, C). Fibrocartilage-like tissue regeneration occurred only in the KGN+PRP treated group (Fig. 2A). These results were further confirmed by qPCR, which showed significantly higher expression of cartilage-related genes (Data not shown). Furthermore, the cartilage-like transition zone in the KGN+PRP group also stained positive for Col-I, Col-II, Scx and Sox-9 indicating the regeneration of fibrocartilage tissues in the KGN+PRP treated TBJ (Fig. 3A, B). Mechanical testing showed that the KGN+PRP (47±12 N) and PRP (50±13 N) groups had significantly higher ultimate strength than the controls (39±16 N). There was no significant difference in ultimate strength between the KGN+PRP group and PRP group. Conclusion: In this study, we have shown that KGN injection enhanced healing of the wounded TBJ by promoting the formation of a fibrocartilage transition zone. Thus, KGN can be used as a mode of cell free therapy to promote regeneration of the fibrocartilage transition zone in injured TBJs. PRP alone enhanced healing of the TBJ but did not induce fibrocartilage regeneration. PRP + KGN are advantageous due to the numerous growth factors located within PRP and PRPs ability to serve as a KGN carrier. Future research is needed to optimize KGN and PRP dosage regimens and the optimal delivery methods.

Physiologically Distributed Loading Patterns Drive the Formation of Zonally Organized Collagen Structures in Tissue-Engineered Meniscus.

The meniscus is a dense fibrocartilage tissue that withstands the complex loads of the knee via a unique organization of collagen fibers. Attempts to condition engineered menisci with compression or tensile loading alone have failed to reproduce complex structure on the microscale or anatomic scale. Here we show that axial loading of anatomically shaped tissue-engineered meniscus constructs produced spatial distributions of local strain similar to those seen in the meniscus when the knee is loaded at full extension. Such loading drove formation of tissue with large organized collagen fibers, levels of mechanical anisotropy, and compressive moduli that match native tissue. Loading accelerated the development of native-sized and aligned circumferential and radial collagen fibers. These loading patterns contained both tensile and compressive components that enhanced the major biochemical and functional properties of the meniscus, with loading significantly improved glycosaminoglycan (GAG) accumulation 200-250%, collagen accumulation 40-55%, equilibrium modulus 1000-1800%, and tensile moduli 500-1200% (radial and circumferential). Furthermore, this study demonstrates local changes in mechanical environment drive heterogeneous tissue development and organization within individual constructs, highlighting the importance of recapitulating native loading environments. Loaded menisci developed cartilage-like tissue with rounded cells, a dense collagen matrix, and increased GAG accumulation in the more compressively loaded horns, and fibrous collagen-rich tissue in the more tensile loaded outer 2/3, similar to native menisci. Loaded constructs reached a level of organization not seen in any previous engineered menisci and demonstrate great promise as meniscal replacements.

Implications of the calf musculature and Achilles tendon architectures for understanding the site of injury

Clinically the sites of Achilles Tendon (AT) overuse conditions can be divided into the tendon mid-portion and osteotendinous attachment. Purpose: We propose an anatomical analysis of the triceps surae musculotendon unit that could provide a possible anatomic explanation for these 2 sites of injury. Method: Twelve cadavers (age 74±7 years) were studied. In both legs, calf muscles (lateral gastrocnemius (LG), medial gastrocnemius (MG) and soleus) were dissected and their volumes measured. Fine saw cuts were made in the sagittal plane, either side of the midline of the calcaneus. Each strip contained the distal part of the tendon and its insertion, together with the superior tuberosity of the calcaneus. Trabecular architecture was analyzed from X-rays taken with Faxitron radiography. Histological sections of the enthesis and the thickness of the uncalcified fibrocartilage and the subchondral plate were evaluated. A finite element model of tendon coupled to a rupture index was developed to investigate the AT response to mechanical load. Results: Muscle volume was highest for the soleus, followed by the MG, and LG. Within the AT, the soleus fibers occupy the antero-medial parts, the MG fibers form the posterior lateral layer, yet the LG head fibers retain the antero-lateral part. The quantity of bone and the apparent trabecular thickness at the enthesis were greatest in the central part of the enthesis. Thickness of calcified fibrocartilage tissue was significantly greater in the central part than medially (P=0.04) and laterally (P=0.03). Uncalcified fibrocartilage was significantly thicker medially than laterally (P=0.02). Finally, finite element analysis showed that AT mechanical stress increased with muscle load and converged at 4.6–7.9cm of the enthesis. Conclusion: Our data suggest that the triceps surae musculotendon unit is composed of anatomically distinct parts that undergo non-uniform mechanical loading. There are two sites where potentially tendon mechanical stress increases, the medial/central portions of the enthesis and the tendon midportion.

Miscible Two Diseases in Old Age: Hypopituitarism and Pseudogout

Calcium pyrophosphate dihydrate deposition disease is a dis- ease characterized by collecting of crystals in hyaline carti- lage, fibrocartilage and soft tissues, related to many endo - crine and metabolic conditions, and seen more commonly in old age. Hypopituitarism may occur in old age with fatigue, decrease in libido, weight loss and musculoskeletal symptoms as a major cause of the overall condition impairment. As one gets older, the signs of these two diseases may progress even without any significant disease. The natural process which comes with old age, and pseudogout and hypopituitarism can mask one to another clinically and the symptoms of three situ- ation may be overlapping. This can lead to difficulty and delay in diagnosis. Our case was an elderly female patient diagnosed with hypopituitarism and pseudogout.

Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres.

Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.

Mechanobiological Assessment of TMJ Disc Surfaces: Nanoindentation and Transmission Electron Microscopy.

ABSTRACTObjectivesTemporomandibular disc is a mechanically robust fibrocartilage tissue exhibiting highly elastic compressive, shear, and tensile moduli with structurally dense extracellular matrix that supports functional loading of the joint. The aim of this study was to illustrate structural complexities of the superior and inferior disc surfaces, to demonstrate the robust mechanical ability of the disc as a whole may be due to depth-dependent regional/layered variation, and also to provide characterization data imperative for future tissue engineering efforts focused on restoring function to the joint.Material and MethodsNanoindentation was used to assess tissue zones in conjunction with detailed Transmission Electron Microscopy to define structural attributes that influence the temporomandibular disc function.ResultsThe disc architecture adjacent to the superior surface was shown to have three distinct regional segments within the interface layer: 1-a surface peripheral layer; 2-subsurface region; and 3-a layer of helical matrix bundles. The inferior surface displayed an interface layer (20 µm) that showed limited cell populations with little depth-dependent structural variation, a stiffer elastic modulus and reduced energy dissipation compared to the superior surface. These data indicate that the primary function of the inferior surface is resistance to compression rather than load distribution during joint motion.ConclusionsThese are the first works that demonstrate that the superior central surface of the he temporomandibular disc is structured in depth-dependent isometric layers, each of which provides different mechanical function supporting the bulk tissue’s properties. From a clinical perspective these data have potential to define regions susceptible to fatigue that may translate to diagnostic criteria to better define the stages of dysfunction.

Preparation and Characterization of a Novel Decellularized Fibrocartilage "Book" Scaffold for Use in Tissue Engineering.

At the tendon-to-bone insertion, there is a unique transitional structure: tendon, non-calcified fibrocartilage, calcified fibrocartilage, and bone. The reconstruction of this special graded structure after defects or damage is an important but challenging task in orthopedics. In particular, reconstruction of the fibrocartilage zone has yet to be successfully achieved. In this study, the development of a novel book-shape scaffold derived from the extracellular matrix of fibrocartilage was reported. Specifically, fibrocartilage from the pubic symphysis was obtained from rabbits and sliced into the shape of a book (dimensions: 10 mm × 3 mm × 1 mm) with 10 layers, each layer (akin to a page of a book) with a thickness of 100-μm. These fibrocartilage “book” scaffolds were decellularized using sequentially 3 freeze-thaw cycles, 0.1% Triton X-100 with 1.5 M KCl, 0.25% trypsin, and a nuclease. Histology and DNA quantification analysis confirmed substantial removal of cells from the fibrocartilage scaffolds. Furthermore, the quantities of DNA, collagen, and glycosaminoglycan in the fibrocartilage were markedly reduced following decellularization. Scanning electron microscopy confirmed that the intrinsic ultrastructure of the fibrocartilage tissue was well preserved. Therefore, the results of this study suggest that the novel “book” fibrocartilage scaffold could have potential applications in tissue engineering.

Macrophage-specific metalloelastase (MMP-12) immunoexpression in the osteochondral unit in osteoarthritis correlates with BMI and disease severity

Background: Metalloproteinase 12 (MMP-12) is induced in chondrocytes during fetal development and malignant transformation. Objectives: The aim of our study is to examine the expression of MMP-12 in the cartilage and the subchondral bone of patients with osteoarthritis (OA) and to correlate its expression with disease severity and anthropometric characteristics. Methods: Overall, 60 sections from 20 patients with idiopathic OA, were examined for the immunolocalization of MMP-12. As controls, we used the femoral heads of 4 patients treated with seniarthroplasty after fracture. Demographic characteristics and Body Mass Index (BMI) were calculated for all subjects. Results: Specimens were divided into four groups based on the Mankin histological severity score. The immunohistochemical study showed MMP-12 expression in the cartilage and subchonral bone of OA patients, while there was no expression in normal controls. At the moderate OA changes (Mankin score: 6–7), MMP-12 was detected mainly at the matrix of fibrocartilage tissue. During disease progression, MMP-12 was expressed at the sides of the cartilage and bone erosion and in the bone cysts. Furthermore, it was traced in the osteocytes of the subchondral bone. Osteoblast-like cells and bone lining cells express MMP-12 during the stage of severe OA (Mankin: ≥8). Osteoclasts expressing MMP-12 were also detected in the group of severe OA. Interestingly, MMP-12 expression was positively correlated with the age and the BMI of OA patients. Conclusion: The increased expression of MMP-12 in the bone-cartilage unit of OA patients suggests a possible role in OA pathogenesis and progression. Level of evidenceIII, prospective comparative study.

Association Between Individual Calf Muscle Volume And Achilles Tendon Enthesis Morphology

Although chronic Achilles tendinopathy (AT) is one of the most frequent injuries in sport, it is generally difficult to treat as the etiology and background to the pain mechanisms has not yet been clarified. Anatomically, as calf muscles travel distally, the AT internally rotate, consequently the initially posterior fibers of the soleus insert mainly on the medial aspect of the AT enthesis whereas those of the gatsrocnemuis insert mainly on the lateral aspect. Recently, it has been suggested that the AT undergoes non-uniform deformation under in vivo loading conditions PURPOSE: We investigated whether there are regional differences in calf muscle volumes, in the trabecular architecture of the calcaneus or in the histology of the AT enthesis that could suggest unequal force transmission from muscle to tendon and from tendon to bone. METHOD: We studied 12 cadavers (age 74 ± 7 years). For both legs, individual calf muscles (lateral gastrocnmius (LG), medial gastrocnemius (MG) and soleus (SL) were dissected and their volumes measured. Fine saw cuts were made in the sagittal plane, either side of the midline of the calcaneus, so that approximately 8-mm-wide strips of tissue were sampled from the insertion site. Each strip contained the distal part of the tendon and its insertion, together with the superior tuberosity of the calcaneus. Trabecular architecture was analysed from X-rays taken with a Faxitron radiography. Histological sections were made at the enthesis and regional differences in the thickness of the uncalcified fibrocartilage and the subchondral plate were evaluated. RESULTS: The radiological studies showed that the quantity of bone and the apparent trabecular thickness in the calcaneus were greatest in the center. The thickness of calcified fibrocartilage tissue was significantly greater in the central part of the enthesis than medially (P = 0.04) and laterally (P = 0.03). Uncalcified fibrocartilage was significantly thicker medially than laterally (P = 0.02). Muscle volume was highest (P <0.05) for the SL, followed by the GM, and GL. CONCLUSION: Overall, the results indicate that mechanical stress at the AT enthesis is asymmetrically distributed and greater on the medial than on the lateral side. Thus, we suggest that the functional anatomy of the AT is closely related to regional variations in force transmission, which in turn relates to the site of pain and pathology in AT.

Quantitative tracking of passage and 3D culture effects on chondrocyte and fibrochondrocyte gene expression.

Success in cartilage and fibrocartilage tissue engineering relies heavily on using an appropriate cell source. Many different cell sources have been identified, including primary and stem cells, along with experimental strategies to obtain the required number of cells or to induce chondrogenesis. However, no definitive method exists to quantitatively evaluate the similarity of the resulting cell phenotypes to those of the native cells between candidate strategies. In this study, we develop an integrative approach to enable such evaluations by deriving, from gene expression profiles, two quantitative metrics representing the nearest location within the range of native cell phenotypes and the deviation from it. As an example application to evaluating potential cell sources for cartilage or meniscus tissue engineering, we examine phenotypic changes of juvenile and adult articular chondrocytes and fibrochondrocytes across multiple passages and subsequent 3D culture. A substantial change was observed in cell phenotype due to the isolation process itself, followed by a clear progression toward the outer meniscal cell phenotype with passage. The new metrics also indicated that 3D culture moderately reduced the passage-induced deviation from the native meniscal phenotypes for juvenile chondrocytes and adult fibrochondrocytes, which was not obvious through examination of individual gene expressions. However, brief 3D culture alone did not move any of the cells towards an inner meniscal phenotype, the most relevant target for meniscal tissue engineering. This integrative approach of examining and combining multiple gene expressions can be used to evaluate various other tissue-engineering strategies to direct cells toward the desired phenotype. Copyright © 2015 John Wiley & Sons, Ltd.

TGIF1 Gene Silencing in Tendon-Derived Stem Cells Improves the Tendon-to-Bone Insertion Site Regeneration

Background/Aims: The slow healing process of tendon-to-bone junctions can be accelerated via implanted tendon-derived stem cells (TDSCs) with silenced transforming growth interacting factor 1 (TGIF1) gene. Tendon-to-bone insertion site is the special form of connective tissues derivatives of common connective progenitors, where TGF-β plays bidirectional effects (chondrogenic or fibrogenic) through different signaling pathways at different stages. A recent study revealed that TGF-β directly induces the chondrogenic gene Sox9. However, TGIF1 represses the expression of the cartilage master Sox9 gene and changes its expression rate against the fibrogenesis gene Scleraxis (Scx). Methods: TGIF1 siRNA was transduced or TGIF1 was over-expressed in tendon-derived stem cells. Following suprapinatus tendon repair, rats were either treated with transduced TDSCs or nontransduced TDSCs. Histologic examination and Western blot were performed in both groups. Results: In this study, the silencing of TGIF1 significantly upregulated the chondrogenic genes and markers. Similarly, TGIF1 inhibited TDSC differentiation into cartilage via interactions with TGF-β-activated Smad2 and suppressed the phosphorylation of Smad2. The area of fibrocartilage at the tendon-bone interface was significantly increased in the TGIF1 (-) group compared with the control and TGIF1-overexpressing groups in the early stages of the animal model. The interface between the tendon and bone showed a increase of new bone and fibrocartilage in the TGIF1 (-) group at 4 weeks. Fibrovascular scar tissue was observed in the TGIF1-overexpressing group and the fibrin glue only group. Low levels of fibrocartilage and fibrovascular scar tissue were found in the TDSCs group. Conclusion: Collectively, this study shows that the tendon-derived stem cell modified with TGIF1 gene silencing has promising effects on tendon-to-bone healing which can be further explored as a therapeutic tool in regenerative medicine.

The Effect of Rotating Collector Design on Tensile Properties and Morphology of Electrospun Polycaprolactone Fibres

Electrospinning is a technique that can produce fibres in the nanoscale range. This process is useful for many applications, including fabrication of fibrous scaffolds for fibrocartilage tissue engineering. For this application, cell attachment and tissue development is influenced by fibre morphology and mechanical properties. This electrospinning study investigated the influence of rotating collector design on morphology and mechanical properties of electrospun polycaprolactone fibre. The experiment employed 4 mandrel designs: 1) full surface of aluminium; 2) with gap feature; 3) with gap feature and teflon support; 4) with gap feature and tape support. The highest elastic modulus was obtained from mandrel with gap and tape support, which was 24.6 MPa and significantly higher compared to fibres acquired from other collector designs. Fibre diameter attained was identical across the different collectors, ranging from 0.5 - 2 μm. Gap introduction showed enhanced alignment in the resultant fibre. It can be concluded that fibre alignment and tensile properties can be improved by simply modifying the collector design. This improved fibre mat can be developed as a biomaterial for fibrocartilage tissue engineering scaffolds.

Injectable scaffold as minimally invasive technique for cartilage tissue engineering: in vitro and in vivo preliminary study.

Cartilage is a tissue with limited repair capacity and also sparse population of cells entrapped within a dense extracellular matrix, therefore, delivery of the cells to site of damaged cartilage can improve its healing potential. Synthetic biomaterials such as poly (d,l-lactide-co-glycolide) (PLGA) have been used as both preformed or injectable scaffolds in tissue engineering in order to carry and keep cells in the site of injury with minimal side effects. The injectable biocompatible polymeric scaffolds can reach to effected area via minimally invasive injection without need to open the joint, less painful approach and also having possibility to fill complicated shape defects. In this study, it was hypothesized that PLGA solved in n-methyl pyrrolidine (NMP) may act as a proper carrier for cell delivery to the site of the damage and also supports their growth. The results of in vitro assays including both live/dead (AO/PI) and MTT showed the majority of the cells were remained alive between 3 up to 21 days, respectively. The amount of resealed GAG from the mesenchymal stem cells (MSCs) which were in contact with both PLGA and alginate constructs (used as control) indicated that for day 7 MSCs in contact with alginate secreted more GAG (3.45 ± 0.453 µg/mL for alginate and 2.36 ± 0.422 µg/mL for PLGA matrices), but at longer times (21 days) cells in contact with PLGA elicited more GAG (6.26 ± 0.968 µg/mL for alginate and 8.47 ± 0.871 µg/mL for the PLGA matrices). Sol–gel systems comprising PLGA, NMP, and cells as well as alginate/cells were subcutaneously injected into four nude mice (each mouse had three injection sites). PLGA/NMP was solidify immediately and formed an interconnecting 3-D porous structure that allowed body fluid to penetrate through them. In vivo evaluation showed that PLGA/NMP scaffolds could support injected cells as a fibrocartilage tissue was formed after 6 months of injection. We found that PLGA/NMP system might be a proper minimally invasive therapeutics option for cartilage repair.