Fibrocartilaginous Tissue Research Articles

Annexin A5 Involvement in Bone Overgrowth at the Enthesis.

Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5-deficient (Anxa5-/- ) mice, the sizes of bone ridge outgrowths at the entheses of the tibias and femur were increased after age 7 weeks. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP-expressing cells were observed in the fibrocartilage layer in Anxa5-/- mice than in wild-type (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5-/- mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5-/- mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail-suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5-/- and WT mice at 8 weeks after hindlimb unloading. These results suggest that bone overgrowth at the enthesis requires mechanical forces. We further examined effects of Anxa5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. Anxa5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in Anxa5-/- mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators. © 2018 American Society for Bone and Mineral Research.

Engineering Stem and Stromal Cell Therapies for Musculoskeletal Tissue Repair.

Stem cells and tissue-derived stromal cells stimulate the repair of degenerated and injured tissues, motivating a growing number of cell-based interventions in the musculoskeletal field. Recent investigations have indicated that these cells are critical for their trophic and immunomodulatory role in controlling endogenous cells. This Review presents recent clinical advances where stem cells and stromal cells have been used to stimulate musculoskeletal tissue repair, including delivery strategies to improve cell viability and retention. Emerging bioengineering strategies are highlighted, particularly toward the development of biomaterials for capturing aspects of the native tissue environment, altering the healing niche, and recruiting endogenous cells.

Proliferating Cells in Knee Epiphyses of Lizards Allow for Somatic Growth and Regeneration after Damage

After bone damage, fracture or amputation, lizards regenerate a variable mass of cartilaginous and fibro-cartilaginous tissues, depending from the anatomical site and intensity of inflammation. Aside tail and vertebrae, also long bones and knee epiphyses can regenerate a relative large mass of cartilage after injury. Regeneration is likely related to the persistence of stem cells in growing centers of these bones, localized in the epiphyses of femur, tibia and fibula. The epiphyses form ossified secondary centers in adults but a few progenitor cells remain in the articular cartilage and growth plate, allowing a continuous growth during most lifetime of lizards. The present Review indicates that putative progenitor/stem cells, identified by long labeling retaining of 5-bromo-deoxy-uridine (5BrdU) and immunolocalization of telomerase, remain localized in the articular cartilage and growth plates of the femur and tibia. These cells are re-activated after limited epiphyses damage or amputation of the distal part of the femur or tibia-fibula, and can re-form cartilaginous epiphyses. Regenerating chondrocytes show an intense proliferation and the production of new extracellular matrix components such as collagen VI, chondroitin sulfate proteoglycan, and hyaluronate receptors. The molecular factors at the origin of the chondrogenic potential of the articular cartilage, growth plates, and the periosteum in lizard bones remain to be studied.

The Effects of Bone Morphogenetic Protein‐7 and Interleukin‐1 on Biomechanical Properties of Meniscus Explants

IntroductionThe meniscus is a wedge, C‐shape fibrocartilaginous tissue that is essential for the normal biomechanical function of the knee. Damage to the meniscus leads to degenerative changes and loss of function mechanical function of the knee. Cytokines like interleukin‐1 (IL‐1) can inhibit repair process. However, anabolic factors like bone morphogenetic protein‐7 (BMP‐7) may help aid in the repair process. The goal of this project is to observe the effects of BMP‐7 and IL‐1 on the biomechanical properties of the meniscus using microindentation test.MethodSix‐millimeter porcine meniscus explants were cut using a biopsy punch and cultured in 1% fetal bovine serum media for seven days. Explants were cultured without treatment (control group) or treated with BMP‐7, IL‐1 alpha, and or combined with BMP‐7 and IL‐1 alpha. Microindentation test was used to test the elasticity of the explant tissues.ResultsThe elasticity for the explants treated with BMP‐7 (2.480 MPa) were significant compared to explants treated with either IL‐1 alpha (1.501 MPa; p= .001) or both BMP‐7 and IL‐1 alpha (.866; p ≤ .000). This indicates BMP‐7 and IL‐1 have a significant effect on the meniscus biomechanical properties. BMP‐7 increase the elasticity (but not significant) compared to the control explants (2.072 MPa; p= .065), while IL‐1significantly decrease the elasticity (p= .017).ConclusionThis data shows that BMP‐7 has the potential to exert an anabolic effect on cultured meniscus explants reflected by the presence of increased mechanical compression properties. As a result, the meniscus explant tissues that were cultured with BMP‐7 became more elastic after being cultured for seven days. In addition, meniscus cultured with IL‐1 alpha or a combination of BMP‐7 and IL‐1 alpha became less elastic. Future experiments will continue testing the effect of these two treatments.

3D-Printed Poly(ε-caprolactone) Scaffold Augmented With Mesenchymal Stem Cells for Total Meniscal Substitution: A 12- and 24-Week Animal Study in a Rabbit Model

Background: Total meniscectomy leads to knee osteoarthritis in the long term. The poly(ε-caprolactone) (PCL) scaffold is a promising material for meniscal tissue regeneration, but cell-free scaffolds result in relatively poor tissue regeneration and lead to joint degeneration. Hypothesis: A novel, 3-dimensional (3D)–printed PCL scaffold augmented with mesenchymal stem cells (MSCs) would offer benefits in meniscal regeneration and cartilage protection. Study Design: Controlled laboratory study. Methods: PCL meniscal scaffolds were 3D printed and seeded with bone marrow–derived MSCs. Seventy-two New Zealand White rabbits were included and were divided into 4 groups: cell-seeded scaffold, cell-free scaffold, sham operation, and total meniscectomy alone. The regeneration of the implanted tissue and the degeneration of articular cartilage were assessed by gross and microscopic (histological and scanning electron microscope) analysis at 12 and 24 weeks postoperatively. The mechanical properties of implants were also evaluated (tensile and compressive testing). Results: Compared with the cell-free group, the cell-seeded scaffold showed notably better gross appearance, with a shiny white color and a smooth surface. Fibrochondrocytes with extracellular collagen type I, II, and III and proteoglycans were found in both seeded and cell-free scaffold implants at 12 and 24 weeks, while the results were significantly better for the cell-seeded group at week 24. Furthermore, the cell-seeded group presented notably lower cartilage degeneration in both femur and tibia compared with the cell-free or meniscectomy group. Both the tensile and compressive properties of the implants in the cell-seeded group were significantly increased compared with those of the cell-free group. Conclusion: Seeding MSCs in the PCL scaffold increased its fibrocartilaginous tissue regeneration and mechanical strength, providing a functional replacement to protect articular cartilage from damage after total meniscectomy. Clinical Relevance: The study suggests the potential of the novel 3D PCL scaffold augmented with MSCs as an alternative meniscal substitution, although this approach requires further improvement before being used in clinical practice.

Biological Augmentation of Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) injury is a common sports injury. Generally, arthroscopic ACL reconstruction is performed because of the poor self-healing ability of the injured ACL. In the healing process after ACL reconstruction, tendon graft remodeling and incorporation at the tendon-bone junction are important factors for healing success. It is commonly accepted that the strength of the tendon graft attenuates immediately after ACL reconstruction and gradually increases thereafter. This gradual maturation of the grafted tendon is considered a cause for graft failure or elongation after ACL reconstruction. On the other hand, the bone-tendon junction is the weakest region after ACL reconstruction. In native anatomy, fibrocartilaginous tissue binds the ACL and bone, but this tissue does not undergo remodeling during reconstruction. A fibrous scar-like tissue often binds the grafted tendon and bone, ultimately affecting the stability of the graft in the bone tunnel and contributing to ACL rerupture. To solve this and other issues related to ACL reconstruction, extensive basic research has been conducted in recent years regarding biological or tissue augmentation, including studies on stem cells, growth factors, cytokines, and synthetic grafts. Alternatives for quicker graft maturation and more robust tendon-bone junction remodeling have been reported in animal models. These investigative results show promise for more aggressive rehabilitation techniques in the near future that will facilitate an earlier return to sports for patients with ACL injury.

Сurrent concepts in physiological and reparative osteogenesis

Studies conducted in the recent years by biologists strongly suggest that physiological and reparative osteogenesis, as well as of the functional, adaptive, and post-traumatic reconstruction of bone tissues are based on common and stereotypical molecular and cellular mechanisms. Our experimental studies have shown that all stages of the bone microstructure morphogenesis are synchronously and continuously associated with focal and stereotypical angiogenesis (capillarogenesis). A powerful factor in the implementation of reparative osteogenesis is the osteoinductive interaction of the ends of the damaged bone segments, which positively shows itself even in cases of large diastasis between the fragments (provided that the fragments are steadily fixed). After any kind of stable osteosynthesis, by ensuring the stability of the bone fragments for the entire period of consolidation, an endosteal cortical bone regeneration by direct osteogenesis (i.e. without fibro-cartilaginous tissue) is observed in the minimum amount at the shortest time period. Periosteal bone formation in this case is actually a reserve source of bone formation, which becomes effective during insufficiently stable conditions. The instability of the bone damage area particularly that of the metal implants results in the most severe destructive consequences.

Efficacy of mesenchymal stem cells in healing of meniscus tear

Background: The meniscus is a semilunar fibro-cartilaginous tissue performing various function including knee joint stability, shock absorption, load distribution, joint lubrication, and joint congruity. Menisectomy often leads to articular cartilage degeneration and ends up in osteoarthritis of knee. Authors described use of mesenchymal stem cells for evaluation of healing of meniscus tear. Purpose: To evaluate the efficacy of mesenchymal stem cells in healing of meniscal tear. Study design: Prospective, randomised, case-control double blinded study. Methods: A total number of 30 patients with meniscus tear of knee, were selected on the basis of pre-defined inclusion and exclusion criteria after informed written consent. Both cases and controls were 15 in numbers. No case were lost to follow- up.MRI was done twice pre and post intervention. In case group autologus bone marrow was aspirated,cultured and injected percutaneously under ultrasound guidance. Whereas in control group normal saline was injected as placebo. All patients received standard physiotherapy. Objective assessment was done by functional scoring systems (WOMAC, KOOS, VAS SCORE) Results: There was significant reduction in pain scores (VAS scores) in case group (p=.001) as compared to control group (p=.166).There was improvement in functional outcome scores such as WOMAC in case group (P=.001) as compared to control group(p=.419) and Global KOOS score in case group (p=.001) compared to control group(p=.910). Significant improvement was noted in MRI signals in case group as compared to control group (p=.013) depicting healing of meniscal tears with injection of mesenchymal stem cells. Conclusions: Mesenchymal stem cells injection is efficacious for healing of meniscal tear which reduces pain, helps in integration of torn meniscus and improves functional outcome.

Combining a novel leucocyte-platelet-concentrated membrane and an injectable collagen scaffold in a single-step AMIC procedure to treat chondral lesions of the knee: a preliminary retrospective study.

Different surgical approaches are currently available to treat knee chondral defects. Microfracture is the most commonly applied, but it often leads to a mechanically inferior fibrocartilaginous tissue. To overcome this shortcoming, the Autologous, Matrix-Induced Chondrogenesis (AMIC) technique has been proposed. To further enhance the outcome of AMIC, the addition of haemoderivatives containing growth factors that stimulate cartilage healing has emerged as a new treatment method. Recently, a novel leucocyte-platelet-concentrated membrane (CLP-MB), highly enriched in platelets, monocytes/macrophages, fibrinogen, CD34+ and CD34+VEGFR-2+CD133+ cells, has been developed. Additionally, an injectable collagen scaffold (Cartifill) has been proposed as a replacement of the AMIC standard collagen membrane. This preliminary study is aimed to evaluate the short-term safety and efficacy of the use of the CLP-MB membrane and injectable collagen scaffold when combined in single-step AMIC procedures for the treatment of knee chondral lesions. Medical records of patients who underwent an AMIC procedure with the CLP-MB membrane combined with Cartifill were reviewed retrospectively. Follow-up assessments were conducted at 6 and 12months after surgery. Clinical function was assessed on the basis of the International Knee Documentation Committee (IKDC) score. Pain was evaluated using the visual analogue scale (VAS). Twenty-five patients were identified as meeting the inclusion criteria. Mean IKDC and VAS scores significantly improved during the follow-up time. The postoperative course was uneventful. AMIC combined with the CLP-MB membrane, and Cartifill seems to be a promising approach to treat knee chondral defects.

Anisotropic Shape-Memory Alginate Scaffolds Functionalized with Either Type I or Type II Collagen for Cartilage Tissue Engineering.

Regenerating articular cartilage and fibrocartilaginous tissue such as the meniscus is still a challenge in orthopedic medicine. While a range of different scaffolds have been developed for joint repair, none have facilitated the development of a tissue that mimics the complexity of soft tissues such as articular cartilage. Furthermore, many of these scaffolds are not designed to function in mechanically challenging joint environments. The overall goal of this study was to develop a porous, biomimetic, shape-memory alginate scaffold for directing cartilage regeneration. To this end, a scaffold was designed with architectural cues to guide cellular and neo-tissue alignment, which was additionally functionalized with a range of extracellular matrix cues to direct stem cell differentiation toward the chondrogenic lineage. Shape-memory properties were introduced by covalent cross-linking alginate using carbodiimide chemistry, while the architecture of the scaffold was modified using a directional freezing technique. Introducing such an aligned pore structure was found to improve the mechanical properties of the scaffold, and promoted higher levels of sulfated glycosaminoglycans (sGAG) and collagen deposition compared to an isotropic (nonaligned) pore geometry when seeded with adult human stem cells. Functionalization with collagen improved stem cell recruitment into the scaffold and facilitated more homogenous cartilage tissue deposition throughout the construct. Incorporating type II collagen into the scaffolds led to greater cell proliferation, higher sGAG and collagen accumulation, and the development of a stiffer tissue compared to scaffolds functionalized with type I collagen. The results of this study demonstrate how both scaffold architecture and composition can be tailored in a shape-memory alginate scaffold to direct stem cell differentiation and support the development of complex cartilaginous tissues.

Augmented repair of radial meniscus tear with biomimetic electrospun scaffold: an in vitro mechanical analysis.

BackgroundLarge radial tears that disrupt the circumferential fibers of the meniscus are associated with reduced meniscal function and increased risk of joint degeneration. Electrospun fibrous scaffolds can mimic the topography and mechanics of fibrocartilaginous tissues and simultaneously serve as carriers of cells and growth factors, yet their incorporation into clinically relevant suture repair techniques for radial meniscus tears is unexplored. The purposes of this study were to (1) evaluate the effect of fiber orientation on the tensile properties and suture-retention strength of multilayered electrospun scaffolds and (2) determine the mechanical effects of scaffold inclusion within a surgical repair of a simulated radial meniscal tear. The experimental hypothesis was that augmentation with a multilayered scaffold would not compromise the strength of the repair.MethodsThree multilayered electrospun scaffolds with different fiber orientations were fabricated–aligned, random, and biomimetic. The biomimetic scaffold was comprised of four layers in the following order (deep to superficial)–aligned longitudinal, aligned transverse, aligned longitudinal, and random–respectively corresponding to circumferential, radial, circumferential, and superficial collagen fibers of the native meniscus. Material properties (i.e., ultimate stress, modulus, etc.) of the scaffolds were determined in the parallel and perpendicular directions, as was suture retention strength. Complete radial tears of lateral bovine meniscus explants were repaired with a double horizontal mattress suture technique, with or without inclusion of the biomimetic scaffold sheath. Both repair groups, as well as native controls, were cyclically loaded between 5 and 20 N for 500 cycles and then loaded to failure. Clamp-to-clamp distance (i.e., residual elongation) was measured following various cycles. Ultimate load, ultimate elongation, and stiffness, were also determined. Group differences were evaluated by one-way ANOVA or Student’s t-test where appropriate.ResultsAligned scaffolds possessed the most anisotropic mechanical properties, whereas random scaffolds showed uniform properties in the parallel and perpendicular directions. In comparison, the biomimetic scaffold possessed moduli in the parallel (68.7 ± 14.7 MPa) and perpendicular (39.4 ± 11.6 MPa) directions that respectively approximate the reported circumferential and radial tensile properties of native menisci. The ultimate suture retention load of the biomimetic scaffold in the parallel direction (7.2 ± 1.6 N) was significantly higher than all other conditions (p < 0.001). Biomimetic scaffold augmentation did not compromise mechanical properties when compared against suture repair in terms of residual elongation after 500 cycles (scaffold: 5.05 ± 0.89 mm vs. repair: 4.78 ± 1.24 mm), ultimate failure load (137.1 ± 31.0 N vs. 124.4 ± 21.4 N), ultimate elongation (12.09 ± 5.89 mm vs. 10.14 ± 4.61 mm), and stiffness (20.8 ± 3.6 vs. 18.4 ± 4.7 N/mm).ConclusionsWhile multilayered scaffold sheets were successfully fabricated to mimic the ultrastructure and anisotropic tensile properties of native menisci, improvements in suture retention strength or adoption of superior surgical techniques will be needed to further enhance the mechanical strength of repairs of radial meniscal tears.

The use of a cartilage decellularized matrix scaffold for the repair of osteochondral defects: the importance of long-term studies in a large animal model

To investigate the effect of decellularized cartilage-derived matrix (CDM) scaffolds, by itself and as a composite scaffold with a calcium phosphate (CaP) base, for the repair of osteochondral defects. It was hypothesized that the chondral defects would heal with fibrocartilaginous tissue and that the composite scaffold would result in better bone formation. After an 8-week pilot experiment in a single horse, scaffolds were implanted in eight healthy horses in osteochondral defects on the medial trochlear ridge of the femur. In one joint a composite CDM-CaPscaffold was implanted (+P), in the contralateral joint a CDM only (-P) scaffold. After euthanasia at 6 months, tissues were analysed by histology, immunohistochemistry, micro-CT, biochemistry and biomechanical evaluation. The 8-week pilot showed encouraging formation of bone and cartilage, but incomplete defect filling. At 6 months, micro-CT and histology showed much more limited filling of the defect, but the CaP component of the+P scaffolds was well integrated with the surrounding bone. The repair tissue was fibrotic with high collagen type I and low type II content and with no differences between the groups. There were also no biochemical differences between the groups and repair tissue was much less stiff than normal tissue (P<0.0001). The implants failed to produce reasonable repair tissue in this osteochondral defect model, although the CaP base in the-P group integrated well with the recipient bone. The study stresses the importance of long-term invivo studies to assess the efficacy of cartilage repair techniques.

Regeneration of Articular Cartilage by Human ESC-Derived Mesenchymal Progenitors Treated Sequentially with BMP-2 and Wnt5a.

The success of cell‐based therapies to restore joint cartilage requires an optimal source of reparative progenitor cells and tight control of their differentiation into a permanent cartilage phenotype. Bone morphogenetic protein 2 (BMP‐2) has been extensively shown to promote mesenchymal cell differentiation into chondrocytes in vitro and in vivo. Conversely, developmental studies have demonstrated decreased chondrocyte maturation by Wingless‐Type MMTV Integration Site Family, Member 5A (Wnt5a). Thus, we hypothesized that treatment of human embryonic stem cell (hESC)‐derived chondroprogenitors with BMP‐2 followed by Wnt5a may control the maturational progression of these cells into a hyaline‐like chondrocyte phenotype. We examined the effects of sustained exposure of hESC‐derived mesenchymal‐like progenitors to recombinant Wnt5a or BMP‐2 in vitro. Our data indicate that BMP‐2 promoted a strong chondrogenic response leading to terminal maturation, whereas recombinant Wnt5a induced a mild chondrogenic response without promoting hypertrophy. Moreover, Wnt5a suppressed BMP‐2‐mediated chondrocyte maturation, preventing the formation of fibrocartilaginous tissue in high‐density cultures treated sequentially with BMP‐2 and Wnt5a. Implantation of scaffoldless pellets of hESC‐derived chondroprogenitors pretreated with BMP‐2 followed by Wnt5a into rat chondral defects induced an articular‐like phenotype in vivo. Together, the data establish a novel role for Wnt5a in controlling the progression from multipotency into an articular‐like cartilage phenotype in vitro and in vivo. Stem Cells Translational Medicine 2017;6:40–50

Role of scaffold mean pore size in meniscus regeneration.

Recently, meniscus tissue engineering offers a promising management for meniscus regeneration. Although rarely reported, the microarchitectures of scaffolds can deeply influence the behaviors of endogenous or exogenous stem/progenitor cells and subsequent tissue formation in meniscus tissue engineering. Herein, a series of three-dimensional (3D) poly(ε-caprolactone) (PCL) scaffolds with three distinct mean pore sizes (i.e., 215, 320, and 515μm) were fabricated via fused deposition modeling. The scaffold with the mean pore size of 215μm significantly improved both the proliferation and extracellular matrix (ECM) production/deposition of mesenchymal stem cells compared to all other groups in vitro. Moreover, scaffolds with mean pore size of 215μm exhibited the greatest tensile and compressive moduli in all the acellular and cellular studies. In addition, the relatively better results of fibrocartilaginous tissue formation and chondroprotection were observed in the 215μm scaffold group after substituting the rabbit medial meniscectomy for 12weeks. Overall, the mean pore size of 3D-printed PCL scaffold could affect cell behavior, ECM production, biomechanics, and repair effect significantly. The PCL scaffold with mean pore size of 215μm presented superior results both in vitro and in vivo, which could be an alternative for meniscus tissue engineering. Meniscus tissue engineering provides a promising strategy for meniscus regeneration. In this regard, the microarchitectures (e.g., mean pore size) of scaffolds remarkably impact the behaviors of cells and subsequent tissue formation, which has been rarely reported. Herein, three three-dimensional poly(ε-caprolactone) scaffolds with different mean pore sizes (i.e., 215, 320, and 515μm) were fabricated via fused deposition modeling. The results suggested that the mean pore size significantly affected the behaviors of endogenous or exogenous stem/progenitor cells and subsequent tissue formation. This study furthers our understanding of the cell-scaffold interaction in meniscus tissue engineering, which provides unique insight into the design of meniscus scaffolds for future clinical application.

Assessment of regeneration in meniscal lesions by use of mesenchymal stem cells derived from equine bone marrow and adipose tissue.

OBJECTIVE To assess the ability to regenerate an equine meniscus by use of a collagen repair patch (scaffold) seeded with mesenchymal stem cells (MSCs) derived from bone marrow (BM) or adipose tissue (AT). SAMPLE 6 female Hispano-Breton horses between 4 and 7 years of age; MSCs from BM and AT were obtained for the in vitro experiment, and the horses were subsequently used for the in vivo experiment. PROCEDURES Similarities and differences between MSCs derived from BM or AT were investigated in vitro by use of cell culture. In vivo assessment involved use of a meniscus defect and implantation on a scaffold. Horses were allocated into 2 groups. In one group, defects in the medial meniscus were treated with MSCs derived from BM, whereas in the other group, defects were treated with MSCs derived from AT. Defects were created in the contralateral stifle joint but were not treated (control samples). RESULTS Both types of MSCs had universal stem cell characteristics. For in vivo testing, at 12 months after treatment, treated defects were regenerated with fibrocartilaginous tissue, whereas untreated defects were partially repaired or not repaired. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that MSCs derived from AT could be a good alternative to MSCs derived from BM for use in regenerative treatments. Results also were promising for a stem cell-based implant for use in regeneration in meniscal lesions. IMPACT FOR HUMAN MEDICINE Because of similarities in joint disease between horses and humans, these results could have applications in humans.

Unravelling the Role of Mechanical Stimuli in Regulating Cell Fate During Osteochondral Defect Repair.

We have previously developed a computational mechanobiological model to explore the role of substrate stiffness and oxygen availability in regulating stem cell fate during spontaneous osteochondral defect repair. This model successfully simulated many aspects of the regenerative process, however it was unable to predict the spatial patterns of endochondral bone and fibrocartilaginous tissue formation observed during the latter stages of the repair process. It is hypothesised that this was because the mechanobiological model did not consider the role of tissue strain in regulating specific aspects of chondrocyte differentiation. To test this, our mechanobiological model was updated to include rules whereby intermediate levels of octahedral shear strain inhibited chondrocyte hypertrophy, while excessively high octahedral shear strains resulted in the formation of fibrocartilage. This model was used to simulate spontaneous osteochondral defect repair, where it correctly predicted the experimentally observed patterns of bone formation. Overall the results suggest that oxygen availability regulates chondrogenesis and endochondral ossification during the early phases of osteochondral defect repair, while direct mechanical cues play a greater role in regulating chondrocyte differentiation during the latter stages of this process. In particular, these results suggest that an appropriate loading regime can assist in promoting the development of stable hyaline cartilage during osteochondral defect repair.

Applying Electrospun Gelatin/Poly(lactic acid-co-glycolic acid) Bilayered Nanofibers to Fabrication of Meniscal Tissue Engineering Scaffold.

The menisci are fibrocartilaginous tissues composed primarily of an interlacing network of collagen fibers with nanoscale diameter. Electrospinning is a suitable process of producing nanoscale fibers that mimic collagen fibers. In this study, a bilayered scaffold (group B), which consists of a gelatin nanofiber mesh and a PLGA nanofiber mesh, has been fabricated through an electrospinning method. At the same time, we electrospun pure PLGA fibrous mesh (group A) and gelatin/PLGA composite fibrous mesh (group C) as control groups. In order to compare all scaffold morphologies, the scaffolds were imaged by SEM and some parameters were measured and analyzed as following: Diameters of fibrils are from the smallest of less than average 0.14 μm for group C to the biggest of nearly average 0.38 μm for group B. The scaffolds pore diameters are from average 4.9 μm for group A to average 11.2 μm for group B. Porosity rates show that the group B has the highest porosity rate at about 91%. The scaffolds' properties were compared and analyzed, including hydrophilicity property (water contact angle) and mechanical properties (tensile strength). The results of water contact angle showed the group B is the most hydrophil among the groups. The results of tensile strength showed the tensile strength of group C is the weakest among the groups. All the results showed significant differences between the groups. Finally, in vitro, the meniscal cells derived from New Zealand white rabbits menisci were seeded in the scaffolds. We observed the cells proliferation behavior in the scaffolds. All above demonstrates that a bi-layered gelatin/PLGA scaffold reveals not only concurrent effects of mechanics and cytocompatibility in a fibrous context, but also a promising scaffold for future meniscal repair strategies.

Micro-precise spatiotemporal delivery system embedded in 3D printing for complex tissue regeneration

Three dimensional (3D) printing has emerged as an efficient tool for tissue engineering and regenerative medicine, given its advantages for constructing custom-designed scaffolds with tunable microstructure/physical properties. Here we developed a micro-precise spatiotemporal delivery system embedded in 3D printed scaffolds. PLGA microspheres (μS) were encapsulated with growth factors (GFs) and then embedded inside PCL microfibers that constitute custom-designed 3D scaffolds. Given the substantial difference in the melting points between PLGA and PCL and their low heat conductivity, μS were able to maintain its original structure while protecting GF’s bioactivities. Micro-precise spatial control of multiple GFs was achieved by interchanging dispensing cartridges during a single printing process. Spatially controlled delivery of GFs, with a prolonged release, guided formation of multi-tissue interfaces from bone marrow derived mesenchymal stem/progenitor cells (MSCs). To investigate efficacy of the micro-precise delivery system embedded in 3D printed scaffold, temporomandibular joint (TMJ) disc scaffolds were fabricated with micro-precise spatiotemporal delivery of CTGF and TGFβ3, mimicking native-like multiphase fibrocartilage. In vitro, TMJ disc scaffolds spatially embedded with CTGF/TGFβ3-μS resulted in formation of multiphase fibrocartilaginous tissues from MSCs. In vivo, TMJ disc perforation was performed in rabbits, followed by implantation of CTGF/TGFβ3-μS-embedded scaffolds. After 4 wks, CTGF/TGFβ3-μS embedded scaffolds significantly improved healing of the perforated TMJ disc as compared to the degenerated TMJ disc in the control group with scaffold embedded with empty μS. In addition, CTGF/TGFβ3-μS embedded scaffolds significantly prevented arthritic changes on TMJ condyles. In conclusion, our micro-precise spatiotemporal delivery system embedded in 3D printing may serve as an efficient tool to regenerate complex and inhomogeneous tissues.

Engineering Human TMJ Discs with Protein-Releasing 3D-Printed Scaffolds

The temporomandibular joint (TMJ) disc is a heterogeneous fibrocartilaginous tissue positioned between the mandibular condyle and glenoid fossa of the temporal bone, with important roles in TMJ functions. Tissue engineering TMJ discs has emerged as an alternative approach to overcoming limitations of current treatments for TMJ disorders. However, the anisotropic collagen orientation and inhomogeneous fibrocartilaginous matrix distribution present challenges in the tissue engineering of functional TMJ discs. Here, we developed 3-dimensional (3D)–printed anatomically correct scaffolds with region-variant microstrand alignment, mimicking anisotropic collagen alignment in the TMJ disc and corresponding mechanical properties. Connective tissue growth factor (CTGF) and transforming growth factor beta 3 (TGFβ3) were then delivered in the scaffolds by spatially embedding CTGF- or TGFβ3-encapsulated microspheres (µS) to reconstruct the regionally variant fibrocartilaginous matrix in the native TMJ disc. When cultured with human mesenchymal stem/progenitor cells (MSCs) for 6 wk, 3D-printed scaffolds with CTGF/TGFβ3-µS resulted in a heterogeneous fibrocartilaginous matrix with overall distribution of collagen-rich fibrous structure in the anterior/posterior (AP) bands and fibrocartilaginous matrix in the intermediate zone, reminiscent of the native TMJ disc. High dose of CTGF/TGFβ3-µS (100 mg µS/g of scaffold) showed significantly more collagen II and aggrecan in the intermediate zone than a low dose (50 mg µS/g of scaffold). Similarly, a high dose of CTGF/TGFβ3-µS yielded significantly higher collagen I expression in the AP bands compared with the low-dose and empty µS. From stress relaxation tests, the ratio of relaxation modulus to instantaneous modulus was significantly smaller with CTGF/TGFβ3-µS than empty µS. Similarly, a significantly higher coefficient of viscosity was achieved with the high dose of CTGF/TGFβ3-µS compared with the low-dose and empty µS, suggesting the dose effect of CTGF and TGFβ3 on fibrocartilage formation. Together, our findings may represent an efficient approach to engineering the TMJ disc graft with anisotropic scaffold microstructure, heterogeneous fibrocartilaginous matrix, and region-dependent viscoelastic properties.

Subchondral bone remodeling: comparing nanofracture with microfracture. An ovine in vivo study

microfracture, providing direct stimulation of chondrogenic mesenchymal stem cells (MSCs) in the subchondral bone, remains the most frequently used primary cartilage repair technique. However, the newly formed type I collagen-rich fibrocartilaginous tissue has poor biomechanical properties and a tendency to degenerate. To overcome these limitations the nanofracture technique was introduced. Our purpose was to compare subchondral bone remodeling 6 months after microfracture versus nanofracture (subchondral needling) treatment in an ovine model. full-thickness chondral lesions were created in the load-bearing area of the medial femoral condyles in four adult sheep. Each animal was then treated on one side with microfracture and on the contralateral side with nanofracture. Subchondral bone remodeling was assessed by micro-CT using a Bruker(®) SKYSCAN and CTVOX 2.7 software (Bruker Corp., Billerica, MA, USA) for image reconstruction; trabecular bone density measurements were performed through a color-representation structure thickness analysis. at the six-month endpoint, the microfracture-treated samples showed limited perforation depth and cone-shaped channels with large diameters at the joint surface. The channel walls displayed a high degree of regularity with significant trabecular bone compaction leading to a sealing effect with limited communication with the surrounding trabecular canals. Condyles treated with nanofracture showed channels characterized by greater depth and smaller diameters and natural irregularities of the channel walls, absence of trabecular compaction around the perforation, remarkable communication with trabecular canals, and neo-trabecular remodeling inside the channels. nanofracture is an effective and innovative repair technique allowing deeper perforation into subchondral bone with less trabecular fragmentation and compaction when compared to microfracture; it results in better restoration of the normal subchondral bone architecture at six months. our data support the use of smaller-diameter and deeper subchondral bone perforation for MSC stimulation; this technique may prove to be an attractive alternative to standard microfracture procedures.