Regenerative Potential of Platelet Concentrate Lysate in Mechanically Injured Cartilage and Matrix-Associated Chondrocyte Implantation In Vitro

Adjuvant therapy in autologous chondrocyte implantation (ACI) can control the post-traumatic environment and guide graft maturation to support cartilage repair. To investigate both aspects, we examined potential chondro-regenerative effects of lysed platelet concentrate (PC) and supplementary interleukin 10 (IL-10) on mechanically injured cartilage and on clinically used ACI scaffolds. ACI remnants and human cartilage explants, which were applied to an uniaxial unconfined compression as injury model, were treated with human IL-10 and/or PC from thrombocyte concentrates. We analyzed nuclear blebbing/TUNEL, sGAG content, immunohistochemistry, and the expression of COL1A1, COL2A1, COL10A1, SOX9, and ACAN. Post-injuriously, PC was associated with less cell death, increased COL2A1 expression, and decreased COL10A1 expression and, interestingly, the combination with Il-10 or Il-10 alone had no additional effects, except on COL10A1, which was most effectively decreased by the combination of PC and Il-10. The expression of COL2A1 or SOX9 was statistically not modulated by these substances. In contrast, in chondrocytes in ACI grafts the combination of PC and IL-10 had the most pronounced effects on all parameters except ACAN. Thus, using adjuvants such as PC and IL-10, preferably in combination, is a promising strategy for enhancing repair and graft maturation of autologous transplanted chondrocytes after cartilage injury.

Multi-layered in vitro 3D-bone model via combination of osteogenic cell sheets with electrospun membrane interlayer

In this study, it was aimed to present an approach for the development of multi-layered tissue engineering constructs by using cell sheet engineering. Briefly, MC3T3-E1 mouse pre-osteoblast cells were cultured in temperature-responsive plates (Nunc Upcell®) in the presence of osteogenic medium and the resulting cell sheets were laminated with electrospun poly(L-lactic acid) (PLLA) membranes to obtain viable three-dimensional, thick constructs. The constructs prepared without PLLA membranes were used as control. The cell viability and death in the resulting structures were investigated by microscopic and colorimetric methods. The in vitro performance of the structures was discussed comparatively. Alkaline phosphatase (ALP) activity, collagen and sulfated glycosaminoglycan (sGAG) content values were calculated. The presented approach shows potential for engineering applications of complex tissues with at least two or more microenvironments such as osteochondral, corneal or vascular tissues.

Preliminary assessment of an injectable extracellular matrix from decellularized bovine myocardial tissue

The goal of this study was to develop an injectable form of decellularized bovine myocardial tissue matrix which could retain high levels of functional ECM molecules, and could gel at physiological temperature. Dissected ventricular tissue was processed by a detergent-based protocol, lyophilized, enzymatically-digested, and neutralized to form the injectable myocardial matrix (IMM). Histochemical analysis, DNA quantification, and agarose gel electrophoresis demonstrated the efficiency of the applied protocol. Chemical, thermal, morphological, and rheological characterization; protein and sulfated glycosaminoglycan (sGAG) content analysis were performed, in vitro biological properties were evaluated. An in vivo histocompatibility and biodegradability study was performed. Histochemistry revealed complete removal of myocardial cells. DNA content analysis revealed a significant decrease (87%) in the nuclear material, while protein and sGAG contents were highly preserved following decellularization. Soluble IMM was capable of turning into gel form at ∼37 °C, indicating selfassembling property. In vitro findings showed the biomaterial was noncytotoxic, nonhemolytic, and supported the attachment and proliferation of mesenchymal stem cells. In vivo study demonstrated IMM was well-tolerated by rats receiving subcutaneous injection. This work demonstrates that the IMM from decellularized bovine myocardial tissue has the potential for use as a feasible regenerative biomaterial in prospective tissue engineering and regenerative medicine studies.

Potential of Soluble Decellularized Extracellular Matrix for Musculoskeletal Tissue Engineering – Comparison of Various Mesenchymal Tissues

BackgroundIt is well studied that preparations of decellularized extracellular matrix (ECM) obtained from mesenchymal tissues can function as biological scaffolds to regenerate injured musculoskeletal tissues. Previously, we reported that soluble decellularized ECMs derived from meniscal tissue demonstrated excellent biocompatibility and produced meniscal regenerate with native meniscal anatomy and biochemical characteristics. We therefore hypothesized that decellularized mesenchymal tissue ECMs from various mesenchymal tissues should exhibit tissue-specific bioactivity. The purpose of this study was to test this hypothesis using porcine tissues, for potential applications in musculoskeletal tissue engineering.MethodsNine types of porcine tissue, including cartilage, meniscus, ligament, tendon, muscle, synovium, fat pad, fat, and bone, were decellularized using established methods and solubilized. Although the current trend is to develop tissue specific decellularization protocols, we selected a simple standard protocol across all tissues using Triton X-100 and DNase/RNase after mincing to compare the outcome. The content of sulfated glycosaminoglycan (sGAG) and hydroxyproline were quantified to determine the biochemical composition of each tissue. Along with the concentration of several growth factors, known to be involved in tissue repair and/or maturation, including bFGF, IGF-1, VEGF, and TGF-β1. The effect of soluble ECMs on cell differentiation was explored by combining them with 3D collagen scaffold culturing human synovium derived mesenchymal stem cells (hSMSCs).ResultsThe decellularization of each tissue was performed and confirmed both histologically [hematoxylin and eosin (H&E) and 4’,6-diamidino-2-phenylindole (DAPI) staining] and on the basis of dsDNA quantification. The content of hydroxyproline of each tissue was relatively unchanged during the decellularization process when comparing the native and decellularized tissue. Cartilage and meniscus exhibited a significant decrease in sGAG content. The content of hydroxyproline in meniscus-derived ECM was the highest when compared with other tissues, while sGAG content in cartilage was the highest. Interestingly, a tissue-specific composition of most of the growth factors was measured in each soluble decellularized ECM and specific differentiation potential was particularly evident in cartilage, ligament and bone derived ECMs.ConclusionIn this study, soluble decellularized ECMs exhibited differences based on their tissue of origin and the present results are important going forward in the field of musculoskeletal regeneration therapy.

Redifferentiation of Articular Chondrocytes by Hyperacute Serum and Platelet Rich Plasma in Collagen Type I Hydrogels

Matrix-assisted autologous chondrocyte transplantation (MACT) for focal articular cartilage defects often fails to produce adequate cartilage-specific extracellular matrix in vitro and upon transplantation results in fibrocartilage due to dedifferentiation during cell expansion. This study aimed to redifferentiate the chondrocytes through supplementation of blood-products, such as hyperacute serum (HAS) and platelet-rich plasma (PRP) in vitro. Dedifferentiated monolayer chondrocytes embedded onto collagen type I hydrogels were redifferentiated through supplementation of 10% HAS or 10% PRP for 14 days in vitro under normoxia (20% O2) and hypoxia (4% O2). Cell proliferation was increased by supplementing HAS for 14 days (p < 0.05) or by interchanging from HAS to PRP during Days 7–14 (p < 0.05). Sulfated glycosaminoglycan (sGAG) content was deposited under both HAS, and PRP for 14 days and an interchange during Days 7–14 depleted the sGAG content to a certain extent. PRP enhanced the gene expression of anabolic markers COL2A1 and SOX9 (p < 0.05), whereas HAS enhanced COL1A1 production. An interchange led to reduction of COL1A1 and COL2A1 expression marked by increased MMP13 expression (p < 0.05). Chondrocytes secreted less IL-6 and more PDGF-BB under PRP for 14 days (p < 0.0.5). Hypoxia enhanced TGF-β1 and BMP-2 release in both HAS and PRP. Our study demonstrates a new approach for chondrocyte redifferentiation.

Osmotic Swelling Responses are Conserved Across Cartilaginous Tissues with Varied Sulfated-Glycosaminoglycan Contents

The interactions between the negatively charged sulfated glycosaminoglycan (sGAG) chains and the ionic interstitial fluid in articular cartilage and meniscal fibrocartilage give rise to an osmotic swelling stress that is critical for the load-bearing capability of both tissues. This osmotic swelling stress is altered when the sGAG content is changed, as during progression of degenerative joint disease; understanding the influence of sGAG concentration on the osmotic swelling stress of cartilage and meniscus is important to enhance our understanding of physiology and disease. This study compared the effect of altered osmotic environments on the confined compression swelling behavior of bovine tissues spanning a range of sGAG concentrations: juvenile articular cartilage, juvenile and adult meniscus, and juvenile cartilage degraded to reduce sGAG content. The transient response to changes in bath conditions was evaluated for explants assigned to one of three compressive offsets (5%, 10%, or 15% strain) and one of three bath conditions (0.1X, 1X, or 10X Phosphate Buffered Saline). Our results show that relative responses to alterations to the osmotic environment are consistent across tissue types, demonstrating that the role of sGAG in the swelling properties of the tissues tested is conserved, even when sGAG is present at low concentrations. Additionally, this study found unexpected correlations across tissue types between sGAG and collagen contents and between the aggregate modulus and both sGAG and collagen contents. These results suggest some conservation of composition-function relationships across a range of tissue types.

Influence of Cartilaginous Matrix Accumulation on Viscoelastic Response of Chondrocyte/Agarose Constructs Under Dynamic Compressive and Shear Loading

A method has been developed to restore cartilage defects by culturing autologous chondrocytes to create a three dimensional tissue and then implanting the cultured tissue. In this kind of approach, it is important to characterize the dynamic mechanical behavior of the regenerated cartilaginous tissue, because these tissues need to bear various dynamic loadings in daily life. The objectives of this study were to evaluate in detail the dynamic viscoelastic responses of chondrocyte-seeded agarose gel cultures in compression and torsion (shear) and to determine the relationships between these mechanical responses and biochemical composition. The results showed that both the dynamic compressive and shear stiffness of the cultured constructs increased during culture. The relative energy dissipation in dynamic compression decreased, whereas that in dynamic shear increased during culture. Furthermore, correlation analyses showed that the sulfated glycosaminoglycan (sGAG) content of the cultured construct showed significant correlations with the dynamic modulus in both compression and shear situations. On the other hand, the loss tangent in dynamic compression, which represents the relative energy dissipation capability of the constructs, showed a low correlation with the sGAG content, whereas this capability in shear exhibited moderate correlation. In conclusion, we explored the dynamic viscoelasticity of the tissue-engineered cartilage in dynamic compression and shear, and determined correlations between viscoelasticity and biochemical composition.