Devitalizing Effect of High Hydrostatic Pressure on Human Cells-Influence on Cell Death in Osteoblasts and Chondrocytes.

Chemical and physical processing of allografts is associated with a significant reduction in biomechanics. Therefore, treatment of tissue with high hydrostatic pressure (HHP) offers the possibility to devitalize tissue gently without changing biomechanical properties. To obtain an initial assessment of the effectiveness of HHP treatment, human osteoblasts and chondrocytes were treated with different HHPs (100-150 MPa, 250-300 MPa, 450-500 MPa). Devitalization efficiency was determined by analyzing the metabolic activity via WST-1(water-soluble tetrazolium salt) assay. The type of cell death was detected with an apoptosis/necrosis ELISA (enzyme-linked immune sorbent assay) and flow cytometry. Field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) were carried out to detect the degree of cell destruction. After HHP treatment, the metabolic activities of both cell types decreased, whereas HHP of 250 MPa and higher resulted in metabolic inactivation. Further, the highest HHP range induced mostly necrosis while the lower HHP ranges induced apoptosis and necrosis equally. FESEM and TEM analyses of treated osteoblasts revealed pressure-dependent cell damage. In the present study, it could be proven that a pressure range of 250-300 MPa can be used for cell devitalization. However, in order to treat bone and cartilage tissue gently with HHP, the results of our cell experiments must be verified for tissue samples in future studies.

Development of a Tissue-Engineered Artificial Ligament: Reconstruction of Injured Rabbit Medial Collateral Ligament With Elastin-Collagen and Ligament Cell Composite Artificial Ligament.

Ligament reconstruction using a tissue-engineered artificial ligament (TEAL) requires regeneration of the ligament-bone junction such that fixation devices such as screws and end buttons do not have to be used. The objective of this study was to develop a TEAL consisting of elastin-coated polydioxanone (PDS) sutures covered with elastin and collagen fibers preseeded with ligament cells. In a pilot study, a ring-type PDS suture with a 2.5 mm (width) bone insertion was constructed with/without elastin coating (Ela-coat and Non-coat) and implanted into two bone tunnels, diameter 2.4 mm, in the rabbit tibia (6 cases each) to access the effect of elastin on the bond strength. PDS specimens taken together with the tibia at 6 weeks after implantation indicated growth of bone-like hard tissues around bone tunnels accompanied with narrowing of the tunnels in the Ela-coat group and not in the Non-coat group. The drawout load of the Ela-coat group was significantly higher (28.0 ± 15.1 N, n = 4) than that of the Non-coat group (7.6 ± 4.6 N, n = 5). These data can improve the mechanical bulk property of TEAL through extracellular matrix formation. To achieve this TEAL model, 4.5 × 106 ligament cells were seeded on elastin and collagen fibers (2.5 cm × 2.5 cm × 80 µm) prior to coil formation around the elastin-coated PDS core sutures having ball-shape ends with a diameter of 2.5 mm. Cell-seeded and cell-free TEALs were implanted across the femur and the tibia through bone tunnels with a diameter of 2.4 mm (6 cases each). There was no incidence of TEAL being pulled in 6 weeks. Regardless of the remarkable degradation of PDS observed in the cell-seeded group, both the elastic modulus and breaking load of the cell-seeded group (n = 3) were comparable to those of the sham-operation group (n = 8) (elastic modulus: 15.4 ± 1.3 MPa and 18.5 ± 5.7 MPa; breaking load: 73.0 ± 23.4 N and 104.8 ± 21.8 N, respectively) and higher than those of the cell-free group (n = 5) (elastic modulus: 5.7 ± 3.6 MPa; breaking load: 48.1 ± 11.3 N) accompanied with narrowed bone tunnels and cartilage matrix formation. These data suggest that elastin increased the bond strength of TEAL and bone. Furthermore, our newly developed TEAL from elastin, collagen, and ligament cells maintained the strength of the TEAL even if PDS was degraded.

Bioengineering strategies for bone and cartilage tissue regeneration using growth factors and stem cells.

Bone and cartilage tissue engineering is an integrative approach that is inspired by the phenomena associated with wound healing. In this respect, growth factors have emerged as important moieties for the control and regulation of this process. Growth factors act as mediators and control the important physiological functions of bone regeneration. Herein, we discuss the importance of growth factors in bone and cartilage tissue engineering, their loading and delivery strategies, release kinetics, and their integration with biomaterials and stem cells to heal bone fractures. We also highlighted the role of growth factors in the determination of the bone tissue microenvironment based on the reciprocal signaling with cells and biomaterial scaffolds on which future bone and cartilage tissue engineering technologies and medical devices will be based upon.

A biphasic, demineralized, and Decellularized allograft bone-hydrogel scaffold with a cell-based BMP-7 delivery system for osteochondral defect regeneration.

The reconstruction of bone and cartilage defects remains a challenge in orthopedics and tissue engineering. In this study, a mimetic natural scaffold based on a demineralized and decellularized bone and collagen type I (Col I) allograft was developed. The resulting hydrogel has the capability of loading allogeneic bone marrow mesenchymal stem cells (BMSCs) resulting in the sustained release of bone morphogenetic protein-7 (BMP-7). BMSCs transfected with lentivirus loaded with BMP-7 gene were used as the BMP-7 delivery system, and then seeded on a demineralized and decellularized allograft bone-collagen biphasic scaffold to enhance bone and cartilage regeneration. The results indicated that, after transfection, BMSCs had a higher expression of the BMP-7 gene and the sustained release of BMP-7 lasted more than 28 days. The preliminary biphasic scaffold promoted cell adhesion and proliferation. After implanting the transfected cells into the knee joints of beagle dogs, enhanced osteochondral defect regeneration was identified at 12 weeks postimplantation. Our results revealed that the new cell-loaded scaffold can avoid the side effects and the short half-life of BMP-7, and promote the reconstruction of bone and cartilage defects. Such a composite system, therefore, shows potential in bone and cartilage tissue engineering applications.

Physical Stimulations for Bone and Cartilage Regeneration.

A wide range of techniques and methods are actively invented by clinicians and scientists who are dedicated to the field of musculoskeletal tissue regeneration. Biological, chemical, and physiological factors, which play key roles in musculoskeletal tissue development, have been extensively explored. However, physical stimulation is increasingly showing extreme importance in the processes of osteogenic and chondrogenic differentiation, proliferation and maturation through defined dose parameters including mode, frequency, magnitude, and duration of stimuli. Studies have shown manipulation of physical microenvironment is an indispensable strategy for the repair and regeneration of bone and cartilage, and biophysical cues could profoundly promote their regeneration. In this article, we review recent literature on utilization of physical stimulation, such as mechanical forces (cyclic strain, fluid shear stress, etc.), electrical and magnetic fields, ultrasound, shock waves, substrate stimuli, etc., to promote the repair and regeneration of bone and cartilage tissue. Emphasis is placed on the mechanism of cellular response and the potential clinical usage of these stimulations for bone and cartilage regeneration.

Physical properties imparted by genipin to chitosan for tissue regeneration with human stem cells: A review.

Genipin is a fully assessed non-cytotoxic crosslinking compound. The chitosan|genipin physical properties such as morphology, roughness, porosity, hydrophilicity, ζ-potential, surface area and surface energy exert control over cell adhesion, migration, phenotype maintenance and intracellular signaling in vitro, and cell recruitment at the tissue-scaffold interface in vivo. For example a therapy using fucose|chitosan|genipin nanoparticles encapsulating amoxicillin, based on the recognition of fucose by H. pylori, leads to sharply improved clinical results. A bioactive scaffold sensitive to environmental stimuli provides an alternative approach for inducing adipose stem cell chondrogenesis: the expression of specific genes, the accumulation of cartilage-related macromolecules and the mechanical properties are comparable to the original cartilage-derived matrix (CDM), thus making the CDM|genipin a contraction-free biomaterial suitable for cartilage tissue engineering. For the regeneration of the cartilage, chitosan|genipin permits to modulate matrix synthesis and proliferation of chondrocytes by dynamic compression; chondrocytes cultured on the composite substrate produce much more collagen-II and sulfated GAG. The main advantages gained in the bone regeneration area with chitosan|genipin are: acceleration of mineral deposition; enhancement of adhesion, proliferation and differentiation of osteoblasts; promotion of the expression of osteogenic differentiation markers; greatly improved viability of human adipose stem cells.