X.D. Zhu

Three‐dimensional nano‐HAp/collagen matrix loading with osteogenic cells in organ culture

Transplantation of osteogenic cells with a suitable matrix is one strategy for engineering bone tissue. Three-dimensional distribution and growth of cells within the porous scaffold are of clinical significance for the repair of large bony defects. A nano-HAp/collagen (nHAC) composite that mimics the natural bone both in composition and microstructure to some extent was employed as a matrix for the tissue engineering of bone. A porous nHAC composite was produced in sheet form and convolved to be a three-dimensional scaffold. Using organ culture techniques and the convolving method, we have developed three-dimensional osteogenic cells/nHAC constructs in vitro. Scanning electron microscopic and histological examination has demonstrated the development of the cells/material complex. Spindle-shaped cells migrating out of bone fragments continuously proliferated and migrated throughout the network of the coil. The porous nHAC scaffold provided a microenvironment resembling that seen in vivo, and cells within the composite eventually acquired a tridimensional polygonal shape. In addition, new bone matrix was synthesized at the interface of bone fragments and the composite.

Formation of calcium phosphate/collagen composites through mineralization of collagen matrix

Several types of calcium phosphate/collagen composites, including noncrystalline calcium phosphate/collagen, poorly crystalline carbonate-apatite (PCCA)/collagen, and PCCA + tetracalcium phosphate/collagen composites, were prepared through the mineralization of collagen matrix. The type I collagen was presoaked with a PO(3-)(4) containing solution and then immersed in a Ca(2+) containing solution to allow mineral deposition. The solution of 0.56 M sodium dibasic phosphate (Na(2)HPO(4)) with a pH of nearly 14 was metastable and its crystallization produced Na(2)HPO(4) and sodium tripolyphosphate hexahydrate (Na(5)P(3)O(10)). 6H(2)O), leading to a controlled release of orthophosphate ions during the subsequent mineral precipitation. The development of the composites was investigated in detail. The mineral contributed up to 60-70% of the weight of the final composites. The strength and Youngs modulus of the composites in tensile tests overlapped the lower range of values reported for bone. When implanted in muscle tissue, the composite showed biodegradability that was partly through a multinucleated giant cell mediated process. In a bone explant culture model it was observed that bone-derived cells deposited mineralizing collagenous matrix on the composite.

Tissue response to nano-hydroxyapatite/collagen composite implants in marrow cavity.

The tissue response to a nano-hydroxyapatite/collagen composite implanted in a marrow cavity was investigated by histology and scanning electron microscopy. A Knoop microhardness test was performed to compare the mechanical behavior of the composite and bone. The ultrastructural features of the composite, especially the carbonate-substituted hydroxyapatite with low crystallinity and nanometer size, made it a bone-resembling material. It was bioactive, as well as biodegradable. At the interface of the implant and marrow tissue, solution-mediated dissolution and giant cell mediated resorption led to the degradation of the composite. Interfacial bone formation by osteoblasts was also evident. The process of implant degradation and bone substitution was reminiscent of bone remodeling. The composite can be incorporated into bone metabolism instead of being a permanent implant. For lack of the hierarchical organization similar to that of bone, the composite exhibited an isotropic mechanical behavior. However, the resistance of the composite to localized pressure could reach the lower limit of that of the femur compacta.

Morphological behaviour of osteoblasts on diamond-like carbon coating and amorphous C–N film in organ culture

Similar to diamond-like carbon (DLC) coating, amorphous carbon nitride (C-N) films can be extremely hard and wear-resistant. They may serve as candidates for the solution to the problem of aseptic loosening of total hip replacements. Morphological behaviour of osteoblasts on silicon, DLC-coated silicon and amorphous C-N film-deposited silicon in organ culture was investigated by scanning electron microscopy. Cells on the silicon wafers were able to attach, but were unable to follow this attachment with spreading. In contrast, the cells attached, spread and proliferated on the DLC coatings and amorphous C-N films without apparent impairment of cell physiology. The morphological development of cells on the coatings and films was similar to that of cells in the control. The preliminary results support the biocompatibility of DLC coating and are encouraging for the potential biomedical applications of amorphous C-N film.

In vitro and in vivo evaluation of degradability of hydroxyapatite coatings synthesized by ion beam-assisted deposition

Abstract Degradability is among the most important properties in the biomedical field, which is crucial to bone apposition on implants, bone-implant bonding and implantation longevity. The present paper evaluated the degradability of ion beam-assisted deposition (IBAD) hydroxyapatite (HA) coatings in vitro and in vivo. In vitro testing showed that IBAD HA coatings degraded little in 37°C sterile Hank’s physiologic (pH 5.2) balanced salt solution throughout the testing time, while the plasma-sprayed HA coating tested in comparison underwent an increasing degradation: the concentration of Ca increased from the initial 1 ml to 2.75 ml at 3 days post-incubation. In vivo testing revealed that no significant degradation occurred throughout the whole implant period (12 weeks). All the results consistently suggest that the IBAD HA coating is slightly degradable. High magnification SEM observation and HRTEM investigations of the coating further pointed out that the low degradability of the IBAD HA coating derives from its dense microstructure and unique properties such as no sharp grain boundaries. The present study demonstrates that IBAD HA coatings are much more chemically stable than plasma-sprayed HA coatings. The IBAD HA coating possesses more superior adhesive properties, as would be of clinical importance in that it may favor a longer longevity of orthopedic implants.

Mechanical properties of skeletal bone in gene-mutated stöpseldtl28d and wild-type zebrafish (Danio rerio) measured by atomic force microscopy-based nanoindentation

An atomic force microscopy (AFM)-based nanoindenter was used to evaluate the mechanical properties of skeletal bones in wild-type and gene-mutated zebrafish (Danio rerio), stöpsel(dtl28d). Both skeletons were isolated from adult zebrafish and tested under a load of 5 mN. It was found that stp/stp bone has a similar nanohardness but significantly greater elastic modulus compared with that of wild-type bone. The residual indenter impressions using AFM and the fracture surfaces of both bones using scanning electron microscopy were examined and showed that the bone of zebrafish becomes more brittle after the stp mutation. This first observation of the alteration of bone mechanical behavior by gene mutation in zebrafish system is of scientific and clinical relevance to many areas of study, such as bone fracture and fragility mechanisms in human heritable disorders and bone-materials fabrication via gene engineering.

MICROSTRUCTURE AND MICROMECHANICAL PROPERTIES OF THE MID-DIAPHYSES OF HUMAN FETAL FEMURS

The microstructure, composition and the micromechanical properties across the thickness of femoral mid-diaphyses from 14 to 26 week human fetuses have been investigated. Scanning electron microscopy and transmission electron microscopy were employed to examine structural changes with maturation. The fetal bones consist of layers of woven bone. From young to old fetuses and from outer to inner bone layers, the collagen fibrils become more cross-linked, densely packed and change from disordered to an ordered arrangement. The collagen fibril bundles are also more preferentially oriented and change from a chiefly circumferential to longitudinal direction. The sizes of the apatite crystals also increase with age. The Ca/P ratio remains constant around 1.55 for all the bone layers except the outmost layer which is lower than 1.2. An nano-indenter was used to evaluate the microhardness and elastic modulus of each bone layer. The increase of microhardness and elastic modulus correlates with the maturation of bone. The mechanical properties of the mid-diaphyses of human fetal femurs are anisotropic, which is due to the preferential orientation of collagen fibrils.

Microstructural evolution in external callus of human long bone

Abstract Accurate knowledge of bone fracture healing process is of clinical and theoretical importance in bone repair and regeneration, and biomineralization. It is well known that the histological healing occurs via the formation of hematoma, fibrocartilage, bony callus and bone modeling/remodeling. However, the detailed process from fracture to healing at the microstructural level remains unclear. In the present study, an evolutionary model of external callus is proposed, in which five representative stages are presented in terms of the organization of collagen and minerals during the formation of bony callus. The first stage is the formation of loose, disordered collagen fibrils, which is followed by mineralization on some of these individual microfibrils. Then the matrix is characterized by the fusion of mineralized individual fibrils into bundles. In the third stage, the absorption of disordered matrix occurs. This is gradually replaced by ordered collagen in stage four. Finally, completely ordered mineralized tissue is formed. The proper sequence of the process plays an important role in deciding the success of healing. In addition to the common mineral phase of hydroxyapatite (HA), dicalcium phosphate dihydrate (DCPD) phase was also found in early stage of healing, especially in rapid healing (childrens callus). It vanished in the following process of healing. The deposition of DCPD is supposed to be brought about by some non-collagenous protein.