Mechanical properties and hydroxyproline content of connective tissue in porous ceramic implants.

The present study describes a model applying ceramic implants (A1203) for in vivo studies of connective tissue regeneration. Two types of implants have been developed: a one-piece model for histological examination and chemical analyses, and a two-piece implant which can also be used for mechanical testing of connective tissue. When these were implanted subcutaneoulsly on the back of rats, a correlation was found between the mechanical strength and the hydroxyproline content of connective tissue in the implants. The peak synthesis occurred between the 7th and the 14th day after implantation, and a plateau was reached for both strength-increases and hydroxyproline-formation between the 14th and the 21st days. For histological examination, the implants were embedded in plastic materials and prepared as hard tissue specimens. The model presented can be applied to study connective tissue regeneration in normal and pathological conditions, including studies of the effects of various drugs on the connective tissue.     

Early events associated with periodontal connective tissue attachment formation on titanium and hydroxyapatite surfaces.

Endosseous dental implants can support at least three types of biomaterial/connective tissue interfaces: osseointegration, fibro-osseous integration, and periodontal connective tissue attachment. Although a periodontal connective tissue attachment offers distinct advantages, only osseointegration and fibro-osseous integration are at present clinically achievable. Recent studies indicate a periodontal regeneration-competent cell population and an appropriate biomaterial substrate both are required for periodontal connective attachment formation on biomaterial surfaces. We therefore have developed an in vitro model to characterize the effects of various biomaterial substrates on the early events of periodontal connective tissue attachment formation. Primary cultures of periodontal ligament and gingival connective tissue cells were cultured on uncoated (control) and coated (titanium- and hydroxyapatite-coated) tissue culture plastic, and the level of cell proliferation, collagen, and noncollagen protein synthesis, alkaline phosphatase activity, and expression of a 42 kD cementum extracellular matrix protein were measured over 5, 7, and 9 days in culture. While all three substrates supported cell attachment, proliferation, and protein synthesis, only uncoated and titanium-coated tissue culture plastic supported expression of the cementum extracellular matrix protein after 9 days of culture. In addition, the levels of cell proliferation and collagen and noncollagen protein synthesis for cells grown on hydroxyapatite-coated surfaces lagged behind cells cultured on the control or titanium-coated surfaces at each of the three time points. These data suggest that biomaterial substrates markedly can influence the temporal sequence of extracellular matrix proteins associated with periodontal connective tissue attachment formation. In addition to surface composition (titanium versus hydroxyapatite), surface properties (e.g., topography) also may have an effect on periodontal connective tissue attachment formation. This model may be of use in designing biomaterials to support the formation of periodontal connective tissue attachment in vivo.     

Fabrication and mechanical and tissue ingrowth properties of unidirectionally porous hydroxyapatite/collagen composite

This study investigated the effects of the three-dimensional (3-D) pore structure of a porous hydroxyapatite/collagen (HAp/Col) composite on their mechanical properties and in vivo tissue ingrowth. The unique 3-D pore structure, comprising unidirectionally interconnected pores, was fabricated by the unidirectional growth of ice crystals by using a cooling stage and a subsequent freeze-drying process. The unidirectional pores had a spindle-shaped cross section, and their size gradually increased from the bottom to the upper face. The porous composite showed an elastic property and anisotropic compressive strength for the pore directions. While the strength and modulus parallel to the pore axis were 1.3- and twofold higher than those of the porous composite with spherical pores formed randomly, the strength and modulus perpendicular to the pore axis showed the lowest values. The subcutaneous implantations revealed that when compared with the random pores, the unidirectional pores promote the ingrowth of the surrounding tissues into the pores.     

Electromechanical and physicochemical properties of connective tissue

This review has dealt primarily with the electromechanical and transport properties of the extracellular matrix, which generally contains ionized charged groups under physiological conditions. Connective tissues are not electrically "active" in the sense of nerve or muscle; that is, electrical signals do not propagate as waves within the tissue. However, we have attempted to show the importance of "passive" electromechanical coupling and the coupling of passive transport mechanisms to the functional health of connective tissues. The effect of mechanical and electrical stresses on cell growth and biosynthesis is a relatively new and exciting area of research that should provide important clues concerning the interactions between cells and the extracellular matrix. While the role of cells in connective tissues is beyond the scope of this review, it is well known that environmental stresses have a direct effect on the structure and composition of connective tissues. Studies have shown that changes in the chemical and mechanical environment of cells can significantly alter cell synthesis of polysaccharide and protein components of the matrix. For example, Gillard et al. studied the glycosaminoglycan and collagen composition of the flexor digitorium profundus tendon of the rabbit. In regions where the tendon is subject to tensile forces, the tissue GAG content is approximately 0.2% of the dry weight, a value not unlike other tendons. However, in the small sesamoid region where the tendon hooks around the heel bone, the tendon is subjected to high compressional stresses. In this region, the GAG concentration is 15 to 20 times higher and the GAG composition is similar to that of articular cartilage. Gillard et al. found that manipulation of the tendon so as to release the compressional forces lead to a decrease in GAG content by more than 60%. Subsequent replacement of the tendon to its original position caused a concomitant increase in the GAG content. These results can be interpreted to be directly linked to the influence of mechanical forces on cell synthesis. The recent finding that cell synthesis is also affected by imposed electrical fields may suggest that electrical, mechanical and chemical signals are somehow interpreted by the cells along common pathways. The fact that electrical potentials are naturally produced near cells by deformation of the extracellular matrix provides additional support for such hypotheses.     

Synthesis and properties of hydroxyapatite/poly-L-lactide composite biomaterials

Calcium hydroxyapatite (HAp) and poly-L-lactide (PLLA) were synthesized chemically. The obtained HAp was of high purity and, after special thermal treatment, of high crystallinity as well. Synthesis of PLLA was performed using L-lactide as a monomer and nontoxic initiator. In this way a polymer of large molar weight (about 400,000) was obtained. The HAp and PLLA obtained were used as constituents of the HAp/PLLA composite biomaterial, a potential material for implants. The composite was obtained by mixing completely dissolved PLLA with granules of HAp. The composite was compacted by cold and hot pressing at pressures of 49.0-490.5 MPa and temperatures of 20-184 degrees C. The material obtained at optimum process parameters had a density of 99.6% and compressive strength of 93.2 MPa.     

Antigenic properties of a non-collagenous reticulin component of normal connective tissue

Rabbit antisera raised against a saline-insoluble non-collagenous reticulin component (NCRC) of pig and human kidney gave immunofluorescent staining of basement membranes, stroma of liver and kidney and newly formed blood vessels in pig, rat and human tissue. The staining patterns closely resembled those reported for anti-reticulin antibodies, and both species-specific and species-shared determinants could be distinguished. Although the antisera reacted least with rat glomeruli in cryostat sections, rabbit immunoglobulin localized persistently but harmlessly in the renal glomeruli of rats given the antisera intravenously. Absorption with NCRC of sera from patients with gluten-sensitive enteropathy in most cases removed the anti-reticulin antibody characteristic of this group of diseases.     

The effect of a strongly-interacting macromolecular probe on the swelling and exclusion properties of loose connective tissue.

Poly(L-lysine), a cationic polypeptide known to undergo specific interactions with acid mucopolysaccharides, is used as a macromolecular probe in an investigation into the structure of loose connective tissue. The polypeptide apparently binds, via an electrostatic mechanism, to the mucopolysaccharide component of the tissue, leading to a reduction in the swelling of the tissue. The polypeptide does not, however, have a deswelling effect on the tissue; the ultimate reduction in swelling is influenced by the absolute amount of polybase which enters the tissue during the swelling process. Dextran 2000 is found to have a dwelling effect on the tissue which is independent of the action of the polypeptide; this is suggestive of a large degree of independence between the collagen network and the mucopolysaccharide-containing matrix. The polypeptide, moreover, causes a reduction in the excluded volume of the tissue.     

In vivo evaluation of a porous hydroxyapatite/poly-DL-lactide composite for bone tissue engineering

As reported previously, a porous composite of uncalcined hydroxyapatite (u-HA) and poly-DL-lactide (PDLLA) showed excellent osteoconductivity and biodegradability as a bone substitute in rabbit model. In this study, to investigate the usefulness of this composite as a scaffold loaded with cells, we estimated whether this material showed osteogenesis on implantation to extraosseous site. On loading with syngeneic bone marrow cells and implantation into rat dorsal subcutaneous tissue, osteogenesis with enchondral ossification was seen both on and in the material at 3 weeks after implantation. The osteogenesis in the u-HA/PDLLA had progressed, and newly formed bone tissue was found in the material by 6 weeks. To investigate the osteoinductive properties of the material, we implanted this porous composite material into extraosseous canine dorsal muscle. At 8 weeks, osteogenesis was seen in the pores of the material. Newly formed bone could be observed adjacent to the material. In addition, cuboidal osteoblasts adjacent to the newly formed bone were evident. Neither cartilage nor chondrocytes were found. These results might indicate that the material induced osteogenesis by intramembranous ossification. Conversely, similar porous PDLLA did not induce osteogenesis during the observation period. Therefore, porous HA/PDLLA, which has osteoconductive and osteoinductive properties, might be a useful material for use as a bone substitute and cellular scaffold.     

Nano-scaled hydroxyapatite/polymer composite IV. Fabrication and cell adhesion properties of a three-dimensional scaffold made of composite material with a silk fibroin substrate to develop a percutaneous device

Nano-scaled sintered hydroxyapatite (HAp) particles with an a-axis length of 87 ± 23nm, a c-axis length of 236 ± 81nm, and a mean aspect ratio (c/a) of 2.72 were covalently linked onto a silk fibroin (SF) substrate chemically modified by graft polymerization with -methacryloxypropyl trimethoxysilane (MPTS). Graft polymerization with poly(MPTS) on SF was conducted by free-radical initiation in a water solvent with pentaethylene glycol dodecyl ether as a nonionic surfactant. The alkoxysilyl groups of the graft polymers avoided hydrolysis and maintained their activity in coupling with the hydroxyl groups on the HAp surface despite the use of water as the reaction solvent. The weight gain of poly(MPTS) on SF increased with increasing the reaction time, eventually reaching a plateau value of about 15wt% after 50min of reaction time. After HAp covalent coating, the particles separated or aggregated into several crystals, as shown by scanning electron microscopic observation. L929 fibroblast cells adhered more plentifully on HAp-coated SF compared to untreated SF and hydrolyzed poly(MPTS)-grafted SF during 24h or 48h of incubation. The cells adhered only on the HAp surface but not at all on the dehydrated grafted surface of SF without HAp. A button-shaped prototype for a percutaneous device was manufactured by transplantation of HAp-coated SF fibers of about 100µm in length onto silicone moldings using an adhesive, and the device showed good cell adhesiveness.     

Autoantibodies to malondialdehyde-modified epitope in connective tissue diseases and vasculitides.

Tuberculosis is characterized by fever, weight loss, a prolonged acute-phase protein response and granuloma formation. These characteristics may partly be due to action of proinflammatory cytokines tumour necrosis factor (TNF), IL-6 and IL-8. We investigated plasma concentrations of these cytokines before and after ex vivo lipopolysaccharide stimulation of whole blood leucocytes from 41 Zambian patients with tuberculosis, 32 of whom were also HIV+. Although patients had a reduced weight, were more anaemic and had higher erythrocyte sedimentation rate compared with controls (all P < 0.0005), clinical and laboratory measurements of disease state were similar in those who died and survivors. In contrast, plasma IL-6 and IL-8 concentrations were higher in patients who died (P < 0.05). There was no detectable cytokine mRNA in unstimulated leucocytes. There was reduced secretion of TNF (P < 0.005 at 2 h), IL-6 (P < 0.005 at 8 h) and IL-8 (P < 0.005 at 24 h) after ex vivo stimulation of whole blood leucocytes from patients who died compared with survivors. This was partly due to a soluble inhibitory factor present in plasma. The only additional effect of concurrent infection by HIV with Myco. tuberculosis was decreased IL-6 secretion following ex vivo stimulation of leucocytes. Reduced proinflammatory cytokine release may represent a critical impairment of host immune defences important in determining outcome in tuberculosis.     

Effect of formalin fixation on the ultrastructure of connective tissue components

Electron microscopic examinations of fibrillar components and the ground substance of human skin derma fixed in formaldehyde showed good preservation of the fibrillar structures. The typical changes in fixed collagen fibrils in negative staining include the lack of transversal lines in the zone A and poor manifestation of microfibrillarity in the zone B. Alterations in the ground substance are more significant. Staining with rutenium red reveals no reticular structure here, but floccular formations appear in the amorphous interfibrillar substance. Other species of rutenium-positive structures (sheaths of elastic fibers and collagen fibrils as well as lines of thin cross-striation of the latter) are well preserved. All these alterations should be taken into consideration in ultramicroscopical examinations of formalin-fixed connective tissue.     

Effect of collagen on the mechanical properties of hydroxyapatite coatings

In this study, the mechanical properties of bioactive coatings on Ti6Al4V substrates were investigated using instrumented nanoindentation. The aim was to observe the differences in the mechanical properties before and after immersion in collagen solution. The hydroxyapatite coatings were prepared through two processes: self-assembly in simulated body fluid and a hydrothermal method. Sintered hydroxyapatite disks were used as controls. The test samples were then incubated in a dilute collagen solution for 24 hours to produce composite coatings. The materials were investigated using XRD, SEM and nanoindentation. The results showed that the grain sizes of the hydroxyapatite coatings formed using two processes were 1?μm and 10?μm, respectively. The Young's modulus of the pure hydroxyapatite, the disk and the coatings, was 3.6 GPa. After collagen incubation treatment, the composites had a Young's modulus of 7.5 GPa. The results also showed that the strengthening phenomena of collagen were more obvious for homogeneous and small-grain hydroxyapatite coatings. These results suggest that there are similarities between these HAp/collagen composited and natural composite materials, such as teeth and bones.     

纳米羟基磷灰石/聚磷酸钙纤维/聚乳酸骨组织工程复合支架的特性*

背景：近年来国内外在骨与软骨组织支架复合材料方面进行了广泛的研究,取得了积极的成果,但仍存在许多问题。 目的：观察纳米羟基磷灰石/聚磷酸钙纤维/聚乳酸(HAP/CPP/PLLA)骨组织工程支架复合材料的特性。 方法：采用溶媒浇铸、粒子滤取技术与气体发泡相结合的方法制备出纳米HAP/CPP/PLLA骨组织工程支架复合材料,测试该支架复合材料的物理力学性能,并用扫描电子显微镜对其微观结构进行观察。 结果与结论：结果表明,纳米HAP/CPP/PLLA支架复合材料具有三维、连通、微孔网状结构,并具有较高的孔隙率和较好的压缩模量,是理想的骨组织工程支架材料。