Determination of the orientation of collagen fibers in human bone.

A human calcaneus bone, consisting of hydroxyapatite and collagen fibers, was successively sliced into samples in a direction perpendicular to the long axis of the bone and parallel to the long axis of the human lower limb. The transmitted microwave intensities of 12 GHz, reflecting the dielectric property, were measured for the slice samples using Osaki's microwave method (Tappi J., 1987; 70:105-108). The complex dielectric constant of 12 GHz of the collagen fiber film was much greater than that of hydroxyapatite disc, which demonstrated that the dielectric anisotropy observed for the sliced bone was mainly affected by the collagen fibers. The angular dependence of the transmitted microwave intensity gives the orientation angle reflecting the direction of the collagen-fiber orientation, and the degree of orientation reflecting the anisotropic property of collagen fibers. The orientation angle and the degree of orientation for the slice samples changed with changing position along the long axis of the calcaneus bone. The direction of orientation deviated to the lateral side at the heel part of the left calcaneus, and to the medial side at the middle part. The degree of orientation is relatively high at the heel part and low at the middle. Such results give a two-dimensional distribution of collagen-fiber orientation in the left calcaneus, and suggest that the direction and degree of orientation are closely related to the direction and magnitude of the stress applied to the bone, respectively.     

An osteoconductive collagen/hyaluronate matrix for bone regeneration.

A new type of collagen-hyaluronate (COL/HA) matrix was synthesized by cross-linking collagen fibers with modified hyaluronate polymers bearing active formyl groups. The resulting matrix is a three-dimensional scaffold consisting of interconnected pores with an average size of 40 microm and a high pore volume/surface area ratio. The covalent nature of the bond between the collagen fibers and the modified hyaluronate was demonstrated by extended elution with phosphate buffered saline and by extraction in increasing ionic gradients. The fraction of covalently bound hyaluronate in the matrix ranged from 5 to 25 w%. The total hyaluronate content of the COL/HA matrix affected both the in vitro non-enzymatic and enzymatic degradation as well as the in vivo turnover. When implanted in cranial defects in rats, the COL/HA matrix demonstrated good biocompatibility and exhibited greater osteoconductive potential than matrices composed of either cross-linked collagen or cross-linked hyaluronate alone.     

Characterization of collagen orientation in human dermis by two-dimensional second-harmonic-generation polarimetry

We have proposed an optical probe that can be used to characterize the orientation of collagen fibers in human dermis. A specific probing ability for collagen results from the use of second-harmonic-generation (SHG) light induced by collagen molecules in the tissue. Based on the concept of collagen SHG light, a reflection-type polarization measurement system (named SHG polarimetry) with a probe light spot of 15 microm in diameter has been constructed, and the human reticular dermis has been measured using this system. Resultant data exhibit that the reticular dermis possesses approximately uniaxial orientation of the collagen fibers. Furthermore, we demonstrated a nondestructive measurement of the collagen orientation in the papillary dermis across an epidermis layer. For distribution measurement of the collagen fiber orientation in the reticular dermis, we have extended the SHG polarimetry to one- (1-D) and two-dimensional (2-D) measurement. By the macroscopic 2-D SHG polarimetry, we have observed that the orientation angle and organization degree of collagen fibers vary widely depending on the discrete probing positions in the reticular dermis. Furthermore, microscopic 1-D SHG polarimetry indicated a swell of the orientation angle and a large variance of the organization degree in the collagen fibers in the microscopic region. These results imply that the reticular dermis posses a tangled structure of collagen fibers, which is highly consistent with the result of the anatomical examination of the skin. The proposed method will be a powerful tool for monitoring the microscopic distribution of the collagen fiber orientation in the human dermis.     

Collagen-based matrices with axially oriented pores

The aim of this work was the implementation of a simple technique for the production of cylindrical collagen-based scaffolds with axially oriented pore channels. Matrices with this particular porous structure have the potential to improve the regeneration of peripheral nerves and spinal cord by physically supporting and guiding the growth of neural structures across the site of injury. The regenerative potential may be further enhanced when the collagen scaffold is used as a delivery vehicle for exogenous cells and growth factors. The scaffold manufacturing technique described here is based on unidirectional freezing of a collagen suspension and subsequent freeze-drying, which produces nearly axially oriented pores. The mean pore size is dependent on both the concentration of collagen in suspension and the temperature of freezing. Environmental scanning electron microscopy and light microscopy were used to assess qualitatively and quantitatively the pore size and the pore orientation. In particular the definition of an orientation index (OI) was employed as a means to quantify the orientation of the pore channels inside the scaffolds.     

Intensity profiles of linearly polarized light backscattered from skin and tissue-like phantoms

Anisotropy of mouse and human skin is investigated in vivo using polarized videoreflectometry. An incident beam (linearly polarized, wavelength 650 nm) is focused at the sample surface. Two types of tissuelike media are used as controls to verify the technique: isotropic delrin and highly anisotropic demineralized bone with a priori knowledge of preferential orientation of collagen fibers. Equi-intensity profiles of light, backscattered from the sample, are fitted with ellipses that appear to follow the orientation of the collagen fibers. The ratio of the ellipse semiaxes is well correlated with the ratio of reduced scattering coefficients obtained from radial intensity distributions. Variation of equi-intensity profiles with distance from the incident beam is analyzed for different initial polarization states of the light and the relative orientation of polarization filters for incident and backscattered light. For the anisotropic media (demineralized bone and human and mouse skin), a qualitative difference between intensity distributions for cross- and co-polarized orientations of the polarization analyzer is observed up to a distance of 1.5 to 2.5 mm from the entry point. The polarized videoreflectometry of the skin may be a useful tool to assess skin fibrosis resulting from radiation treatment.     

Collagen distribution in the rat demonstrated by a specific anti-collagen antiserum

A specific anti-collagen antiserum was prepared in rabbits using as antigen a rat tail collagen preparation free of serum protein reactants. This antiserum reacted with several forms of isolated collagen and with rat urine, but not with rat serum. It was coupled to fluorescein and used to label sites of collagen in frozen and dried cryostat sections of rat tissues. The indirect method, with uncoupled serum, was also used routinely. Tubular and glomerular basement membranes, Bowman's capsule, basement membrane of skin and lung, reticular fibers of spleen and the connective tissue framework of muscle were strongly stained, as was also new bone on trabecular borders and around osteocytes. The supporting fibers of normal hyaline cartilage, calcified bone, most tendons and the deeper fibers of skin were less stained or unstained under identical conditions and are presumably masked or blocked in situ. Cytoplasm or cytoplasmic processes in some fibroblasts of skin and tendon, cytoplasm of osteocytes and some cartilage cells in elastic cartilage were stained. Reaction and masking in fibers are presumably related to the modes of collagen linkage both within the fiber and externally with the ground substance matrix. Activity could be elicited in tendon by a brief heating at 60°C sufficiently to abolish the collagen birefringence. These collagen linkages are believed to be of importance in determining its behavior in overall processes of tissue disaggregation.     

Laser-perforated membranous biomaterials induced pore size-dependent bone induction when used as a new BMP carrier

Previously we found that laser perforation of a collagen membrane (35 microm thickness, Koken Co., Tokyo) produced an effective bone morphogenetic protein (BMP) carrier, if the created pore sizes were larger than 0.5 mm. In this study we applied the same technique to create pores of 0.2 and 1.0 mm in a thicker (1.2 mm thickness) porous biodegradable membrane made of polylactic acid and an epsilon-caprolactone copolymer (PLA-CL) to obtain an effective membranous BMP carrier with higher mechanical strength. Pieces of PLA-CL (0.5 x 1.0 x 0.12 cm) combined with rhBMP-2 (5 microg) were implanted subcutaneously into rats and processed for analyses at 1-3 weeks. The laser-perforated PLA-CL membranes equipped with 1.0 mm pores induced mineralization beginning from the margins of the pores judging from the X-ray patterns, but bone formation seemed to proceed irregularly inside the pores. In the perforated PLA-CL membrane with 1.0-mm pores bone formation did not significantly increase compared with the nonperforated one. This was due to the fact that the PLA-CL membrane was already a porous structure (85% porosity). In contrast with laser-perforated PLA-CL 0.2 mm pores, bone was induced on the collagen fibers and fiber bundles inside the pores. The different patterns of bone formation between the PLA-CL membranes with 1.0 and 0.2 mm pores seemed to be related to the active formation of perpendicular collagen fibers through the 0.2 mm pores.     

Laser-Perforated Membranous Biomaterials Induced Pore Size-Dependent Bone Induction When Used as a New BMP Carrier

Previously we found that laser perforation of a collagen membrane (35 &#119 m thickness, Koken Co., Tokyo) produced an effective bone morphogenetic protein (BMP) carrier, if the created pore sizes were larger than 0.5 mm. In this study we applied the same technique to create pores of 0.2 and 1.0 mm in a thicker (1.2 mm thickness) porous biodegradable membrane made of polylactic acid and an &#108 -caprolactone copolymer (PLA-CL) to obtain an effective membranous BMP carrier with higher mechanical strength. Pieces of PLA-CL (0.5 &#50 1.0 &#50 0.12 cm) combined with rhBMP-2 (5 &#119 g) were implanted subcutaneously into rats and processed for analyses at 1-3 weeks. The laser-perforated PLA-CL membranes equipped with 1.0 mm pores induced mineralization beginning from the margins of the pores judging from the X-ray patterns, but bone formation seemed to proceed irregularly inside the pores. In the perforated PLA-CL membrane with 1.0-mm pores bone formation did not significantly increase compared with the nonperforated one. This was due to the fact that the PLA-CL membrane was already a porous structure (85% porosity). In contrast with laser-perforated PLA-CL 0.2 mm pores, bone was induced on the collagen fibers and fiber bundles inside the pores. The different patterns of bone formation between the PLA-CL membranes with 1.0 and 0.2 mm pores seemed to be related to the active formation of perpendicular collagen fibers through the 0.2 mm pores.     

Preparation and characteristics of hybrid scaffolds composed of beta-chitin and collagen

Hybrid scaffolds composed of beta-chitin and collagen were prepared by combining salt-leaching and freeze-drying methods. The chitin scaffold used as a framework was easily formed into desired shapes with a uniformly distributed and interconnected pore structure with average pore size of 260-330 microm. The mechanical strength and the rate of biodegradation increased with the porosity, which could be modulated by the salt concentration. In addition, atelocollagen solution was introduced into the macropores of the chitin scaffold to improve cell attachment. Web-like collagen fibers fabricated between pores of chitin were produced by a 0.1 wt% collagen solution, whereas a 0.5 wt% collagen solution only coated the surface of the chitin scaffold. After 3 days of culture, fibroblasts cultured in collagen-coated scaffolds were attached at the place where the collagen was fabricated, whereas cells did not attach and aggregate on the scaffold of chitin alone. After 14 days, the fibroblasts showed a good affinity to and proliferation on all collagen-coated chitins.     

Collagen microgel-assisted dexamethasone release from PLLA-collagen hybrid scaffolds of controlled pore structure for osteogenic differentiation of mesenchymal stem cells

Directed stem cell differentiation over three-dimensional porous scaffolds capable of releasing bioactive instructive cues is an important tool in tissue engineering. In this research, we have prepared dexamethasone (Dex)-releasing collagen microbead-functionalized poly(L-Lactide)-collagen hybrid scaffolds as an osteoinductive platform for human bone marrow-derived mesenchymal stem cells (MSCs). The scaffolds were prepared by a combined method of emulsion freeze-drying and porogen-leaching using pre-prepared ice collagen particulates as a porogen material. Dex release from the hybrid scaffolds was studied at 37?°C under shaking condition and the impact of released Dex towards osteogenic lineage differentiation was investigated by 3?week in vitro culture of MSCs. The results showed that hybrid scaffolds had controlled pore structure and interconnected pores deposited with collagen fibers. The hybrid scaffold facilitated cell seeding and the spatial localization of Dex/collagen microbeads facilitated a microgel-assisted spatio-temporal control of Dex release. The released Dex was useful for osteogenic differentiation of MSCs, which was confirmed from the elevated expression of osteogenic-specific gene-encoded proteins. The hybrid scaffolds should be useful for regeneration of a functional bone tissue.     

Characterization of collagen fibers by means of texture analysis of second harmonic generation images using orientation-dependent gray level co-occurrence matrix method

Collagen is the most prominent protein in the human body, making up 30% of the total protein content. Quantitative studies have shown structural differences between collagen fibers of the normal and diseased tissues, due to the remodeling of the extracellular matrix during the pathological process. The dominant orientation, which is an important characteristic of collagen fibers, has not been taken into consideration for quantitative collagen analysis. Based on the conventional gray level co-occurrence matrix (GLCM) method, the authors proposed the orientation-dependent GLCM (OD-GLCM) method by estimating the dominant orientation of collagen fibers. The authors validated the utility of the OD-GLCM method on second harmonic generation (SHG) microscopic images of tendons from rats with different ages. Compared with conventional GLCM method, the authors' method has not only improved the discrimination between different tissues but also provided additional texture information of the orderliness of collagen fibers and the fiber size. The OD-GLCM method was further applied to the differentiation of the preliminary SHG images of normal and cancerous human pancreatic tissues. The combination of SHG microscopy and the OD-GLCM method might be helpful for the evaluation of diseases marked with abnormal collagen morphology.     

Novel collagen sponge reinforced with polyglycolic acid fiber produces robust, normal hair in murine hair reconstitution model

The hair reconstitution assay is a useful system for studying cell-cell and epithelial-mesenchymal interaction. The current method consists of transplantation of both epidermal and dermal cells, using a silicone chamber placed on an athymic nude mouse. However, because of leakage and tilting of the grafted cells, the rate and area of hair growth vary depending on the chamber. We modified this method by using a collagen sponge as a scaffold and compared two types of collagen sponges, each having different tensile strengths. A conventional collagen sponge disturbed normal hair follicle formation; in contrast, a collagen sponge containing polyglycolic acid (PGA) fiber supported proper restructuring of skin and hair follicles. These data suggested the usefulness of PGA fiber-containing collagen sponges for hair reconstitution in research and clinical applications.     

Distribution map of collagen fiber orientation in a whole calf skin

It is important to obtain information about the collagen fiber orientation in biological fibrous tissues such as human and animal skins because the collagen fiber orientation in the skins may closely relate to the motional functions of the body. However, no reports are yet available on the distribution of collagen fiber orientation in a whole skin. The microwave method recently developed by the author was applied for giving the distribution of collagen fiber orientation. A map representing the distribution of collagen fiber orientation was obtained for a leather sheet prepared from the entire skin of a calf. It was found that the direction of collagen fiber orientation was, on an average, parallel to the calf's spinal column and limbs, corresponding to the direction perpendicular to that of skin motions due to eating and walking, and that the degree of orientation was large in the parts where the skin motions were marked. These findings suggest that the distribution map of fiber orientation in calf leather significantly affects the motional functions of its different parts and also hopefully gives useful information for performing an appropriate transplant of skins. Anat Rec 254:147–152, 1999. © 1999 Wiley‐Liss, Inc.     

Wound dressings composed of copper-doped borate bioactive glass microfibers stimulate angiogenesis and heal full-thickness skin defects in a rodent model

There is a need for better wound dressings that possess the requisite angiogenic capacity for rapid in situ healing of full-thickness skin wounds. Borate bioactive glass microfibers are showing a remarkable ability to heal soft tissue wounds but little is known about the process and mechanisms of healing. In the present study, wound dressings composed of borate bioactive glass microfibers (diameter = 0.4–1.2 μm; composition 6Na₂O, 8K₂O, 8MgO, 22CaO, 54B₂O₃, 2P₂O₅; mol%) doped with 0–3.0 wt.% CuO were created and evaluated in vitro and in vivo. When immersed in simulated body fluid, the fibers degraded and converted to hydroxyapatite within ∼7 days, releasing ions such as Ca, B and Cu into the medium. In vitro cell culture showed that the ionic dissolution product of the fibers was not toxic to human umbilical vein endothelial cells (HUVECs) and fibroblasts, promoted HUVEC migration, tubule formation and secretion of vascular endothelial growth factor (VEGF), and stimulated the expression of angiogenic-related genes of the fibroblasts. When used to treat full-thickness skin defects in rodents, the Cu-doped fibers (3.0 wt.% CuO) showed a significantly better capacity to stimulate angiogenesis than the undoped fibers and the untreated defects (control) at 7 and 14 days post-surgery. The defects treated with the Cu-doped and undoped fibers showed improved collagen deposition, maturity and orientation when compared to the untreated defects, the improvement shown by the Cu-doped fibers was not markedly better than the undoped fibers at 14 days post-surgery. These results indicate that the Cu-doped borate glass microfibers have a promising capacity to stimulate angiogenesis and heal full-thickness skin defects. They also provide valuable data for understanding the role of the microfibers in healing soft tissue wounds.     

Promoted growth of murine hair follicles through controlled release of vascular endothelial growth factor

The objective of this study is to investigate whether or not the controlled release of vascular endothelial growth factor (VEGF) is effective in promoting the hair follicle growth of mice in second anagen of hair cycle. VEGF was incorporated into a biodegradable collagen hydrogel for its controlled release. Following implantation of the collagen hydrogel incorporating 0 or 2 microg of VEGF and injection of 0 or 2 microg of VEGF in the solution form into the back subcutis of mice, the hair follicle growth was evaluated photometrically and histologically in terms of the skin color of reverse side of the implanted or injected site, the skin thickness, and the area occupied by hair follicle tissue. Ten days later, the skin color of mice implanted with the collagen hydrogel incorporating 2 microg of VEGF was significantly darker than that injected with 2 pg of VEGF. The collagen hydrogel incorporating VEGF increased the hair follicle area at the implanted site to a significantly greater extent than other agents while significant angiogenetic effect in the skin tissue was observed. VEGF-free, empty collagen hydrogels did not affect the skin darkness, hair follicle growth, and the angiogenesis. Moreover, the hair shaft length was significantly elongated by the collagen hydrogel incorporating VEGF, in marked contrast to other agents. Immunohistolchemicalstaining with proliferating cell nuclear antigen revealed that the collagen hydrogel incorporating VEGF promoted the proliferation of cells around the hair follicle more frequently than free VEGF. We concluded that the controlled release of VEGF more positively acted on the hair growth cycle of mice for hair growth than the injection of free VEGF.     

A microfabricated porous collagen-based scaffold as prototype for skin substitutes

An important element of artificial skin is a tissue scaffold that allows for fast host regeneration. We present a microfabrication strategy, based on gelling collagen-based components inside a microfluidic device, that produces well-controlled pore sizes inside the scaffold. This strategy can produce finely patterned tissue scaffolds of clinically relevant dimensions suitable for surgical handling. Compared to porous collagen-based sponges produced by lyophilization, microfabricated tissue scaffolds preserve the fibrous structure and ligand density of natural occurring collagen. A fibroblast migration assay revealed fast cellular migration through the pores, which is desired for rapid tissue ingrowth. Finally, we also demonstrate a strategy to use this microfabrication technique to build anatomically accurate, multi-component skin substitutes in a cost-effective manner.     

基于聚己内酯纤维的组织工程支架研究进展

聚己内酯(PCL)是一种生物相容性好、可吸收、易加工改性的聚酯,材料基于PCL纤维的组织工程支架,具有比表面积高、机械性能良好与孔径、孔隙率和纤维取向等结构特征易调控等特点,被广泛应用于组织工程领域。重点综述PCL纤维的组织工程支架的主要应用缺陷(包括细胞亲和力差、降解速度过慢及机械强度低)及改进手段,同时针对基于PCL纤维的组织工程支架在皮肤、血管、神经、肌腱、韧带和软骨等组织再生的最新进展进行归纳总结,发现目前多数研究集中于通过引入生物活性物质或药物以改善细胞-支架相互作用和调控支架降解行为,或通过选取不同的纺丝工艺和参数以改变支架的物理结构,调控支架机械性能与细胞诱导行为。此外,目前多数研究仍停留在实验室阶段,利用基于PCL纤维的组织工程支架低成本、易加工的优势以加快其临床转化是未来重要的发展方向。     

Collagen fibrillar networks as skeletal frameworks: a demonstration by cell-maceration/scanning electron microscope method

A cell-maceration/scanning electron microscope (SEM) method was employed to demonstrate the arrangement of the collagen fibrillar network of various tissues. Immersion of fixed tissues in NaOH (25 degrees C) for 3-7 days, followed by rinsing in distilled water successfully removed the cellular elements, exposing collagen fibrils which were identified as such by transmission electron microscopy in their natural locations. SEM observations of the preparations are able to demonstrate the three-dimensional architecture of collagen fibrils much more precisely than other methods, including the silver impregnation method. Collagen fibrils, forming sheaths for housing individual cardiac myocytes, fused together, thus ensuring an equal stretch of contiguous myocytes and preventing the slippage of adjacent cells. Individual skeletal muscle fibers and nerve fibers were ensheathed by the meshwork of collagen fibrils running in two opposite helices. Such structures seem to play an important role in resisting the stretching impetus. At the epithelial-connective tissue junction of the tongue and fingertip skin, interwoven collagen fibrils formed numerous microridges which probably provide a broad anchorage for the epithelium. In the intestinal mucosa, the collagen fibrillar network immediately below the basal laminae of the villous epithelium possessed heterogeneous pores. As the collagen fibrillar network shows morphological features specific to individual organs and tissues, it is suggested that such formations not only constitute the skeletal framework but also provide those cells which are housed there with a microenvironment suitable for their activities.     

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.