Anterior cruciate ligament reconstruction with the patellar tendon augmented by absorbable artificial materials. A histological and molecular biological study in rabbits

An experimental study was performed to investigate the histological and molecular biological properties of reconstructed anterior cruciate ligaments (ACL) using the patellar tendon augmented by absorbable artificial material in rabbits. Thirty rabbits underwent ACL reconstruction with the patellar tendon (non-augmented group), and 30 with the patellar tendon augmented by a polyglactin 910 mesh (augmented group). They were respectively sacrificed at at 1, 2, 3, 4, 6, 8 and 12 weeks after surgery for histological and molecular biological examination. Histologically, the fibrocytes were disappeared in deep portion of transplanted graft at 2 weeks after surgery in both groups. The graft was replaced with granulated tissues at 6 weeks in the augmented group, but it took 8 weeks in the non-augmented group. At 12 weeks, the graft showed hypocellularity with regularly oriented fibers in the augmented group, while it still showed hypercellularity with disoriented fibers in the non-augmented group. Molecular biologically, after the reconstruction at one week, the relative radioactivity in the graft was 1.05 in the non-augmented group, and 2.10 in the augmented group. Additionally the level of the alpha 2 (I) collagen mRNA was also significantly higher in the augmented group than that in the non-augmented group. These results suggested that a polyglactin 910 mesh had an effect on the process of reorganization and remodeling in the transplanted patellar tendon during the post-operative period in rabbit.     

Effects of in situ freezing and stress-shielding on the ultrastructure of rabbit patellar tendons

The effects of in situ freezing and the combination of in situ freezing and stress-shielding on the microstructure and ultrastructure of the patellar tendon were studied with use of 20 mature rabbits. The patellar tendon was frozen in situ with liquid nitrogen to kill fibroblasts and then was completely released from stress by chronically pulling a stainless-steel wire installed between the patella and the tibial tubercle. Microstructurally, the freezing treatment induced separation of collagen fiber bundles and fibroblast necrosis at 3 weeks, although the separation disappeared at 6 weeks. Ultrastructurally, small collagen fibrils with a diameter of less than 90 nm were predominant; at 6 weeks, the area occupied by collagen fibrils had decreased. In the frozen-shielded tendon, numerous large spaces were observed in the matrix at 3 weeks. This treatment increased the number of fibrils with a diameter greater than 360 nm and decreased the number of collagen fibrils per unit of area and the area occupied by collagen fibrils at 3 weeks. This study demonstrated that in situ freezing and the combination of in situ freezing and stress-shielding leads to a smaller volume of collagen fibrils per unit of cross section of the patellar tendon by mechanisms that remain to be defined.     

Delayed engraftment of nonobese diabetic/severe combined immunodeficient mice transplanted with ex vivo-expanded human CD34(+) cord blood cells

The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when cord blood (CB) is used as a source of hematopoietic graft. With the aim of defining the in vivo repopulating properties of ex vivo-expanded CB cells, purified CD34(+) cells were subjected to ex vivo expansion, and equivalent proportions of fresh and ex vivo-expanded samples were transplanted into irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic intervals after transplantation, femoral bone marrow (BM) samples were obtained from NOD/SCID recipients and the kinetics of engraftment evaluated individually. The transplantation of fresh CD34(+) cells generated a dose-dependent engraftment of recipients, which was evident in all of the posttransplantation times analyzed (15 to 120 days). When compared with fresh CB, samples stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor (SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of recipients was associated with the transplantation of the ex vivo-expanded versus the fresh CB cells (CD45(+) repopulation in NOD/SCIDs BM: 3. 7% +/- 1.2% v 26.2% +/- 5.9%, respectively, at 20 days posttransplantation; P <.005). An impaired short-term engraftment was also observed in mice transplanted with CB cells incubated with IL-11/SCF/FLT-3 ligand (3.5% +/- 1.7% of CD45(+) cells in femoral BM at 20 days posttransplantation). In contrast to these data, a similar repopulation with the fresh and the ex vivo-expanded cells was observed at later stages posttransplantation. At 120 days, the repopulation of CD45(+) and CD45(+)/CD34(+) cells in the femoral BM of recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%, respectively, and no significant differences of engraftment between recipients transplanted with fresh and the ex vivo-expanded samples were found. The analysis of the engrafted CD45(+) cells showed that both the fresh and the in vitro-incubated samples were capable of lymphomyeloid reconstitution. Our results suggest that although the ex vivo expansion of CB cells preserves the long-term repopulating ability of the sample, an unexpected delay of engraftment is associated with the transplantation of these manipulated cells.     

Histological and biochemical analysis of the fibrous tissue induced by implantation of synthetic ligament (Dacron): an experimental study in a rat model

We conducted a comparative study to evaluate the quality and true nature of the fibrous tissue formed around synthetic grafts when used in ligament replacement. In one group of Lewis rats, a patellar ligament was replaced with a Dacron prosthesis; the comparison group received a tail tendon isograft. Two-, 4-, 8-, 12-, and 24-week comparisons showed histological and biochemical changes in the Dacron group alone that were consistent with foreign-body reaction. Specifically, the Dacron group showed infiltration by large numbers of macrophages and foreign-body, multinucleated giant cells. In addition, the capsule of fibrous tissue that developed around the Dacron ligaments was characterized by lower collagen solubility, a higher content of noncollagenous protein, and a higher proportion of type III collagen than that comprising the isografted tendons. The results of this study call into question the suitability of synthetics for ligament replacement.     

Evidence that carbon monoxide stimulates prostaglandin endoperoxide synthase activity in rat hypothalamic explants and in primary cultures of rat hypothalamic astrocytes

Freezing or freeze-drying and gamma-irradiation are techniques currently used for processing tendon allografts. However, it is still unknown how these processing methods affect graft remodeling. In this study, we used a rat patellar tendon transplantation model to investigate the effect of various processing methods on remodeling by quantifying loss of collagen labeled with a radioactive isotope. The grafts were divided into the following four groups according to the processing method: fresh-frozen, freeze-dried, fresh-frozen and gamma-irradiated, or freeze-dried and gamma-irradiated. The percentage of donor collagen, calculated from hydroxyproline content and radioactivity level, was used as an indicator of graft remodeling. At 2 weeks, the level of donor collagen in the fresh-frozen group was 62%; in the freeze-dried group, 59%; in the fresh-frozen and irradiated group, 57%; and in the freeze-dried and irradiated group, 44%. At 4 weeks, the percentage of donor collagen remaining in grafts decreased to 38% in the fresh-frozen group, 19% in the freeze-dried group, 27% in the fresh-frozen and irradiated group, and 12% in the freeze-dried and irradiated group. Finally, at 12 weeks, the levels were 19% in the fresh-frozen group, 20% in the freeze-dried group, 15% in the fresh-frozen and irradiated group, and 6% in the freeze-dried and irradiated group. The percentages of donor collagen in the freeze-dried and the fresh-frozen and irradiated groups were significantly lower than that in the fresh-frozen group at 4 weeks. The values for the freeze-dried and irradiated group were significantly lower than those for the fresh-frozen and irradiated group at 4 and 12 weeks. These data suggest that freeze-drying, freeze-drying followed by gamma-irradiation, and fresh-freezing followed by gamma-irradiation temporarily accelerate graft remodeling.     

Effect of targeted mutation in collagen V alpha 2 gene on development of cutaneous hyperplasia in tight skin mice

Collagen V plays a major regulatory role in the formation of heterotypic fibers of the dermis and cartilaginous tissues as well as in the assembly of extracellular matrix. The pN/pN mouse, which is defective in collagen V alpha 2 gene, exhibits skeletal abnormalities, skin fragility, and alterations in the collagen fiber organization, whereas the TSK/+ mouse, which is defective in fibrillin-1, the major component of microfibrils present in the extracellular matrix, develops cutaneous hyperplasia and autoimmunity. We have studied the role of collagen V in the formation of heterotypic collagen fibers in F1 mice, which are obtained by breeding pN/pN with TSK/+ mice. Our results show that F1 progeny neither develop cutaneous hyperplasia nor produce anti-topoisomerase I autoantibodies, unlike TSK/+ mice. The diameter of the collagen fibrils in the skin is also comparable to that found in control mice. Thus, the phenotypic changes observed in the TSK mouse could be reversed by genetic complementation with a collagen V-defective mouse.     

Fibrillogenesis in tendon healing: an experimental study

The aim of this study was the histochemical, immunohistochemical and ultrastructural analysis of reparative fibrillogenesis in experimental lesions of Achilles' tendon. Subtotal tenotomy of Achilles' tendon was performed in twenty Wistar rats. The scar tissue was analysed 2, 4, 7, 14, 21, 30, 45 and 60 days post-operatively. Histochemical, (resorcin-fuchsin, aldehyde-fuchsin, iron haematoxylin and Fullmer and Lillie's methods) immunohistochemical (antibody against collagen I, II and elastin) and ultrastructural analyses were performed. Three phases in the healing process were distinguished: 1) inflammatory, 2) proliferative, and 3) remodelling phase. The inflammatory phase was characterised by haematoma, fibrin deposition, inflammatory cells, fibroblasts, beginning of collagen fibrillogenesis (200-400 A ? fibrils) and oxytalan fibrils. The proliferative phase was characterised by angiogenesis and fibroblast proliferation. Collagen fibres displayed a random arrangement and had a diameter of 400-600 A. Immature elastic fibres reached maximum tissutal concentration. In the remodelling phase, hypocellularity, normal vascularisation, tendon crimps, collagen fibres (800-1,000 A ?), elastic fibres with increased elastin deposition and reduction in oxytalan fibres were observed. In the course of the healing process collagen and elastic fibre fibrillogenesis exhibited consistent quantitative and qualitative variations (i.e. differences in the type and diameter of fibrils). The present study suggests that, together with other matrix macromolecules, also elastic fibres (oxytalan, elaunin and mature) are synthesised in significantly higher amounts during reparative fibrillogenesis and play a role in cell-matrix interaction.     

Correlation of remaining patellar tendon width with quadriceps strength after autogenous bone-patellar tendon-bone anterior cruciate ligament reconstruction

One hundred twenty-one patients were prospectively studied to determine whether the different remaining patellar tendon widths after central 10-mm bone-patellar tendon-bone graft harvest influenced the rate and level of quadriceps strength achieved during rehabilitation. Size of the patellar tendon width, measured at the same location in each patient, ranged from 24 to 35 mm. For this study, patients were grouped according to their remaining tendon size into small (14 to 17 mm; mean, 15.8), medium (18 to 20 mm; mean, 19.2), and large (21 to 25 mm; mean, 22.5) widths. Postoperatively, the patient's isokinetic quadriceps scores were determined at 6 weeks, 3 months, 6 months, and 1 year. At 6 weeks, the small- and medium-width tendon groups were significantly weaker than the large-width tendon group. At 3 months, only the small-width tendon group continued to be significantly weaker than the large-width tendon group. At and beyond 6 months, no statistically significant differences were seen between remaining patellar tendon width groups and their isokinetic quadriceps scores. A constant-sized autogenous patellar tendon graft may be harvested for anterior cruciate ligament reconstruction without compromising ultimate postoperative quadriceps strength recovery.     

A method for preparing collagen graft materials

The aim of this research was to develop a method of local production of collagen graft materials which are presently imported. The following methods were used to produce collagen membrane and sponge from human placentas and rat tail tendons. Collagen type I was isolated from human placenta and rat tail tendon by acetic acid extraction and characterised by SDS-PAGE. The collagen sponge was prepared by dissolving the collagen in HCl. The resulting dispersion was poured into a glass container, freeze-dried and then cross-linked by immersion in glutaraldehyde solution. It was then washed with distilled water and freeze-dried again. The collagen membrane was also similarly prepared by dispersing lyophilized collagen in HCl but then mixed with glutaraldehyde, exposed to U.V. light and later air dried.     

Collagen organization in the branching region of human brain arteries

BACKGROUND AND PURPOSE: Unruptured saccular aneurysms are relatively common, occurring in 4% to 9% of autopsies. Their development at the apex region of brain artery bifurcations is attributed to a combination of structural factors and the effect of blood pressure. Collagen is a primary tension-bearing fabric of the vessel wall, and our purpose was to examine its 3-dimensional alignment at arterial branches. METHODS: Sixteen segments of arteries from the circle of Willis, including bifurcations, were pressure distended, fixed, and sectioned in 1 of 3 orthogonal planes. We measured the 3-dimensional organization of collagen at the flow divider by using the polarized light microscope. An electron microscopy study performed in tandem provided measurements on the collagen fibril diameters and packing density. RESULTS: Orientation data of the collagen fabric were obtained from sections from 3 different cutting planes. The tunica media of all bifurcations had an alignment that was primarily circumferential, and the medial gap (medial defect) was distinguishable at the apex of all bifurcations. The subendothelial layer was thin at the apex but thicker and more disorganized distally. Adventitial collagen showed little organization except for a high degree of alignment along the apex. Results from the electron microscopy study showed densely packed collagen fibrils of uniform diameter at the apex, compared with slightly smaller and less densely packed fibrils nearby. CONCLUSIONS: In the region of the medial gap, a narrow band of highly aligned tendonlike collagen running in the direction of the ridge of the flow divider was a consistent finding. This structure would provide strength and stability to the vessel and is inconsistent with the concept of an inherent defect in the structure of bifurcations.     

Morphology and matrix composition during early tendon to bone healing

This study outlines the early morphologic phenomenon of tendon to bone healing in the rabbit model. Twelve skeletally mature, male New Zealand White rabbits received transplantation of the hallucis longus tendon into a 2-mm calcaneal bone tunnel. The morphologic characteristics of the healing tendon to bone interface were evaluated at 1, 2, 4, and 6 weeks after surgery by the use of conventional histology and immunohistochemical localization of collagen Types I, II, and III. Histologic analysis illustrated progressive maturation and reorganization of the tendon to bone interface with subsequent development of tissue collagen fiber continuity between the tendon and bone. Initially, diffuse immunolocalization of all three collagen types was observed within the scar tissue filling the space between the tendon and bone. During a 6-week period, reorganization of the scar tissue into an interface occurred, similar to an indirect insertion. Although a definitive fibrocartilage region did not form, Type II collagen was localized at the remodeling insertion site throughout the first 6 weeks of repair. In addition, Type III collagen fibers, resembling Sharpey's fibers, were noted to span this interface. The characterization of the insertion between tendon and bone is important to the understanding of healing in commonly used orthopaedic grafting procedures, such as anterior cruciate ligament reconstructions.     

Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel

Full-thickness articular cartilage defects are a major clinical problem; however, presently there is no treatment available to regeneratively repair these lesions. The current therapeutic approach is to drill the base of the defect to expose the subchondral bone with its cells and growth factors. This usually results in a repair tissue of fibrocartilage that functions poorly in the loaded joint environment. The use of phenotypically appropriate chondrocytes embedded in a collagen gel delivery vehicle may provide a method that could be used to repair full-thickness articular cartilage defects with functionally satisfactory hyaline cartilage. Allograft articular chondrocytes embedded in a type I collagen gel were transplanted into large (6 x 3 x 3 mm), full-thickness articular cartilage defects in condylar and patellar weight-bearing surfaces to develop clinically applicable methods to repair articular cartilage defects. Chondrocytes were isolated from the articular cartilage of 4-week-old New Zealand rabbits and embedded in type I collagen gels. This composite was transplanted into a full-thickness defect on the medial femoral condyle and patellar groove of adolescent host rabbits. The repair cartilage was assessed histologically by a semiquantitative scoring system and biomechanically with a microindentation technique of specimens 4-48 weeks after chondrocyte transplantation. Defects in both locations were repaired with histologically apparent hyaline cartilage observed from as early as 4 weeks until 48 weeks after transplantation. The repair cartilage in the medial femoral condyle was more irregular than in the patellar groove, but in all other respects was similar. The grafted tissue did not remodel and differentiate into the morphological zones seen in normal articular cartilage. No tidemark or subchondral bony plate formed even 48 weeks after transplantation. Biomechanically, the repaired cartilage demonstrated indentation values similar to normal articular cartilage 12 weeks after transplantation and remained the same 48 weeks after transplantation. By contrast, the control (i.e., empty) defects healed with tissue that exhibited very poor metachromatic staining and exhibited very high indentation values. Incomplete bonding of the repair tissue to the normal cartilage was seen, and the surface was significantly irregular with major discontinuities. These observations provide the basis for considering the use of allograft articular chondrocytes to repair articular cartilage defects in the weight-bearing regions of the knee.     

The effect of a preparation from the horns of the saiga antelope on the goal-directed drinking behavior of rats

We analyzed 79 consecutive patients with aneurysms and found a patient who lacked type III collagen. Collagen was extracted from the skin, and the lack of type III collagen was determined by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Only 1 of the 79 patients was found to lack type III collagen. This patient was a 55-year-old man who had sustained an abdominal aortic aneurysm and aortic dissection. He did not show any of the typical clinical symptoms of Ehlers-Danlos syndrome type IV, such as hyperextensible skin and joints. He had none of the usual risk factors nor any clear family history of the syndrome. Furthermore, his collagen fibrils demonstrated a homogeneous appearance. This case may represent a new form of type III collagen deficiency.     

Ehlers-Danlos syndrome type VIIB. Morphology of type I collagen fibrils formed in vivo and in vitro is determined by the conformation of the retained N-propeptide

Previously we showed that fibrils generated from collagen and pNcollagen-ex6 from fibroblasts of an individual with Ehlers-Danlos syndrome (EDS) type VIIB were hieroglyphic in cross-section and all N-propeptides were located at the fibril surface. Hieroglyphs were resolved to near-cylindrical fibrils (that were similar in appearance to the fibrils seen in the tissues of individuals with EDS type VIIB) by treatment with N-proteinase which cleaved the pN alpha 1(I) chains but not the pN alpha 2(I)-ex6 chains (Watson, R. B., Wallis, G. A., Holmes, D. F., Viljoen, D., Byers, P. H., and Kadler, K. E. (1992) J. Biol. Chem. 267, 9093-9100). Here, quantitative scanning transmission electron microscopy (STEM) showed that N-propeptides in hieroglyphs were in a "bent-back" conformation and thus located exclusively in the overlap zone of the fibril D-period (D = 67 nm). In contrast, STEM of fibrils from the dermis of an individual with EDS type VIIB showed that partially cleaved N-propeptides (in which cleaved pN alpha 1(I) remained in noncovalent association with pN alpha 2(I)-ex6 chains) were distributed equally between the gap and overlap zones of the fibrils. Comparison of experimental data with theoretical mass distributions of the fibril based on amino acid sequence data gave a consistent value of 33 nm for the total axial extent for the N-propeptides in hieroglyphic and tissue fibrils irrespective of the location of N-propeptides to the gap or overlap zone. These data exclude the possibility that N-propeptides adopt a random configuration, but rather, that they locate to specific sites in the gap and overlap zones. The results demonstrated that cleavage of pN alpha 1(I) chains in vivo releases the N-propeptides from the constraints of the bent-back conformation. Co-distribution of partially cleaved N-propeptides between gap and overlap zones allows a higher surface packing density of N-propeptides and explains how circularity of large diameter fibrils can be achieved despite the retention of N-propeptides in tissues of individuals with EDS type VIIB.     

Kinking deformities in collagen (author's transl)

The affect of stretch on collagen was investigated. Alterations of mechanical dimensions and thermostability of fibrils were measured and changes in fine structure determined by x-ray diffraction and electronmicroscopy. Collagen underwent changes both in tensile strength and fine structure following stretch beyond the physiological range. The severity of these changes depended both on the degree of stretch and the cross-link density of the collagen Fibrils either became split into bundles consisting of subfibrillar units or showed circumscribed kinking deformities. The mechanism producing kinking was investigated. It is possible that a connection exists between fibrillar kinking and tendon rupture.     

Treatment of non-obese diabetic (NOD)/Severe-combined immunodeficient mice (SCID) with flt3 ligand and interleukin-7 impairs the B-lineage commitment of repopulating cells after transplantation of human hematopoietic cells

Until recently, the identification of cellular factors that govern the developmental program of human stem cells has been difficult due to the absence of repopulation assays that detect human stem cells. The transplantation of human bone marrow (BM) or cord blood (CB) into non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) mice has enabled identification of primitive human cells capable of multilineage repopulation of NOD/SCID mice (termed the SCID-repopulating cell [SRC]). Here, we examined the effect of long-term in vivo treatment with various combinations of human cytokines on the developmental program of SRC. Detailed flow cytometric analysis of engrafted mice indicated that the vast majority of the human graft of untreated mice was comprised of B lymphocytes at various stages of development as well as myeloid and primitive cells; T cells were not reproducibly detected. Many studies, including murine in vitro and in vivo data and human in vitro experiments, have suggested that flt3 ligand (FL) and/or Interleukin-7 (IL-7) promotes T- and B-cell development. Unexpectedly, we found that treatment of engrafted mice with the FL/IL-7 combination did not induce human T- or B-cell development, but instead markedly reduced B-cell development with a concomitant shift in the lineage distribution towards the myeloid lineage. Effects on lineage distribution were similar in engrafted mice transplanted with highly purified cells indicating that the action of the cytokines was not via cotransplanted mature cells from CB or BM cells. These data show that the lineage development of the human graft in NOD/SCID mice can be modulated by administration of human cytokines providing a valuable tool to evaluate the in vivo action of human cytokines on human repopulating cells.     

Biochemical and molecular homogeneity in the patellar tendon of the immature pig

Patellar tendon is widely used for reconstruction of the anterior cruciate ligament. However, few studies have investigated the tendon's homogeneity, a characteristic often assumed of it in experiments. In this study, the assumption that the patellar tendon is homogeneous was tested by dividing the central half of the tendon into six sections along its length and width and comparing commonly measured biochemical parameters and patterns of gene expression among these sections. No significant differences were found between the sections for any of the studied parameters: water content (p > 0.5), DNA content (p > 0.9), total collagen content (p > 0.8), amount of type I collagen (p > 0.7) or type-III collagen (p > 0.7), or expression of mRNA (p > 0.9). For all parameters, the minimum power value for statistical analyses was greater than 0.80. It was concluded that the central half of the tendon is homogeneous in terms of all of the measured parameters. The results provide important information for the many experiments that sample part of the patellar tendon to infer the characteristics of the whole tendon, e.g., biopsy studies.     

Study of aging rat tail collagen using atomic force microscope

Collagen structure of young and old rats was examined by using atomic force microscope (AFM) images. Rat tail tendons of eight and twenty-four month-old Wistar rats were digested enzymatically (pepsin), and allowed to refibrillate for 24 hours at 37 degrees C. The samples were examined using a Nanoscope III (Digital Instruments, Santa Barbara, CA, U.S.A.) with a J scanning head and a 200 microns silicon nitride cantilever. The study was performed in air and without filters. The AFM inspection of refibrillated collagen produced images showing long fibrils with relatively homogeneous heights and widths, characterized by clear banding with a periodic interval (D band) of 67 nm. With respect to collagen extracted from young rats, collagen extracted from old rats revealed fibrils exhibiting the same band interval, but with lower widths and heights. Furthermore, the depth of gap between two overlaps showed a higher mean value in the aged rats. These data are consistent with biochemical reports of collagen modifications during aging; we suggest that post-synthetic reactions might be responsible for this as they interfere with the refibrillation process and also modify the three-dimensional structure of fibrils.     

Association of type VI collagen with D-periodic collagen fibrils in developing tail tendons of mice

The process of the arrangement of D-periodic collagen fibrils and their growth in maturing tail tendon of mice were studied with the association of type VI collagen, from fetal day 10 to 10 weeks after birth. In tail tendons, the amount of collagen fibers gradually increased along with the diameters of D-periodic collagen fibrils during maturation. Type VI collagens first appeared on fetal day 10, when D-periodic collagen fibrils were not recognizable. Type VI collagens were observed around the fibroblastic cells in early stages of development, but were among thick collagen fibrils in the adult tendon. While the periodic distances of type VI collagen fibrils were over 100 nm at fetal days, they were packed to 80-90 nm after birth. The periodic bands were stained well with ruthenium red in adult but not in young tendons, indicating the close association of proteoglycans or glycosaminoglycans (PGs/GAGs) with maturing type VI collagens. Since type VI collagen in native form is known to associate with D-periodic collagen fibrils via PGs/ GAGs, ruthenium red-stainability on the surface of D-periodic collagen fibrils was also examined; results showed that ruthenium red-stainable elements were D-periodically associated. When the surface morphology of D-periodic collagen fibrils in adult animals was examined by atomic force microscopy, a large depth of the groove between elevated and depressed surfaces became prominent when the fibril surface was digested with hyaluronidase. Thus, it is possible to observe topologically the association of PGs/GAGs and probably that of type VI collagens with D-periodic collagen fibrils.     

Expression of collagens I, III, IV and V mRNA in excimer wounded rat cornea: analysis by semi-quantitative PCR

A semi-quantitative polymerase chain reaction (PCR) methodology was used to evaluate the kinetic changes occurring in collagens I, III, IV and V mRNA in rat cornea following excimer laser keratectomy. cDNA was synthesized from RNA extracted from rat cornea at various times following excimer laser photoablative keratectomy. Collagen cDNA sequences were subsequently amplified using specific sets of oligonucleotide primers. Competitive PCR amplification was carried out using an internal standard so that a semi-quantitative analysis of message for synthesis of collagen types I, III, IV and V could be performed and time course dynamics of message for these collagens studied. There was a biphasic increase in the levels of collagens III, IV and V mRNA following excimer laser keratectomy. Collagen I mRNA levels demonstrated a more sustained increase and were still elevated at 6 weeks following wounding. Collagens IV and V mRNA showed the largest increase with an approximate three fold increase over controls between 4 days and 1 week. Our results demonstrate that upregulation of stromal collagens I, III, and V mRNA and basement membrane collagen IV mRNA occurs in rat cornea following excimer laser keratectomy.