Bone block augmentation from the iliac crest for treatment of deep osteochondral defects of the knee resembles biomechanical properties of the subchondral bone.

PURPOSE: Bone block augmentation from the iliac crest can be used for reconstruction of the osteochondral unit to restore the underlying subchondral bone upon restoration of the cartilaginous layer via matrix-induced chondrocyte transplantation. To critically understand the successful restoration of the defect, biomechanical and histological analysis of the implanted bone blocks is required. The aim of the study was to analyse the ability of the bone block technique to restore huge bone defects by mimicking the physiological subchondral zone. METHODS: The experiments were performed using lateral femoral condyles and iliac crest bone grafts from the same cadavers (n = 6) preserved using the Thiel method. CT scans were made to evaluate bone pathology. Bone mineral density of all specimens was evaluated in the femoral head prior to testing. A series of tests were conducted for each pair of specimens. A static compression test was performed using an electro dynamic testing machine with maximal strength and failure behavior analyzed. Biomechanical tests were performed in the medial-lateral direction for iliac crest and for femoral condyles with and without removal of the cartilage layer. Histological analysis was performed on decalcified specimens for comparison of the condyle at lesion site and the graft. RESULTS: No significant difference in failure load could be found for iliac crest (53.3-180.5 N) and femoral condyle samples upon cartilage removal (38.5-175.1 N) (n.s.). The femoral condyles with an intact cartilage layer showed significantly higher loads (118.3-260.4N) compared to the other groups indicating that native or regenerated cartilage can further increase the failure load (p < 0.05). Bone mineral density significantly influenced failure load in all study groups (p < 0.05). Histological similarity of the cancellous bone in the femoral condyle and in the iliac crest was observed. However, within the subchondral zone, there was a higher density of sponge like organized trabeculae in the bone samples from the iliac crest. Tide mark was only detected at the osteochondral interface in femoral condyles. CONCLUSION: This study demonstrated that, bone blocks derived from the iliac crest allow a biomechanical appropriate and stable restoration of huge bony defects by resembling the subchondral zone of the femoral condyle. Therefore, bone augmentation from the iliac crest combined with matrix-induced autologous chondrocyte transplantation seems to be a reasonable method to treat these challenging injuries.

Dynamic hydrostatic pressure enhances differentially the chondrogenesis of meniscal cells from the inner and outer zone.

This study analyses the influence of dynamic hydrostatic pressure on chondrogenesis of human meniscus-derived fibrochondrocytes and explores the differences in chondrogenic differentiation under loading conditions between cells derived from the avascular inner zone and vascularized outer region of the meniscus. Aggregates of human fibrochondrocytes with cell origin from the inner region or with cell origin from the outer region were generated. From the two groups of either cell origin, aggregates were treated with dynamic hydrostatic pressure (1Hz for 4h; 0.55-5.03MPa, cyclic sinusoidal) from day 1 to day 7. The other aggregates served as unloaded controls. At day 0, 7, 14 and 21 aggregates were harvested for evaluation including histology, immunostaining and ELISA analysis for glycosaminoglycan (GAG) and collagen II. Loaded aggregates were found to be macroscopically larger and revealed immunohistochemically enhanced chondrogenesis compared to the corresponding controls. Loaded or non-loaded meniscal cells from the outer zone showed a higher potential and earlier onset of chondrogenesis compared to the cells from the inner part of the meniscus. This study suggests that intrinsic factors like cell properties in the different areas of the meniscus and their reaction on mechanical load might play important roles in designing Tissue Engineering strategies for meniscal repair in vivo.

RAP-PCR fingerprinting reveals time-dependent expression of matrix-related molecules following stem-cell based TGFß1-induced chondrocyte development.

Different approaches of engineering cartilage to treat defects in the articulating surfaces of the joints have been designed, which mainly use mesenchymal stem cells or autologous chondrocytes for in situ transplantation. However, these cells are poorly characterized with respect to viability, degree of differentiation and morphology or production of extracellular matrix. At present, one of the key approaches to generate chondrocytes is the stimulation of stem cells with transforming growth factor (TGF) ß1. To characterize the molecular alterations occurring during the cellular transformation induced by TGF-ß1 exposure, the differentiation process of bone marrow-derived stem cells into chondrocytes was investigated using an in vitro chondrogenesis model and the RNA arbitrarily primed PCR (RAP-PCR) fingerprinting technique. Distinct genes were found to be differentially regulated during chondrocyte development beginning on day 1: collagen type I, non-muscle myosin MYH9, followed by manganese superoxide dismutase and sodium-potassium ATPase on day 7. The results suggest that using RAP-PCR for differential display fingerprinting is a useful tool to investigate the differentiation process of bone marrow-derived stem cells following TGF-ß1-stimulation.

Treatment of human mesenchymal stem cells with pulsed low intensity ultrasound enhances the chondrogenic phenotype in vitro.

This study examined the effects of low intensity pulsed ultrasound (LIPUS) on human bone marrow-derived mesenchymal stem cells undergoing chondrogenic differentiation. Aggregates of mesenchymal stem cells and mesenchymal stem cells seeded in three dimensional matrices were cultured in a defined chondrogenic medium and subjected to LIPUS for the first 7 days of culture. At 1, 7, 14 and 21 days, samples were harvested for histology, immunohistochemistry, RT-PCR, and quantitative DNA and matrix macromolecule analysis. Cell aggregates with daily treatment for 20 minutes showed no significant differences for proteoglycan and collagen content during chondrogenic differentiation. However ultrasound application for 40 minutes daily resulted in a statistically significant increase of the proteoglycan and collagen content after 21 days in culture. Aggregates treated for 20 minutes daily showed decreased expression of chondrogenic genes at all time points. In contrast, 40 minutes of daily treatment of aggregates resulted in a significant increase of chondrogenic marker genes after an initial decrease at day 7 with time in culture. Ultrasound treated cell-scaffold constructs showed a significant increase of chondrogenic marker gene expression and extracellular matrix deposition. This study indicates that LIPUS can be used to enhance the chondrogenesis of mesenchymal stem cells in cell aggregates and cell-scaffold constructs. We have found a dependency on the specific treatment parameters. We hypothesize that LIPUS can be used for an improved in vitro preparation of optimized tissue engineering implants for cartilage repair. Furthermore this non-invasive method could also be of potential use in vivo for regenerative therapy of cartilage in the future.

Mechanobiological conditioning of stem cells for cartilage tissue engineering.

Articular cartilage possesses little capacity for endogenous repair after having been damaged by disease or trauma. Various surgical procedures depending on ingrowth of mesenchymal stem cells into the defects showed repair with fibrocartilage which is of minor quality and less resistant against physical forces. New treatment options using Tissue Engineering strategies for cartilage repair showed intriguing results. Human mesenchymal stem cells (MSC) isolated from bone marrow are becoming increasingly recognized for their potential to generate different cell types and thereby function effectively in vitro or in vivo in tissue repair. Incorporation of MSCs in suitable tissue engineering scaffolds and culture in chondrogenic medium can produce cartilage-like tissue. MSCs can be harvested from bone marrow by a small puncture of the iliac crest of patients. In contrast to chondral based repair this small procedure creates no additional harvest defect in the knee joints of the patient. Numerous publications show the beneficial influence of mechanobiological conditioning (e.g. mechanical compression, hydrostatic pressure, osmotic, shear, ultrasound) on the chondrogenic differentiation of dedifferentiated chondrocytes. In contrast to chondrocytes and cartilage explants there are few studies that examine the influence of mechanobiological stress on mesenchymal progenitor cells undergoing chondrogenesis. Using an in vitro aggregate culture system enhanced chondrogenesis of mesenchymal progenitor cells, detected by an increased extracellular matrix deposition of collagen and aggrecan, could be shown under repeated cyclic hydrostatic pressure. Similar results, with an increase in chondrogenic differentiation of mesenchymal progenitor cells could be detected, when the cells were loaded in three-dimensional matrices and subjected to cyclic, compressive load or low-intensity pulsed ultrasound. This review will summarize the current state of knowledge in the field of mechanobiological conditioning of mesenchymal stem cells and its possible clinical application.

Engineering of osteochondral tissue with bone marrow mesenchymal progenitor cells in a derivatized hyaluronan-gelatin composite sponge.

The aim of this study was to investigate the potential of a composite matrix, containing esterified hyaluronic acid and gelatin, to facilitate the osteochondral differentiation of culture-expanded, bone marrow-derived mesenchymal progenitor cells. The cell loading characteristics and the effects of the matrix on cell differentiation were examined in vitro and in vivo. Empty and cell-loaded composites were cultivated for up to 28 days in a chemically defined medium with or without transforming growth factor-beta1 (TGF-beta1). A type II collagen-rich extracellular matrix was produced by cells loaded in the matrix and cultured in the presence of TGF-beta1. Empty and cell-loaded matrices were also implanted subcutaneously in immunodeficient mice. Three types of implant were used: empty (group I), cell-loaded matrices (Group II), and cell-loaded matrices cultured for 14 days in vitro in defined medium with TGF-beta1 (group III). No osteochondral differentiation was found in implanted empty matrices; however, the matrix supported osteochondrogenic cell differentiation in the cell-loaded implants. Preculture in vitro in a chondrogenic medium increased the percentage of osteochondral tissue found in the constructs after 3 weeks. These results indicate the potential use of this composite matrix for delivery of bone marrow-derived mesenchymal progenitor cells for the repair of chondral and osseous defects. The results also indicate that this composite matrix is useful for in vitro tissue engineering.

[Improvement of the amplification rate of human chondrocytes with IGF-I and RGD]. / Verbesserung der Amplifikationsrate humaner Chondrozyten unter dem Einfluss von IGF-I und RGD.

Human chondrocytes were incubated under following conditions: Group 1 (control group): Incubation in 25 cm2 cell culture flasks (Costar) with RPMI-medium (6%-AB-serum, L-Glutamin, Hepes-buffer and antibiotics); Group 2: Different concentrations of IGF-I (1 ng/ml, 10 ng/ml) were added to the RPMI-medium; Group 3: Incubation (like control group) with additional coating of the cell culture flasks with different concentrations of RGD (5 mg/ml; 7.5 mg/ml; 10 mg/ml; 20 mg/ml); Group 4: Combination of coating with RGD (5 mg/ml; 10 mg/ml) and addition of IGF-I (1 ng/ml; 10 ng/ml) to the medium. The cells of the control group could be doubled within 2 weeks. The amplification rate of the groups 2 and 3 was improved in comparison to group 1 with the following maxima: Group 2 (5 mg/ml RGD) 3.1 times and group 3 (1 ng/ml IGF-I) 2.6 times of the number of the cells in the beginning. Group 4 (RGD and IGF-I) showed additive effects, for 4.1 times of the number of the cells in the beginning could be counted after 14 days. RGD and IGF-I (groups 2 to 4) made possible an earlier dedifferentiation and adhesion of the cells to the bottom of the cell culture flasks. By using both growth factors (RGD and IGF-I), the number of the cells could be enhanced more than 2 times in comparison to the control group within the same time. So less than half of the autologous patient's cartilage is necessary for cultivation of hyaline cartilagee.

Immunohistochemical demonstration of nerve growth factor receptor in bovine testis.

Nerve growth factor receptor (low-affinity form) was demonstrated immunohistochemically in bovine testis by using a monoclonal mouse anti-human antibody. In the 7-month-old fetus and in the early postnatal testis, the peritubular and intertubular fibroblast-like mesenchymal cells showed a strong reaction. Following differentiation of these cells into Leydig and myoid peritubular cells, the nerve growth factor receptor was no longer expressed. However, peritubular and intertubular testicular fibroblasts/fibrocytes, which are also derived from mesenchymal precursors, remained positive. Additionally, the nerve growth factor receptor was demonstrated in postnatal prespermatogonia, A-spermatogonia, I-spermatogonia and members of the spermatogonia precursor cell line; B-spermatogonia remained negative. In A-spermatogonia and I-spermatogonia, the expression of the nerve growth factor receptor was cell-cycle-dependent and was mostly observed during G1-phase. Pre-embedding ultrahistochemistry with gold-conjugated antibody followed by silver-enhancement revealed that the nerve growth factor receptor was localized at the outer cell surface. The metal granules showed a regular distribution in positive spermatogonia. In testicular fibroblasts/fibrocytes the long narrow processes were preferentially decorated.

Immunohistochemical study of seminiferous epithelium in adult bovine testis using monoclonal antibodies against Ki-67 protein and proliferating cell nuclear antigen (PCNA).

The distribution pattern of proliferating cell nuclear antigen (PCNA) and Ki-67 protein was studied in adult bovine seminiferous epithelium by means of immunohistochemistry using monoclonal antibodies. Tailoring the methodological protocol for each of the two proliferation markers was a necessary prerequisite for obtaining optimal results in tubular sections and whole-mounts. A-, I- and B-spermatogonia displayed PCNA-positive nuclei, except during meta-, ana- and telophases of mitosis. PCNA-negative nuclei in the basal tubular compartment, excluding those from non-cycling Sertoli cells, belonged to the spermatogonia precursor cell line. However, only about 30%, 45% and 47% of the respective A-, I-, B-spermatogonia had positive nuclei after exposure to the MIB-1 antibody directed against the Ki-67 protein. Spermatogonia with MIB-1-negative nuclei represented cells in the G1-phase. Both antibodies reacted intensely with the nuclei of preleptotene primary spermatocytes. PCNA reactivity was also present during leptotene through pachytene. Ki-67 protein expression was absent during leptotene and zygotene but was again encountered during pachytene and meiosis I and II. Anti-PCNA/anti-protein gene product 9.5 double-labelling indicated that the transition from spermatogonia precursor cells into A1-spermatogonia is not strictly synchronized in a given tubular segment, a possible reason for the flexibility in A-spermatogonial propagation seen in bovine seminiferous tubules.

Evolution and ultrastructure of the bovine spermatogonia precursor cell line.

The spermatogonial stem cell line in prepubertal and adult bovine testis was studied by electron microscopy and protein gene product 9.5 immunohistochemistry. Three successive spermatogonia precursor cell configurations were observed. Small basal stem cells were found to possess a spherical shape and nuclei with two to three nucleoli. They were observed in prepubertal testes (25 and 30 weeks) and in low numbers during all the stages of the seminiferous epithelial cycle in the adult. Aggregated spermatogonia precursor cells are the dominating germ cell type in the 25-week-old and 30-week-old calf. In the adult seminiferous epithelium, they cause expansion of the basal tubular compartment as they form dense groups containing up to 15 cells. These groups are observed concomitantly with cycling A-spermatogonia and preleptotenes at the beginning of spermatocytogenesis. At the end of A-spermatogonia propagation, the aggregated spermatogonia precursor cells separate and intermingle with cycling A-spermatogonia. The spermatogonia precursor cells can later be found together with I-spermatogonia as members of an interconnected cellular network of medium-sized cells. When the I-spermatogonia divide to form the smaller B-spermatogonia, the precursor cells, which stay connected with the cycling spermatogonial population, pass through a growth phase. They can now be considered as committed spermatogonia precursor cells and are continuously being transformed into A1-spermatogonia to start a new round of spermatocytogenesis. Ultrastructurally, all members of the precursor cell line are similar. However, a number of features have been found to show a quantitative increase (endoplasmic reticulum, mitochondria) or to exhibit a rising degree of complexity (nucleolus) during the progression from basal stem cells to committed spermatogonia precursor cells.

Immunolocalization of the neural cell adhesion molecule L1 in non-proliferating epithelial cells of the male urogenital tract.

The localization of the neural cell adhesion molecule L1 in the male urogenital tract (including seminal vesicles and prostate) of the mouse and bull was investigated using immunocytochemical and immunochemical methods in order to better understand the function of this glycoprotein in non-neural tissues. L1 antibodies labeled non-myelinated nerves in all portions of the urogenital tract investigated. However, L1 immunoreactivity was also found between epithelial cells of several regions of the urogenital system including epididymal tail, deferent duct, ejaculatory duct and seminal vesicles. Some L1 immunoreactivity was also demonstrated between epithelial cells of murine urinary bladder and urethra. The specificity of the immunoreaction was verified by western blots. There was no correlation between L1 expression and proliferating activity as revealed by double immunocytochemistry using various markers of cell proliferation. This unexpected expression of L1 in nonneural tissues is mainly restricted to non-proliferating epithelia of those portions of the urogenital tract that are derived from the Wolffian duct. It is suggested that L1 in these epithelia could enhance the mechanical resistance and reduce transepithelial permeability.

Configuration and distribution of bovine spermatogonia.

The configuration and distribution of bovine spermatogonia, preleptotene primary spermatocytes and Sertoli cells in the basal seminiferous tubular compartment have been studied by means of whole-mount preparations, immunohistochemistry and quantitative morphology. Three types of spermatogonia (Sg) can be identified. Large A-spermatogonia are irregularly distributed in the tubular periphery. Following the period of propagation of the A-spermatogonia, an interconnected meshwork of medium-sized spermatogonia with different cytogenetic potency is observed. Although the majority of the medium-sized spermatogonia are kinetically of the I type and divide to produce small B-spermatogonia, some members of the medium-sized population are seen in a growth phase and differentiate into large A-spermatogonia. These mark the beginning of a new round of spermatocytogenesis. Only one generation of B-spermatogonia divides into preleptotene primary spermatocytes. The architectural arrangement of multiplying spermatogonia in circles or rows is primarily the result of the distribution of the Sertoli cells. Spermatogonial multiplication is not strictly coordinated with the stages of the seminiferous epithelial cycle. Spermatogonial degeneration amounts on average to 3.6% and has therefore no decisive impact on the yield of primary spermatocytes.

Distribution pattern of F-actin, vimentin and alpha-tubulin in the bovine testis during postnatal development.

The distribution of F-actin, vimentin and alpha-tubulin was studied immunohistochemically in bovine seminiferous and straight testicular tubules, rete testis and intertubular tissue during postnatal development. Sites of antigenicity were detected by ABC immunoperoxidase technique and visualized by metal-enhanced deposition of diaminobenzidine. Within the seminiferous epithelium, F-actin appears at 20 weeks and is found in adult Sertoli cells as part of specialized cell contacts. In peritubular cells, F-actin increases gradually from 4 to 30 weeks when the adult concentration is achieved. After 20 weeks, subepithelial fibroblasts of the mediastinum testis start to express F-actin and at 52 weeks, a thick layer of positive myofibroblasts is seen beneath the epithelia of rete testis and straight testicular tubules. Testicular macrophages and light intercalated cells (LIC) are also characteristically decorated following F-actin immunoreaction. Vimentin is localized in perinuclear position in pre-Sertoli cells of 4-20 weeks and in adult Sertoli cells. During the period of transformation from pre-Sertoli to Sertoli cells, the perinuclear vimentin coat is absent. The epithelia of rete testis and straight tubules exhibit a strong vimentin immunoreaction in their basal parts. This specific pattern does not change from 4 weeks to adulthood. Alpha -tubulin is absent in 4-week-old seminiferous tubules. At 8 weeks, the perinuclear area of pre-Sertoli cells reacts positive. The alpha-tubulin content increases in these cells continuously, and from 30 weeks on nearly the entire supranuclear cytoplasm of Sertoli cells is heavily decorated. The epithelial of rete and straight tubules display a growing number of alpha-tubulin-positive cells from 4 to 40 weeks. From then on, nearly all epithelial cells contain alpha-tubulin, particularly in a narrow zone beneath their lateral cell borders.

The bovine tubouterine junction: general innervation pattern and distribution of adrenergic, cholinergic, and peptidergic nerve fibers.

The innervation of the bovine tubouterine junction was studied in sexually mature heifers using antisera against various neuronal markers and a modified acetylcholinesterase method. The vast majority of the nerve fibres in the bovine tubouterine junction belongs to the sympathetic nervous system; peptidergic and cholinergic fibers are restricted to characteristic locations. The endosalpinx in the adovarian portion of the terminal tubal segment is poorly innervated. The mucosa of the aduterine portion and of the tubouterine transitional region proper receives a strikingly dense innervation, which is observed mainly in combination with a strong vascularisation of specialised mucosal structures. In the endometrium, perivascular nerves accompany the ascending spiral arteries but sporadic contacts between nerve fibres and uterine glands are also observed. From the muscular coat the inner longitudinal layer of the terminal tubal segment is more richly supplied by nerve fibres than the intermediate circular and outer longitudinal layers of the tubouterine junction. No changes in the innervation pattern were seen during the different stages of the sexual cycle.

Expression of the proliferation-associated Ki-67 antigen in bovine testicular tubular cells during the seminiferous epithelial cycle, demonstrated with the MIB-1 antibody.

Ki-67 expression in the seminiferous tubule of the bovine testis was studied by immunohistochemistry during the seminiferous epithelial cycle using the monoclonal antibody MIB-1. Spermatogonial proliferation is most obvious in stages 5-7, and 8, when B-spermatogonia divide. A lower rate of spermatogonial propagation is observed preceding or during meiosis in stages 1-4. The MIB-1 antibody also gives positive results with some post-spermatogonial tubular cells. Preleptotenes passing through S-phase in stage 1 reveal positive nuclei. During prophase of meiosis I pachytenes react strongly, diplotenes react in an attenuated manner, while leptotenes and zygotenes stay negative. Secondary spermatocytes seen in stage 4 are positive as are the chromosomes during meta- and anaphase of the meiotic divisions. Post-meiotic spermatids are also decorated but stop Ki-67 expression abruptly at the end of stage 4. Sertoli cells are negative.

The innervation of the bovine ductus deferens: comparison of a modified acetylcholinesterase-reaction with immunoreactivities of cholinacetyltransferase and panneuronal markers.

The innervation pattern of the bovine deferent duct was studied by acetylcholinesterase (AChE)-histochemistry and by immunohistochemical methods. Using antibodies against protein gene product-9.5 (PGP-9.5) and neuron specific enolase (NSE) the complete innervation pattern can be visualized. Thick nerve bundles in the periductal connective tissue supply the two-layered muscular coat. The inner, mainly circularly arranged muscle bundles are innervated by a particularly dense plexus, whereas the nervous network of the more longitudinally running outer musculature is somewhat looser. Additionally, nerve fibres were observed in the subepithelial space in connection with blood vessels and in close proximity to the basal lamina. An innervation pattern analogous to that of the two panneuronal markers was displayed in the immunoreaction against dopamine-beta-hydroxylase (DBH), indicating that the innervation of the bovine deferent duct is predominantly adrenergic. However, the positive reaction with a monoclonal antibody against cholinacetyltransferase (ChAT) specifically demonstrated for the first time the presence of a cholinergic nerve plexus, restricted to the inner muscular layer and the subepithelial space. A modified, direct-colouring AChE-method is presented, which uses copper chloride as source of cupric ions, acetylthiocholine chloride as substrate and 2-morpholinoethanesulphonic acid (MES) as buffer. After short incubation (1-2 h) our modified method allows the specific visualization of cholinergic nerves, comparable to the results of ChAT-immunoreactivity; following a long incubation time (24 h), it reliably illustrates the autonomous innervation pattern as completely as immunohistochemical panneuronal markers.

The bovine tubouterine junction: general organization and surface morphology.

The bovine tubouterine junction is composed of three parts (terminal tubal segment, transition region proper, uterine apex) and follows a sigmoidal course displaying a tubal and an uterine curvature. In the terminal tubal segment, 4-8 primary longitudinal folds and a system of lower secondary folds, ridges and chords project into the centrally located lumen. The transition region proper possesses a slit-like lumen because of the existence of a thick mucosal pad containing the first uterine glands. The longitudinal primary folds of the tube broaden, flatten and start to diverge when they reach the transition region proper. The mucosal pad and broadened folds are heavily vascularized. A system of lateral outpocketings with blind ends pointing in an ampullary direction develops between the primary and secondary folds, the ridges and chords of the terminal tubal segment and transition region proper. From the bottom of these outpocketings, short tubulo-alveolar crypts originate. The mucosa of the uterine apex forms low transversal ridges. The musculature of the bovine tubouterine junction is divided into a continuous circular or spiral intermediate layer, flanked by inner and outer longitudinal layers. The outer longitudinal layer is incomplete in the terminal tubal segment but increases in thickness to form a continuous stratum in the uterine apex. An inner longitudinal layer occurs only in the terminal tubal segment where it is best developed in the bases of the primary longitudinal folds. The simple columnar surface epithelium of the tubouterine junction contains ciliated and non-ciliated cells. The former undergo cyclical changes, and increase during estrus and postestrus. During proestrus, groups of non-ciliated cells display bulbous apical protrusions. During proestrus and estrus, circumscribed epithelial lesions expose the underlying basal lamina.

Ultrastructure and innervation of water buffalo (Bubalus bubalis) seminal vesicle.

The lining epithelium of secretory end pieces and central glandular duct in the seminal vesicle of the water buffalo (Bubalus bubalis) consists of columnar principal and small polymorphous basal cells. A system of intercellular and even intracellular canaliculi enlarges the secretory surface. The most prominent organelle of the columnar principal cells is the granular endoplasmic reticulum, forming large aggregates of parallel lamellae. Using antibodies against the neural cell adhesion molecule L1 and the neural marker protein gene product 9.5 (PGP 9.5), the innervation pattern of the seminal vesicle becomes evident. The muscular layer surrounding the propria contains a dense network of unmyelinated fibers. Thicker bundles traverse the muscular layer to reach the propria. Around glandular secretory tubules and below the epithelial lining of the glandular duct a tightly woven subepithelial plexus is observed which sends short penetrating branches into the basal zone of the epithelium. These intraepithelial nerves are devoid of Schwann cells and basal lamina (naked axons) and are situated within the intercellular spaces between principal and basal cells. Acetylcholinesterase histochemistry with short (1-2 h) incubation times, dopamine-beta-hydroxylase immunohistochemistry and ultrastructural study of transmitter-containing vesicles was performed. The results suggest that muscular contraction in the seminal vesicle is predominantly under the influence of the sympathetic nervous system, whereas secretory epithelial function is regulated by both sympathetic and parasympathetic fibers.

The microangiographic pattern of the rotator cuff of the dog.

The spontaneous degeneration of the human rotator cuff seems to have mechanical and nutritive causes. Until now it was not known whether other species without an acromion had a vascularization of the rotator tendons which is similar to that of man. We therefore investigated the rotator cuff of dogs microangiographically. The results show a characteristic vascular pattern in each of the rotator tendons which is similar to that of man. Most important is the fact that the supraspinatus tendon shows an area of hypovascularity close to its insertion. Thus, the dog may be used for experimental purposes relating to the rotator cuff.