Paracrine Factors in Adult Rat Testis Gonadotrophin Control of Opioids and LHRH Like Peptide

A paracrine regulation involves agents which are produced by one cell type and act on an other one within an organ. In rodent testis, local control mechanisms modulate the actions of the gonadotrophins according to local requirements. Two groups of peptides-opioids and testicular LHRH are defined as paracrine factors and in vivo they are both modified by HCG. In vitro, after HCG exposure, we first localized an opioid like material in Sertoli cells cytoplasma by immunohistochemistry. This material is detected in freeze dried homologous culture media using a dot immunobinding technique. With a longer HCG exposure, an LHRH like material is then visualized in the basal compartment of the Sertoli cells and it is detected in freeze dried homologous culture media by the same technical procedure than for opioid material. By adding synthetic enkephalins to culture medium, we obtain the same results as with the endogenous opioid material, excreted after HCG addition. If naloxone a potent opiate antagonist, is added to the culture medium previously to HCG or enkephalins, the Sertoli cells cytoplasma are no more immunoreactives with the anti-enkephalin serum and no LHRH material is neither visualized by immunohistochemical technique neither detected in culture media. We conclude that testicular opioids, synthetized by the Leydig cells and which have specific Sertoli cells receptors are one Leydig-Sertoli paracrine communication factor. One way of response to their receptor fixation is the synthesis and excretion by Sertoli cells of testicular LHRH. This one is known to act on Leydig cells via specific receptors and it is one Sertoli-Leydig cells paracrine communication factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen receptors in fracture healing.

Estrogen has profound effects on the regulation of bone metabolism, but its role in fracture healing is unknown. Several recent reports have documented the presence of estrogen receptors in vitro and in diseased tissue. The authors investigated estrogen receptors in nonneoplastic skeletal tissues by immunohistochemical and radioligand binding techniques. Using a fibular osteotomy model of fracture healing in New Zealand rabbits, radioligand binding detected specific, high affinity, saturable estradiol binding sites at low levels throughout fracture healing, with a trend towards a bimodal distribution. Peaks occurred three and 16 days after osteotomy. No estrogen receptor was found by either method in later fracture callus, growth plate, or periosteum. These findings suggest a possible role for estrogen in the early inductive phase and later phase of endochondral ossification in fracture healing.     

Specific mechanism of the knee joint preventing tumor invasion

Tumor extension from the anterior aspect of the distal femur into the knee joint was examined histologically and with MRI in 10 patients with primary malignancy of bone. Histologically, 1 tumor had invaded the joint with destruction of the transitional area between the articular cartilage and bone. In another case, the transitional area had been destroyed by mechanical traction of the periosteum by the tumor without invasion. The fatty connective tissue and superficial synovial layer were detached from the femoral cortex and elevated upwards by tumor growth without destruction in 7 tumors but were unchanged in 3 tumors. Tl-weighted MRI clearly revealed high signal intensity of the connective tissue and synovium in the 7 tumors with elevation. The average elevation of this detachment of the fatty connective tissue and synovium from the femoral cortex calculated from MR images was 12 mm. Destruction of the transitional area between the articular cartilage and bone was detected in 2 tumors on Tl-weighted MR images. Our results indicate that fatty connective tissue and the superficial synovial layer are easily detached and elevated by tumors. This mechanism probably prevents tumor extension into the joint space.     

Intraarticular and periarticular opioid binding in inflamed tissue in experimental canine arthritis.

Small-dose (1 mg) intraarticular morphine has been used successfully in many studies to provide long-lasting analgesia after arthroscopic knee surgery. We used radioligand binding to determine whether these effects could be mediated by opioid binding sites in the joint, particularly after the induction of inflammation. Inflammation was induced by the injection of oleyl alcohol (20 microL) in sterile peanut oil (0.25 mL) into the left radiocarpal joint of 27 dogs, and the dogs were euthanized at 12 h. The articular and periarticular tissues from both treated and control joints were collected, and membranes were prepared for equilibrium binding assays. The density of specific opioid binding was markedly enhanced (P < 0.05) in homogenates prepared from the treated relative to those from the control joint. The binding affinities (KD values) for morphine and naloxone (mean +/- SEM) were approximately one one-hundredth (79 +/- 17 nM and 124 +/- 5.5 nM, respectively) that of the corresponding published affinities in brain tissue. However, the binding site densities were approximately one hundred times larger (Bmax = 1032 +/- 265 and 543 +/- 51 fmol/mg of protein) than the respective published values in brain tissue. These findings imply that the opioid binding sites, found in the inflamed articular and periarticular tissues in this study, are similar to those of putative mu 3-opioid binding sites that appear to be present on cultured thymocytes and in the airways of rats and humans. Implications: The high density of opioid binding sites found in inflamed canine joint tissue supports the clinical use of intraarticular opioids in the treatment of postoperative and chronic inflammatory joint pain.     

Purification and quantification of opioid peptides in bone and joint tissues--a methodological study in the rat.

The occurrence of methionine-enkephalin-Arg(6)-Phe(7) (MEAP) and dynorphin B (DYNB) representing two main precursors of opioids was analyzed in specimens from rat cortical bone, periosteum, bone marrow and joint tissue by radioimmunoassay (RIA). MEAP and DYNB were extracted in a solution of 4% EDTA in 2 M acetic acid previously proven suitable for extraction of sensory and autonomic neuropeptides in bone and joints. In crude extracts of cortical bone, the immunoreactive (ir) levels of both opioids were under the detection limit of RIA. As for DYNB this also applied to crude extracts of joints and periosteum. Therefore, two purification methods were tested and compared, i.e. reverse phase C 18 and ion exchange chromatography. RIA of the elution fraction disclosed a significant difference between the two methods in terms of recovery, i.e. <5% and 50%, respectively. Thus, purification by ion exchange chromatography prior to RIA appeared to be the most suitable by providing measurable levels of both MEAP and DYNB in all tissues analyzed (highest in bone marrow, lowest in cortical bone). The described method offers a means of quantifying opioid peptides in bone and joints, which may be utilized in the analysis of regulatory mechanisms of nociception, growth and immune responses in different conditions.     

体外培养成骨细胞的研究进展

随着体外细胞培养技术的发展,人们已经从许多动物的颅骨、骨髓基质、骨膜及骨外组织中成功培养出了具有典型成骨细胞特性的细胞,研究表明培养出的成骨细胞具有良好的生物学特性,在不同环境下可以形成骨组织,联合支架的应用,构建组织工程骨,将其植入体内修复骨缺损.现就成骨细胞的来源、分化调控因子、复合移植及中医药方面的研究进展作一综述.     

Role of mesenchymal stem cells in bone regeneration and fracture repair: a review

Mesenchymal stem cells (MSCs) are non-haematopoietic stromal stem cells that have many sources, such as bone marrow, periosteum, vessel walls, adipose, muscle, tendon, peripheral circulation, umbilical cord blood, skin and dental tissues. They are capable of self-replication and of differentiating into, and contributing to the regeneration of, mesenchymal tissues, such as bone, cartilage, ligament, tendon, muscle and adipose tissue. The homing of MSCs may play an important role in the repair of bone fractures. As a composite material, the formation and growth of bone tissue is a complex process, including molecular, cell and biochemical metabolic changes. The recruitment of factors with an adequate number of MSCs and the micro-environment around the fracture are effective for fracture repair. Several studies have investigated the functional expression of various chemokine receptors, trophic factors and adhesion molecules in human MSCs. Many external factors affect MSC homing. MSCs have been used as seed cells in building tissue-engineered bone grafts. Scaffolds seeded with MSCs are most often used in tissue engineering and include biotic and abiotic materials. This knowledge provides a platform for the development of novel therapies for bone regeneration with endogenous MSCs.     

阿片类药物外周镇痛的研究进展

当前治疗各种急、慢性疼痛的所用药物中,阿片类制剂仍然扮演了极为重要的角色,随之而来的是全身用药产生的副作用,如呕吐、呼吸抑制、成瘾等,限制了阿片类药物的应用,外周局部小剂量的应用阿片类药物可产生有效的外周镇痛,从而为临床上控制疼痛提供了一条新的途径.     

Loss of sympathetic nerve fibers in vital intertrochanteric bone cylinders lateral to osteonecrosis of the femoral head

ObjectivesAlthough etiology in osteonecrosis of the femoral head mainly depends on alterations of bone blood flow, vasoregulatory nerve fibers of the sympathetic and sensory nervous system have never been investigated in bone of osteonecrosis patients. This study aimed to demonstrate density of sympathetic and sensory nerve fibers in femoral head and, for comparison, adjacent periosteum, and synovium of the hip joint in patients with osteonecrosis.MethodsImmunofluorescence staining techniques were applied using specific nerve fiber markers. A total of 10 patients with early femoral head osteonecrosis (ARCO I-II), 10 with late femoral head osteonecrosis (ARCO III-IV), and 10 patients with osteoarthritis of the hip were investigated.ResultsIn the bone of the femoral head, density of sympathetic nerve fibers was lower in early and late osteonecrosis compared to osteoarthritis. There was a marked preponderance of sympathetic over sensory nerve fibers in bone of osteoarthritis patients, which was opposite in early and late femoral head osteonecrosis. In periosteum, density of sympathetic nerve fibers was similar in all three groups but density of sensory nerve fibers and cellularity were higher in early osteonecrosis compared to the other two groups.Discussion/ConclusionsDue to the different affinity of norepinephrine for α-adrenoceptors (high affinity) and β-adrenoceptors (low affinity), the loss of sympathetic nerve fibers relative to sensory nerve fibers in femoral head osteonecrosis might change the femoral head blood flow (towards α-adrenergic vasoconstriction). Higher density of sensory nerve fibers and cellularity in periosteum might indicate an inflammatory response in early osteonecrosis.     

髋关节骨性关节炎组织学计量与炎性因子表达

目的 探讨骨性关节炎各组织成分中细胞冈子、金属基质蛋白酶等炎性物质表达水平与疾病的关系.方法 获取行OA组关节置换及创伤性股骨颈骨折组患者手术时的软骨、滑膜组织和软骨下骨,苏木素-伊红(HE)染色行软骨下骨组织形态计量分析,应用放射免疫测定肿瘤坏死因子(TNF)-α与门细胞介素(IL)-1β,以酶联免疫吸附试验(ELISA)测金属基质蛋白酶(MMP)-9蛋白表达水平,并将两组结果进行t检验,软骨下骨组织计量值与细胞因子水平进行相关分析.结果 OA组较非OA对照组软骨下骨骨形成增加,骨质硬化骨小梁数目增加并错乱;滑膜组织中MMP-9表达是调;软骨组织中MMP-9及IL-1 β较对照组提高;而在软骨下骨,MMP-9、TNF-α及IL-β均有增高,并且与软骨下骨组织学改变相关联.结论 证实了OA促炎症条件的病理学基础,并且炎性改变与软骨硬化及关节软骨退变相关.     

Pain and the immune system: friend or foe?

When tissue is destroyed, pain arises. Tissue destruction as well as wound healing are associated with an inflammatory reaction. This leads to activation of nociceptors ("pain receptors") which can cross-communicate with the inflammatory infiltrate. The following review will concentrate on pain-exaggerating (hyperalgesic) and pain-ameliorating (analgesic) mediators which arise from immune cells or the circulation during the inflammation. In the early stages of inflammation endogenous hyperalgesic mediators are produced, including the proinflammatory cytokines IL-1, IL-6 and TNF-alpha, nerve growth factor as well as bradykinin and prostaglandins. Simultaneously, analgesic mechanisms are activated. Opioid peptides such as endorphins, enkephalins and dynorphins are produced by immune cells and can be released locally in the inflamed tissue on stimulation with IL-1 or corticotropin releasing factor. Analgesia is elicited by binding of the opioid peptides to receptors on peripheral sensory neurons. During the course of an inflammatory process, peripheral opioid-mediated analgesia increases. In parallel, antiinflammatory cytokines such as IL-4, IL-10, IL-13 and IL-1ra are produced and reduce hyperalgesic effects of the proinflammatory cytokines initially produced. Inflammatory pain, therefore, is the result of an interplay between hyperalgesic and analgesic mediators. Drugs such as immunosuppressants influencing this interplay may also impair endogenous hyperalgesic and analgesic mechanisms.     

Detection of relaxin receptor in the dorsoradial ligament,synovium, and articular cartilage of the trapeziometacarpal joint

Basilar thumb osteoarthritis (OA) is postulated to occur due to ligament attenuation of the trapeziometacarpal (TM) joint. Relaxin is a peptide hormone, which loosens ligaments before childbirth, through remodeling of the extracellular matrix via upregulation of matrix metalloproteases (MMPs). We postulated that relaxin family peptide receptor 1 (RXFP‐1), the receptor for circulating relaxin, was present in tissues of the TM joint. Ligaments and synovium were sampled from 15 patients during surgery for TM arthritis. We obtained trapezial cartilage from two autopsy donors and four patients. Tissues were fixed, paraffin embedded, and sectioned at 5 µm, then were immunostained for RXFP‐1, as well as MMP‐1, and MMP‐13, using rabbit anti‐human polyclonal antibodies. Eight DRL samples showed positive immunostaining for relaxin receptor, with 14/15 positively stained in synovium. Greater staining was seen in specimens obtained from women with more severe TM arthritis. Trapezial cartilage demonstrated receptor staining within chondrocytes in the middle and deep zones. Immunostaining for MMPs co‐localized with relaxin receptor staining. Relaxin receptors are present at the ligament, cartilage, and synovium of the TM joint, indicating that it is a potential target for relaxin. This suggests that circulating relaxin may impact joint stability. The role of relaxin in cartilage and synovium may be related to its role in collagen regulation as a possible tissue response to OA. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1061–1067, 2014.     

Human renal cell cancer proliferation in tissue culture is tonically inhibited by opioid growth factor

PURPOSE: Peptide growth factors alter cellular events by binding to specific receptors. One group of peptides, the endogenous opioids, is important in the growth of normal and neoplastic tissue. [Met5]enkephalin, also termed opioid growth factor (OGF), is a tonically active inhibitory factor that interacts with the OGF receptor, OGFr, formerly identified as Greek zeta (zeta) and appears to be autocrine produced by human cancer cells. This study examined the hypothesis that OGF directly inhibits proliferation of renal cell carcinoma in tissue culture. MATERIALS AND METHODS: Human renal cancer cells (Caki-2) were grown using routine tissue culture techniques. A variety of natural and synthetic opioids including OGF, opioid antagonists, and opioid antibodies were added to renal cancer cell cultures to determine role of these peptides in renal cell carcinoma. The experiments were repeated in serum-free media, and with 4 other human renal cancer cell lines: Caki-2, A498, SN12C, and ACHN. Immunocytochemistry was performed to examine the presence of OGF and its receptor. RESULTS: OGF was the most potent opioid peptide to influence human renal cell carcinoma. OGF depressed growth within 12 hours of treatment, with cell numbers subnormal by up to 48% of control levels. OGF action was receptor mediated, reversible, not cytotoxic, neutralized by antibodies to the peptide, and detected in the human renal cell carcinoma lines examined. OGF appeared to be autocrine produced and secreted, and was constitutively expressed. Both OGF and its receptor were detected in these cells. CONCLUSION: OGF tonically inhibits renal cancer cell proliferation in tissue culture, and may play a role in the pathogenesis and management of human renal cell cancer.     

Skeletal Cell Fate Decisions Within Periosteum and Bone Marrow During Bone Regeneration

Bone repair requires the mobilization of adult skeletal stem cells/progenitors to allow deposition of cartilage and bone at the injury site. These stem cells/progenitors are believed to come from multiple sources including the bone marrow and the periosteum. The goal of this study was to establish the cellular contributions of bone marrow and periosteum to bone healing in vivo and to assess the effect of the tissue environment on cell differentiation within bone marrow and periosteum. Results show that periosteal injuries heal by endochondral ossification, whereas bone marrow injuries heal by intramembranous ossification, indicating that distinct cellular responses occur within these tissues during repair. Next, lineage analyses were used to track the fate of cells derived from periosteum, bone marrow, and endosteum, a subcompartment of the bone marrow. Skeletal progenitor cells were found to be recruited locally and concurrently from periosteum and/or bone marrow/endosteum during bone repair. Periosteum and bone marrow/endosteum both gave rise to osteoblasts, whereas the periosteum was the major source of chondrocytes. Finally, results show that intrinsic and environmental signals modulate cell fate decisions within these tissues. In conclusion, this study sheds light into the origins of skeletal stem cells/progenitors during bone regeneration and indicates that periosteum, endosteum, and bone marrow contain pools of stem cells/progenitors with distinct osteogenic and chondrogenic potentials that vary with the tissue environment.     

Free periosteal autografting in repair of articular cartilage: an experimental study and clinical application

38 white rabbits in 4 groups were used in this experiment to investigated the metaplastic process and the outcome of the autografted periosteum on cartilage defect created artificially on acetabulum and femoral condyle. The specimens were scrupulously surveyed with conventional methods and analysis of their trace elements and amino acids, which was the means first used in evaluating the nature of metaplastic tissue. Conclusions drown from this experiment are as follows: (1) The autografted periosteum processes a metaplastic potential of forming articular cartilage under certain conditions. (2) Continuous passive movement of the grafted joint accelerate the metaplastic process, forming cartilage. (3) Synovial fluid plays certain role in survival of the transplanted periosteum and its metaplasia. However, excision of the synovium did not affect the result much, probably due to its rapid and powerful regeneration.     

Synovial membrane receptors as therapeutic targets: A review of receptor localization,structure, and function

Joint pathology and degeneration is a significant cause of pain. The synovial membrane plays an important role in maintenance of the joint, contributes to the pathology of many arthropathies and may be adversely affected in joint disease. Improving knowledge of the receptors present within the synovium will aid in a better understanding of joint pathology and the development of new treatments for diseases such as osteoarthritis and rheumatoid arthritis. Knowledge of the location and function of synovial membrane receptors (both in healthy and diseased synovium) may provide important targets in the treatment of various arthropathies. Classic pain receptors such as opioid receptors in the synovium are a mainstay in local and systemic management of chronic pain in many species. In addition to these, many other receptors such as bradykinin, neurokinin, transient receptor potential vanilloid, and inflammatory receptors, such as prostanoid and interleukin receptors have been discovered within the synovial membrane. These receptors are important in pain, inflammation, and in maintenance of normal joint function and may serve as targets for pharmacologic intervention in pathologic states. The goal of this review is to outline synovial membrane receptor localization and local therapeutic modulation of these receptors, in order to stimulate further research into pharmacological management of arthropathies at the local level. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1589–1605, 2017.

     

Spatial distribution of CD44 and hyaluronan in the proximal tibia of the growing rat

CD44 has been described as a cell surface hyaluronan receptor present on a variety of different cells, and it is generally assumed to be prevalent in most connective tissues that contain hyaluronan. A major aim of this study was to test that presumption by localizing CD44 and hyaluronan within several tissues of the proximal tibia of the growing rat. Comparison of these profiles would reveal whether CD44 and hyaluronan co-localize with high fidelity, as would be expected if CD44 were a major hyaluronan binding protein. Using in situ hybridization and immunohistochemistry, CD44 was identified on osteoclasts, chondroclasts, osteocytes, hematopoietic marrow cells, synovial cells, and connective tissue fibroblasts (ligaments, tendons, and fascia). Although the majority of osteocytes expressed CD44, reduced expression was observed for osteoblasts and osteoprogenitor cells. Additionally, CD44 was not detected on chondrocytes from epiphyseal and metaphyseal growth cartilages or in meniscal fibrocartilage. Using biotinylated G1 domain from aggrecan and link protein, hyaluronan was observed in the maturational and hypertrophic zones of all growth cartilages, the synovium and other fibroblastic connective tissues, regional areas of the periosteum and endosteum (around osteoblasts, osteoprogenitor cells, and osteoclasts), osteocyte lacunae, and surrounding blood vessels. In regions of co-localization for CD44 and hyaluronan, it seems that CD44 is a likely hyaluronan binding protein in several tissues of the proximal tibia. However, it does not appear to be the predominant hyaluronan binding protein in growing cartilages of the weanling rat.     

外周阿片类药镇痛的研究进展

外周感觉神经的阿片受体受炎症的调控。位于炎性组织范围内的免疫细胞表达阿片肽,与感觉神经上的阿片受体结合,通过抑制这些神经的兴奋性和（或）炎症前神经肽的释放而产生镇痛作用。现简要介绍外周阿片类药镇痛,并探讨其与临床药理学的联系。在临床药理学的研究中,我们要充分应用外周阿片类药镇痛,克服它的局限性,以此推动外周阿片类药应用在临床镇痛的发展。     

阿片类药物延迟预处理心肌保护作用的研究进展

与缺血预处理类似,阿片类药物预处理亦可诱发延迟性心肌保护作用,临床应用麻醉性镇痛药如吗啡等可通过激动阿片受体而减轻缺血/再灌注损伤.研究发现,阿片类药物预处理的延迟性心肌保护作用持续时间较长,所以对预防心肌缺血/再灌注损伤更具临床应用价值.此文综述了阿片类药物预处理延迟性心肌保护作用的生理学基础和机制.