Generation and characterization of monoclonal antibodies to the neural crest.

The generation of monoclonal antibodies (MAbs) specific for quail neural crest may provide valuable tools for studying the differentiation of embryonic precursor cells. Unfortunately, relatively few antibodies are available because of the difficulty in obtaining sufficient cells for in vivo immunization strategies. We have overcome this problem by using intrasplenic immunization with formaldehyde-fixed cells harvested from neural crest cultures. In addition, booster injections of cultured whole-embryo cells were administered intraperitoneally. Following two fusions, a total of 18 hybridomas were generated with antibody reactivity to the cytoplasm of neural crest cells. Furthermore, 32 were reactive against both somite (a noncrest mesodermal control) and crest cultures, whilst 15 were not reactive. Out of those hybridomas reactive with neural crest, six designated 160D, 164D, OE, 12E, 120E and 124E were further characterized. Interestingly MAb supernatants OE, 12E, 120E, and 124E exhibited reactivity against some but not all neural crest cells suggesting that they might recognise subpopulations. Immunoglobulin isotyping of supernatants revealed that 4 (160D, 164D, OE, and 120E) were IgM and 2 (12E and 124E) were IgG(2b). On assessing their reactivity against human tissue sections, all six hybridoma supernatants cross-reacted with neuroendocrine cells within appendix, colon and rectum. These MAbs could provide novel reagents for the understanding of neural crest development.

The hip joint: the fibrillar collagens associated with development and ageing in the rabbit.

The fibrillar collagens associated with the articular cartilages, joint capsule and ligamentum teres of the rabbit hip joint were characterised from the 17 d fetus to the 2-y-old adult by immunohistochemical methods. Initially the putative articular cartilage contains types I, III and V collagens, but when cavitation is complete in the 25 d fetus, type II collagen appears. In the 17 d fetus, the cells of the chondrogenous layers express type I collagen mRNA, but not that of type II collagen. Types III and V collagens are present throughout life, particularly pericellularly. Type I collagen is lost. In all respects, the articular cartilage of the hip joint is similar to that of the knee. The joint capsule contains types I, III and V collagens. In the fetus the ligamentum teres contains types I and V collagens and the cells express type I collagen mRNA; type III collagen is confined mainly to its surface and insertions. After birth, the same distribution remains, but there is more type III collagen in the ligament, proper. The attachment to the cartilage of the head of the femur is marked only by fibres of type I collagen traversing the cartilage; the attachment cannot be distinguished in preparations localising types III and V collagens. The attachment to the bone at the lip of the acetabulum is via fibres of types I and V collagens and little type III is present. The ligament is covered by a sheath of types III and V collagens. Type II collagen was not located in any part of the ligamentum teres. The distribution of collagens in the ligamentum teres is similar to that in the collateral ligaments of the knee. Its insertions are unusual because no fibrocartilage was detected.

The expression of the fibrillar collagen genes during fracture healing: heterogeneity of the matrices and differentiation of the osteoprogenitor cells.

The cells that express the genes for the fibrillar collagens, types I, II, III and V, during callus development in rabbit tibial fractures healing under stable and unstable mechanical conditions were localized. The fibroblast-like cells in the initial fibrous matrix express types I, III and V collagen mRNAs. Osteoblasts, and osteocytes in the newly formed membranous bone under the periosteum, express the mRNAs for types I, III and V collagens, but osteocytes in the mature trabeculae express none of these mRNAs. Cartilage formation starts at 7 days in calluses forming under unstable mechanical conditions. The differentiating chondrocytes express both types I and II collagen mRNAs, but later they cease expression of type I collagen mRNA. Both types I and II collagens were located in the cartilaginous areas. The hypertrophic chondrocytes express neither type I, nor type II, collagen mRNA. Osteocalcin protein was located in the bone and in some cartilaginous regions. At 21 days, irrespective of the mechanical conditions, the callus consists of a layer of bone; only a few osteoblasts lining the cavities now express type I collagen mRNA. We suggest that osteoprogenitor cells in the periosteal tissue can differentiate into either osteoblasts or chondrocytes and that some cells may exhibit an intermediate phenotype between osteoblasts and chondrocytes for a short period. The finding that hypertrophic chondrocytes do not express type I collagen mRNA suggests that they do not transdifferentiate into osteoblasts during endochondral ossification in fracture callus.

Is acid phosphatase activity present in bone matrix at sites of endochondral ossification in rabbit fracture callus?

It has been suggested that acid phosphatase activity is present in newly formed bone matrix at sites of endochondral ossification in rabbit fracture calluses. Because acid phosphatases are usually found intracellularly, it was decided to test this possibility more rigorously. Tissue from 10- and 14-day healing rabbit fractures was subjected to a series of critical tests for acid phosphatases with a pH optimum of 5.0. Fluoride, tartrate and molybdate were used as potential inhibitors of acid phosphatase activity. The effects of several counterstaining protocols were also investigated. A fluoride- and tartrate-resistant acid phosphatase is located in osteoclasts and mononuclear phagocytes. Diffuse staining of the bone matrix is seen, but it is dependent upon the length of incubation in the substrate medium and the distance from the acid phosphatase-reacting cells. It is concluded that the coloration of the bone matrix is probably caused by diffusion of the dye and reaction product and is, therefore, artifactual.

Fetal and postnatal development of the patella, patellar tendon and suprapatella in the rabbit; changes in the distribution of the fibrillar collagens.

The development of the patella, its associated tendons, and suprapatella of the rabbit knee joint is described from the 17 d fetus to the mature adult. The patellar tendon (ligament) with the patella on its posterior surface is seen in the 17 d fetus and is fully developed by 1 postnatal wk. It is composed of bundles of types I and V collagens separated by endotenons of types III and V collagens. Anteriorly there is an epitenon of types III and V collagens while synovium and a fat pad cover its posterior surface. In the 25 d fetus, the patella is cartilaginous and is separated from the femoral condyles. The cartilage contains type II collagen, but types I, III and V collagens are found along the articular surface. Ossification starts 1 postnatal wk and at 6 wk only the articular cartilage remains. In addition to type II, types III and V collagens are located around the chondrocyte lacunae. The long anterior junction between the patella and its tendon is fibrocartilaginous at 1 wk, but as ossification proceeds this is replaced by bone. Types I and V collagens are found in this region. The suprapatella on the posterior surface of the quadriceps tendon is first seen 1 wk postnatally as an area of irregularly organised fibres and chondrocyte-like cells. Types I, II, III and V collagens are present from 3 wk onwards. It is compared with the fibrocartilage of other tendons that are under compression. The arrangement of the collagens in the patellar tendon is discussed in relation to its use as a replacement for injured anterior cruciate ligaments. It is suggested that the structural differences between the patellar tendon and anterior cruciate ligament preclude the translocated tendon acquiring mechanical strength similar to that of a normal cruciate ligament. The designation 'patellar ligament' as opposed to 'patellar tendon' is questioned. It is argued that the term patellar tendon reflects its structure more accurately than patellar ligament.

Development and ageing of the articular cartilage of the rabbit knee joint: distribution of the fibrillar collagens.

The development of the articular cartilage of the rabbit knee joint from the 17-day fetus to the 2-year adult rabbit has been examined. At 17 days, the developing femur and tibia are separated by the interzone. Cavitation occurs around 25 days; the cells of the intermediate layer flatten and move onto those of the chondogenous layers to create the articular surfaces. After birth, growth of the cartilage is mainly the result of matrix production. Ossification of the epiphyses is complete by 6 weeks postpartum. Horizontal zones can be distinguished in the articular cartilage; the superficial cells are aligned parallel to the surface, but in the deep layers the cells are in columns. The tidemark is first seen at 12-14 weeks. The matrix of the interzone in the 17-day fetus contains types I, III and V collagens, but no type II. After cavitation at 25 days, the surface layer of the articular cartilage still contains type I, but no type II collagen. From 6 weeks postnatal onwards, type II collagen is present throughout the cartilage and type I disappears. Type III collagen is initially in the inter-territorial matrix, but later it is mainly pericellular. Type V collagen is pericellular both in the chondrogenous layers and later in the articular cartilage, but is not present in the epiphyseal cartilage below. From 6 weeks onwards, types III and V collagens create a capsule around all the chondrocytes above the tidemark. The relationship of types V and XI collagens is discussed. It is concluded that the articular chondrocytes form a unique subset of cells from the earliest stages of joint formation in the fetal rabbit.

Changes in the distribution of fibrillar collagens in the collateral and cruciate ligaments of the rabbit knee joint during fetal and postnatal development.

The collateral ligaments can be clearly distinguished in the 25-day fetal rabbit knee joint. Type I and V collagens are present in the extracellular matrix between the cells of the lateral and medial collateral ligaments and this distribution persists until the rabbit is skeletally mature. From 8 months onwards type III collagen is also present, particularly around the cells. Type I collagen mRNA is expressed by the cells from the 25-day fetal to 8-month-old adult ligament. The ligament sheath is composed of types III and V collagens. The cruciate ligaments are present between the femur and tibia in the 20-day fetus. The matrix is composed of types I and V collagens from the 25-day fetus until at 12- to 14-weeks postnatal, type III collagen appears in the pericellular regions together with type V. At 8 months and 2 years the amount of type III collagen has increased. All the cells express the mRNA for type I collagen at 12- to 14-weeks, but only isolated cells express this mRNA at 8 months. Thus, both the collateral and cruciate ligaments undergo changes in their complement of collagens during postnatal development and ageing. The implications of these complex interactions of different types of collagen are discussed in relation to healing and the surgical replacement of torn ligaments by tendons.

Changes in the content of the fibrillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing.

The menisci are first seen as triangular aggregations of cells in the 20-day rabbit fetus. At 25-days, a matrix that contains types I, III and V collagens has formed. These collagens are also found in the 1-week neonatal meniscus, but by 3 weeks, type II collagen is present in some regions. By 12 to 14 weeks, typically cartilaginous areas with large cells in lacunae are found and by 2 years, these occupy the central regions of the inner two-thirds of the meniscus. The surface layers of the meniscus contain predominantly type I collagen. From 12 to 14 weeks onwards, there is little overlap between the regions with types I or II collagens, that is, these are discrete regions of type I-containing fibrocartilage and type II-containing cartilage. Types III and V collagens are found throughout the menisci, particularly in the pericellular regions. All the cells in the fetal and early neonatal menisci express the mRNA for type I collagen. At 3 weeks postnatal, cells that express type I collagen mRNA are found throughout the meniscus, but type II collagen mRNA is expressed only in the regions of developing cartilage. At 12- to 14-weeks, only type II collagen mRNA is expressed, except at the periphery next to the ligament where a few cells still express type I collagen mRNA. Rabbit menisci, therefore, undergo profound changes in their content and arrangement of collagens during postnatal development.

Exogenous fibroblast growth factors-1 and -2 do not accelerate fracture healing in the rabbit.

Both fibroblast growth factors-1 (acidic FGF) and -2 (basic FGF) increase the proliferation of osteoblasts and chondrocytes in vitro and FGF-2 stimulates angiogenesis and bone formation in vivo. To test their effects on rabbit tibial fracture-healing under stable and unstable mechanical conditions, 3 micrograms of either FGF-1 or FGF-2 was injected around rabbit tibial fractures on day 4 after fracture. Neither growth factor had a significant effect on either the size of, or the amounts of bone and cartilage in, the 10-day callus irrespective of the mechanical conditions under which the fracture was healing. The 10-day FGF-2-treated calluses were, however, more mature than FGF-1-treated calluses because the cartilage was separated from the periosteum by bone and endochondral ossification had progressed further. In conclusion, the application of FGF-1 or FGF-2 to normally healing fractures of the rabbit tibia does not have a significant effect on the rate of healing.

The expression of collagen mRNAs in normally developing neonatal rabbit long bones and after treatment of neonatal and adult rabbit tibiae with transforming growth factor-beta 2.

Normal transverse growth of long bones is by periosteal appositional bone formation, balanced by endosteal resorption. Changes in the distribution of cells that are expressing collagen mRNAs during growth were determined using digoxigenin-labelled riboprobes. In neonatal rabbit tibiae osteoblasts expressing type I collagen mRNA are found on periosteal, and at early stages on endosteal, bone surfaces and lining peripheral cavities. Occasional osteocytes express type I collagen mRNA very weakly. The pattern is disrupted when transforming growth factor-beta 2 (TGF-beta 2) is injected daily into the periosteum of neonatal animals; there is increased bone, and later cartilage, formation. Three injections of 20 ng TGF-beta 2 onto the tibia of 3-day-old rabbits led to an increase of periosteal osteoblasts that express the mRNA for type I collagen. Some endosteal osteoblasts and osteocytes in newly-formed peripheral woven bone also express the mRNA. After five injections chondrocytes expressing type II collagen mRNA are found around the injection site. Similar injections of TGF-beta 2 in old rabbits induce only fibrous tissue within which some cells express type I collagen mRNA. This precise localization of mRNAs shows that the expression of type I or II collagen mRNA is here restricted to osteoblasts and chondrocytes, respectively.

The effect of exogenous transforming growth factor-beta 2 on healing fractures in the rabbit.

The effects of exogenous TGF-beta 2 on normally healing fractures were investigated to see if healing can be accelerated; TGF-beta s stimulate bone and cartilage formation on calvariae and long bones. TGF-beta 2 (60 or 600 ng) was injected around the developing callus of rabbit tibial fractures healing under stable, or unstable, mechanical conditions 4 days after fracture. The fractures were examined 5, 7, 10, and 14 days after fracture. A large amount of edema developed around the injection sites. The callus of fractures healing under stable mechanical conditions consists almost entirely of bone. The effects of 60-ng injections of TGF-beta 2 are minimal, but 600-ng doses lead to a small increase in the size of the callus. The callus of fractures healing under unstable mechanical conditions has a large area of cartilage over the fracture site with bone on each side. The effects of TGF-beta 2 on unstable fractures are to retard and reduce bone and cartilage formation in the callus. The overall size of the callus is not affected. In conclusion, TGF-beta 2 does not stimulate fracture healing under either stable, or unstable, mechanical conditions during the initial healing phase. It is argued that agents which stimulate bone formation can retard remodeling when treatment is extended into the remodeling phase of healing.

The effects of age on the response of rabbit periosteal osteoprogenitor cells to exogenous transforming growth factor-beta 2.

Additional bone and cartilage are formed if transforming growth factor-beta is injected into the periosteum of calvariae or long bones. To investigate this further, transforming growth factor-beta 2 was injected into the periosteum of the tibia of 3-day-old, 3-month-old and 2-year-old rabbits. In all instances, there was an increase in proliferation of the cells of the cambial layer of the periosteum, that is, the osteoprogenitor cells, and breakdown of the fibrous layer. Oedema was induced in the surrounding connective tissues. Over the experimental period the normal neonatal tibia is undergoing rapid growth; there is periosteal bone formation and endosteal resorption. In the experimental neonatal tibiae, an increase in periosteal bone formation is seen after three injections of 20 ng of transforming growth factor-beta 2, which is accompanied by cartilage after five injections; the amounts of induced bone and cartilage increase with the number of injections. The chondrocytes hypertrophy after 4 days and the cartilage is replaced by bone endochondrally. In contrast, after seven injections of 20 ng transforming growth factor-beta 2, there is only a small amount of new bone on the 3-month-old tibia and none on the 2-year-old tibia. One day after seven injections of 200 ng transforming growth factor-beta 2, there is a small amount of bone formation, while seven days after cartilage is found as small discrete nodules on the 3-month-old tibia, but as small areas within the bone on the 2-year-old tibia. It is concluded that the primary effect of transforming growth factor-beta 2 in this experimental model is to increase the proliferative rate of the osteoprogenitor cells in the periosteum. It is argued that transforming growth factor-beta 2 does not initiate osteoblastic or chondrocytic differentiation of osteoprogenitor cells. It is suggested that their differentiation is controlled by the local environment, in particular, the vascularity and locally circulating growth factors.

The economics of imperialism and health: Malta's experience.

The thesis of this article is that the prevalence of disease and premature death depends more on national, class, and gender relationships than on medical and biological factors. The political and economic realities of life in the British Colony of Malta revealed here clearly determined the severity of both infant mortality rates and the attacks of brucellosis. A brief history sets the background for an in-depth study of the interaction between socioeconomic conditions and disease in the first half of the 20th century. Britain's adherence to imperialist "free" trade policies and refusal to consider Malta's economy beyond its use as a military base had resulted in the "underdevelopment" of Malta's traditional cotton agroindustry and the erosion of household economic stability. Persistently high infant mortality rates and the absence of preventive disease measures were a clear manifestation of continuing exploitative imperialist policies. In this scenario, the devastation of the Second World War became a catalyst for change.

An immunohistochemical study of the collagens of rabbit synovial interstitium.

The interstitial pathway from joint cavity to synovial transport vessels contains a complex extracellular matrix. Rabbit knee synovial intima contains 3 fibrous elements--banded collagen fibrils, microfibrils and broad banded aggregates. The distribution of 4 types of collagen in rabbit knee synovium was investigated immunohistochemically. Types I and III collagens are both present, although the binding of anti-type I antibody was weak. Type V collagen, which forms thin fibrils, or is copolymerized with type I collagen, and type VI collagen, which forms broad banded aggregates and microfibrils, are widely distributed throughout the intimal and subintimal matrices.

Digoxigenin as a probe label for in situ hybridization on skeletal tissues.

Non-specific staining was encountered using digoxigenin-labelled cDNA probes for in situ hybridization on sections of skeletal tissues. This staining was most pronounced in cartilaginous matrices. Experimental procedures indicate that the background staining is caused by antibody-binding to hydrophobic sites in the tissues revealed by proteolytic permeabilization. A protocol for minimizing this background is described.