Characterisation of cells in regenerating cartilage from autotransplanted perichondrium. immunohistochemical expression of smooth-muscle actin, desmin, vimentin, and Ki-67.

Autotransplanted perichondrium from rib and ear sutured to the knee joints of 26 rabbits has been examined with immunohistochemistry and shows certain structural, functional, and proliferative characteristics of regenerating cartilage. Cryostat sections have been examined for the expression of smooth-muscle actin (SMA), desmin, vimentin, and Ki-67. In this rabbit model of perichondrial grafting SMA staining showed vivid vessel regeneration, particularly in the proliferating stage about two to three weeks after grafting, and no vessels in more mature parts one month or more after transplantation. Desmin staining showed expression and distribution similar to SMA. Vimentin staining shows the cytoskeleton of regenerating cartilaginous tissue and makes cellular borders apparent. Immunohistochemical expression of Ki-67 is constantly negative in perichondrial tissue from rib and ear before transplantation, clearly positive in the proliferative stage, but there is no expression in maturing cartilage. The study also shows that all human antibodies used are applicable in a rabbit model.

Expression of platelet-derived growth factor beta receptor in chondrogenesis of perichondrial transplants.

An experimental study was done in two rabbits to establish whether the early stages of cartilage generation involves stimulation via the platelet-derived growth factor beta receptor (PDGFR-beta). Rib perichondrium was dissected off and transplanted to the knee joint. The perichondrial grafts were removed for microscopic investigation at day 0 and day 6 and kept frozen until analysed by immunohistochemistry and in situ hybridisation for the presence of PDGF beta receptor protein and mRNA, respectively. Samples from day 0 showed sparse cells with a positive immunohistochemical reaction. In samples from day 6 there were signs of perichondrial proliferation and chondroid maturation and a more widespread immunohistochemical staining reaction could be seen mainly on proliferating perichondrial cells, but also on some chondrocytes. In situ hybridisation confirmed the expression of PDGF beta receptor mRNA in the same areas of serial sections. These findings may indicate that PDGF beta receptor activity is part of the early response after autotransplantation of perichondrium.

Adrenergic innervation of the human endolymphatic sac.

Adrenergic innervation of the human endolymphatic sac (ES) has not been verified previously. To investigate this question a sensitive histofluorescence method for visualization of catecholamines and serotonin, using a solution composed of sucrose-potassium phosphate-glyoxylic acid (SPG) in cryostat sections, was employed. Three human ES specimens were obtained during surgery for acoustic neuroma. Distinct fluorescence in the subepithelial tissue, indicating the presence of monoaminergic neurones and their axonal varicosities, was observed. SPG-positive terminal nerve fibres around small ES capillaries and subendothelially were also seen. Like the effects of sympathetic stimulation elsewhere in the human body, the ES might respond to such stimulation with, for example, vasoconstriction and increased transepithelial water transport. Since the ES is thought to be responsible for maintaining inner ear fluid homeostasis, adrenergic influence could be important for it to function properly.

Involvement of connections between the brainstem and the sympathetic ganglia in the pathogenesis of rheumatoid arthritis. An anatomical study in rats.

The central connections between the superior cervical ganglion of the sympathetic trunk and the brainstem were investigated in five rats by trans-synaptic transport of lectin-conjugated horse-radish peroxidase. After injection of this tracer into the superior cervical ganglion, labelled cells were seen in both the ventral and dorsal parts of the medullary reticular formation. The neurons were found mainly on the same side as the injection site. Besides retrograde labelling, there was an antegrade termination within the nucleus of the solitary tract. We hypothesise that a nervous mechanism is involved in the pathogenesis of rheumatoid arthritis, and that a supraspinal input to the sympathetic ganglia might influence this mechanism.

Fibrin sealant in prevention of flexor tendon adhesions: an experimental study in the rabbit.

The effect of fibrin sealant on the development of adhesions in flexor tendon surgery was studied in both immobilized and active weight-bearing paws of rabbits. A partial laceration was produced and was treated with sutures alone or with both sutures and glue. Immobilized tendons treated in these ways showed no difference in adhesion formation, but in mobile tendons there was a difference at 6 weeks in gross appearance, scanning electron micrographic appearance, and strength of adhesions as measured when the distally cut tendon was pulled out of its sheath. These observations indicate that, after repair, a mobile flexor tendon heals with a smooth gliding surface and without formation of restricting adhesions when treated with fibrin glue around the suture site.

T-cell receptor V-gene usage in synovial fluid and synovial tissue from RA patients.

The question of whether there is a preferential use of certain V genes in T cells entering an inflamed joint has hitherto been studied mainly using unfractionated cells from synovial fluid and tissue respectively, and no clear answer to the question has yet been provided. Concomitantly, evidence has been provided that the use of V genes may differ considerably between CD4+ and CD8+ T cells, and consequently that detection of biased V-gene expression within an inflammatory lesion may require separate analysis of the two T-cell subsets. In this paper we have therefore studied T-cell receptor V-gene expression in rheumatoid arthritis by means of double stainings of synovial fluid and blood for available anti-TCR monoclonal antibodies and antibodies to CD4 and CD8, respectively. Double stainings were also performed with anti-TCR antibodies and antibodies to activation markers HLA-DR and IL-2R. A certain bias towards the preferential use of certain V genes was seen particularly in the synovial fluid samples within both the CD4+ and CD8+ T-cell populations, but no uniform pattern was evident among the 35 patients investigated.

Origin of sympathetic and sensory innervation of the endolymphatic sac. A retrograde axonal tracing study in the guinea pig.

Lectin-conjugated horseradish peroxidase (WGA-HRP) was used as a retrograde axonal tracer to determine endolymphatic sac (ES) innervation and the nature of such a nervous supply. WGA-HRP placed into the ES of the guinea pig resulted in labelling of neurons in the ipsilateral sympathetic superior cervical ganglion and the sensory trigeminal ganglion. The number of labelled neurons in the cervical superior ganglion varied according to the survival time with peak labelling occurring after 48 h. Sparse labelling of the sensory trigeminal ganglion was also present after 48 h. The results indicate that sympathetic neurons from the superior cervical ganglion and to a certain extent trigeminal somatosensory neurons innervate the ES or perisaccular tissue. If these findings reflect the existence of a sympathetic functional reflex unit remains to be elucidated.

An anatomical study of the origin of sympathetic and sensory innervation of the elbow and knee joint in the monkey.

The origin of sympathetic and sensory innervation of the elbow and knee joint was studied by the intra-axonal transport method. After injection of WGA-HRP into the elbow and knee joint unilaterally in adult monkeys (macaca fascicularis), labelled cells were observed ipsilaterally in both the sympathetic and dorsal root ganglia. Following injection in the elbow joint the neurons were concentrated in the stellate and third to fourth thoracic (T3-T4) sympathetic ganglia, and in the fourth cervical to second thoracic spinal sensory ganglia. The knee joint injection resulted in labelling of sympathetic and spinal sensory ganglia, preferentially at the lumbo-sacral junction--the third lumbar to third sacral (L3-S3) ganglia. The functional implications are discussed in relation to pain mechanisms of joints and to an earlier hypothesis that the nervous system might be involved in the pathogenesis of chronic joint inflammation.

Sympathetic innervation of normal and rheumatoid synovial tissue.

Fresh synovial tissue was taken from two normal subjects and five patients with rheumatoid arthritis, and treated so that catecholaminergic sympathetic nerve fibres could be seen. A fluorescent network of varicose fibres was visible in normal as well as in rheumatoid synovial tissue. In the rheumatoid inflammatory tissue an increased number of fluorescent sympathetic nerve fibres could be seen in close proximity to blood vessels, even in the smallest synovial plica, indicating that the proliferation of inflammatory vessels is accompanied by simultaneous sympathetic nerve proliferation. A nervous component might thus contribute to the inflammatory process in human rheumatoid arthritis.

Origin of sympathetic and sensory innervation of the temporo-mandibular joint. A retrograde axonal tracing study in the rat.

The cells of origin of sensory and sympathetic innervation of the temporo-mandibular joint were studied by the intraaxonal transport method. Horseradish peroxidase or lectin-conjugated horseradish peroxidase was injected into the temporo-mandibular joint unilaterally in adult rats. Labelled cells were observed ipsilaterally in the superior cervical and stellate sympathetic ganglia, in the sensory trigeminal ganglion and in the second to fifth dorsal root ganglia; none were found contralaterally. The results are discussed in relation to the hypothesis that a nervous mechanism might be involved in the pathogenesis of joint inflammation.

The effect of growth factors and synovial fluid on chondrogenesis in perichondrium.

Reconstruction of cartilage with perichondrium depends on the chondrogenic property of the perichondrial fibrocytes. The present investigation concerns the conditions for the differentiation of fibrocytes into chondrocytes both in vivo and in vitro. For the in vivo studies specimens of rib and auricular perichondrium from adult rabbits were wrapped round silicon rods which were enclosed in dialysis bags. One was placed in the suprapatellar pouch of the knee joint and one was placed intraperitoneally in each rabbit. After two months the bags were extracted, the perichondrium prepared for microscopic examination, and the chondrogenesis evaluated. In vitro the perichondrium was divided into small pieces and incubated with tissue culture medium. The medium was supplemented with fetal calf serum, together with epidermal growth factor, platelet derived growth factor, synovial fluid, or with human serum albumin (control group). After three weeks the explants were prepared for microscopy. Chondrogenesis was judged by the degree of cellular enlargement, capsule formation, deposition of matrix, and activation of the outer fibrocytic layer. In vivo, good cartilage development was found in all specimens placed in the knee joint but, in those placed intraperitoneally, little if any chondrogenesis was seen. In vitro profound differentiation occurred in all cultures supplemented with epidermal growth factor and platelet derived growth factor. An equivalent differentiation was found in perichondrium that had been incubated with synovial fluid. We conclude that the differentiation of perichondrial fibrocytes is initiated in vitro by growth factors. In addition, we have shown that synovial fluid contains factors that promote and enhance the development of cartilage from perichondrium.

Perichondrial grafting for total replacement of the patella. Experimental study in rabbits.

In five adult rabbits excision of the patella of one knee joint was carried out. The defects were reconstructed with free grafts taken from rib perichondrium. After survival times of 2.5 and 5 months, neocartilage was seen in all grafts on histological examination. In three of the five, the central part of the reconstructed patella showed bone formation.

Ligament reconstruction using free perichondrial graft. An experimental study. A preliminary report.

In five adult rabbits the medial collateral ligament of one knee joint was replaced by a free perichondrial graft from a rib. When tested after 2 to 5 months the joints were stable in all cases. Histological sections showed a picture of mixed cartilaginous and ligamentous tissue in all specimens. These promising results may open new fields for the use of perichondrium as a ligament substitute for ligament--for example, in the wrist.

A spinal transcommissural connection for symmetrical sympathetic reflex response. Intra-axonal tracing study in the rat.

Wheatgerm agglutinin conjugated horseradish peroxidase was injected into the ganglion cervicale superior of the sympathetic trunk of seven adult rats. Labelled neurons were found in the ipsilateral anterior commissural nucleus of the spinal cord at the C1 level. Transcommissural crossing of labelled fibres and symmetrical labelling of neurons in the contralateral nucleus were also found. Labelled fibres could then be followed contralaterally into the superior cervical ganglion where labelled neurons were also found.

Origin of sympathetic and sensory innervation of the knee joint. A retrograde axonal tracing study in the rat.

The origin of sympathetic and sensory innervation of the knee joint was investigated by the intra-axonal transport method. After injection of horseradish peroxidase (HRP) or horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) into the knee joint unilaterally in adult rats, labelled neurons were found ipsilaterally both in sympathetic and dorsal root ganglia at the lower thoracic and lumbosacral levels. A maximum of 1500 and 1000 peroxidase-positive cells were observed in the sympathetic and dorsal root ganglia, respectively; in the former they were mainly located at the levels of L2-L4, and in the latter mainly at L3-L5. The functional implications of the above observations are discussed in relation to pain mechanisms in joints and to the earlier hypothesis that a nervous component might be involved in the pathogenesis of chronic joint inflammation.

Formation of chondroitin sulphate proteoglycan in cartilage regenerated from free perichondrial graft.

Perichondrium from rabbit auricular or rib cartilage was used as a free autogenous graft and transplanted either to the subcutaneous tissue of the back of the rabbit or to an experimental defect in the femur condyles. Outgrowth of new tissue, morphologically indistinguishable from cartilage, was observed after six weeks. Inorganic 35SO4, administered in vivo, was incorporated into the newly formed tissue. The labelled products were isolated, identified, and compared with those obtained from authentic cartilage of auricular, rib or joint surfaces. The products of newly formed cartilage were similar to those of authentic cartilage. The results support earlier morphological findings, indicating that perichondrium from rib cartilage has a better ability to regenerate than auricular perichondrium. The synovial environment seems to have a positive effect on the generation of cartilage.

Origin of sympathetic and sensory innervation of the elbow joint in the rat: a retrograde axonal tracing study with wheat germ agglutinin conjugated horseradish peroxidase.

After injection of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) into the elbow joint of adult rats, labeled neurons were found in the stellate and the T2-T4 ganglia of the ipsilateral sympathetic trunk, and also in dorsal root ganglia at the C4-T4 levels. Most labeled sympathetic cells, 90% or more, were located in the stellate ganglion. The sensory innervation to the joint originated mainly from the dorsal root ganglia at the levels of C7-T1.

Recurrent herpes simplex virus infections in the adult hand.

Ten adult patients referred to the Department of Dermatology and Venereology at Akademiska Hospital, Uppsala, from different surgical units due to recurrent herpes simplex virus (HSV) infections in the hand, were investigated. At recurrence, virus isolation and typing revealed genital HSV type 2 virus in nine out of ten patients indicating that recurrent HSV infections located in the hand should be looked upon as a part of a venereal disease.

Perichondrial arthroplasty using fibrin glue and early mobilization. An experimental study.

An experimental study was performed in rabbits to find out whether fibrin glue, used to simplify the procedure for graft fixation in perichondrial arthroplasty, would also allow earlier mobilization of the grafted joint, thereby reducing the risk for postoperative stiffness of the joint. The results indicate the possibility of reducing the time used for postoperative fixation from three weeks originally to one week, and still achieve the same results regarding graft healing and cartilage regeneration. Immediate post-operative mobilization caused loosening of the graft and endangered the results.

The early development of articular cartilage after perichondrial grafting.

In an experimental series of 39 rabbits the articular cartilage of the femur condyles was resected and the defect covered by a perichondrial graft from rib cartilage. The graft was fixed to the bony surface by using Tisseal (Immuno AG) and the joint was immobilized for two weeks. Beginning 24 hours postoperatively the neochondrogenesis from the perichondrial graft was analyzed histologically, sacrificing the rabbits with 48 hours interval. The fibrin glue acted as a visible border line between the bony surface and the graft. No vascular proliferations penetrated the fibrin glue, indicating that the perichondrial graft must be nourished by the synovial fluid only. The neochondrogenesis was found to occur in the median and fibrous layer of the graft.