The in vitro growth of human chondrocytes.

Autologous chondrocyte implantation (ACI) for the treatment of articular cartilage defects has been described by other workers, however, relatively few details of the in vitro growth of the cells have been published. Here we describe the release of cells from adult human articular cartilage and their growth characteristics in vitro.Cultures were successfully established from 29 of 30 biopsies taken from patients aged 20-72 year. No significant relationship was found between donor age and initial cell yield following cartilage digest, however, the time to primary confluence increased in direct proportion to age. Thereafter the kinetics of cell proliferation was independent of donor age.The proportion of apoptotic or necrotic cells in the cartilage digest was low and increased with time in culture only in those cells which remained non-adherent. Conversely, entry into cell cycle was restricted to those cells which had become adherent.These results illustrate that previously reported techniques for isolating and culturing chondrocytes are reproducible, that adherent chondrocytes have considerable proliferative potential, and that concern about cell growth and viability need not, in itself, limit the clinical application of ACI to younger patients.

The effect of substance P on proliferation and proteoglycan deposition of cells derived from rabbit intervertebral disc.

STUDY DESIGN: This study is an in vitro investigation of the effects of substance P on intervertebral disc cell metabolism. OBJECTIVES: To determine whether the neuropeptide, substance P, affects cells isolated from the intervertebral disc. SUMMARY OF THE BACKGROUND DATA: Nerve fibers containing substance P are present in the anulus fibrosus and may be released from the nerve terminals as in other tissues. Substance P is mitogenic for a variety of immune and connective tissue cells, and a fragment of the peptide affects the metabolism of articular chondrocytes. METHODS: Cells were isolated enzymically from the anulus fibrosus of intervertebral disc of 8-week-old rabbits. The effects of substance P and the C-terminal pentapeptide fragment SP7-11 on cell proliferation and proteoglycan deposition were determine by crystal violet and Alcian blue staining, respectively. RESULTS: Substance P ((10)-11-(10)-7 mol/l) had a small stimulatory effect on disc cell proliferation. Proteoglycan deposition in the cell layer increased concomitantly. A greater proliferative effect was observed with substance P fragment 7-11 or with the addition of the neutral endopeptidase inhibitor, phosphoramidon. CONCLUSIONS: Substance P has small mitogenic effects on rabbit intervertebral disc cells in vitro. Further investigation is required to establish whether this might have biologic relevance in relation to the maintenance or repair of the intervertebral disc.

Mechanoreceptors in intervertebral discs. Morphology, distribution, and neuropeptides.

STUDY DESIGN: The present study investigated the occurrence and morphology of mechanoreceptors in human and bovine intervertebral discs and longitudinal ligaments. OBJECTIVE: To determine the type and frequency of mechanoreceptors present in intervertebral discs and anterior longitudinal ligaments in two patient groups, those with low back pain and those with scoliosis. Bovine coccygeal discs were examined. SUMMARY OF BACKGROUND DATA: Nerves have been described in intervertebral tissues, but there is little information on the endings of these nerves and their receptors, stimulation of which can cause a nerve impulse. METHODS: The presence of mechanoreceptors were investigated by immunolocalization of nerves and neuropeptides. By examining sequential sections, the frequency of receptors was assessed. RESULTS: Immunoreactivity to neural antigens showed mechanoreceptors in the anulus fibrosus and longitudinal ligaments of bovine and human specimens. Their morphology resembled Pacinian corpuscles, Ruffini endings, and, most frequently, Golgi tendon organs. They were found in 50% of discs investigated from patients with low back pain and in 15% of those with scoliosis. CONCLUSIONS: Mechanoreceptors were found in the outer 2-3 lamellae of the human intervertebral disc and anterior longitudinal ligament. Physiologic studies in other tissues indicate that these provide the individual with sensation of posture and movement, and in the case of Golgi tendon organs, of nociception. In addition to providing proprioception, mechanoreceptors are thought to have roles in maintaining muscle tone and reflexes. Their presence in the intervertebral disc and longitudinal ligament can have physiologic and clinical implications.

Substance P in intervertebral discs. Binding sites on vascular endothelium of the human annulus fibrosus.

The annulus fibrosus of the human intervertebral disc is sparsely innervated, some of the fibers containing substance P. We could demonstrate, by autoradiography, binding sites for substance P localized on the endothelium of small blood vessels in the annulus fibrosus of human intervertebral discs removed during anterior fusion for back pain. In binding inhibition studies, binding of 125I-Bolton Hunter-substance P was inhibited by unlabeled substance P and the related tachykinins neurokinin A and neurokinin B with a rank order of potency substance P > NKA > NKB. Specific binding was reduced > 75 percent by 5'-guanylylimidodiphosphate, indicating G-protein coupling. These features are characteristic of an NK1 receptor through which vascular effects, i.e., vasodilation, plasma extravasation and angiogenesis of substance P, are mediated. The presence of NK1 receptors on blood vessels in the annulus fibrosus may indicate a role for substance P in tissue repair although acute proinflammatory effects may contribute to discogenic pain.

Innervation of the spondylolysis "ligament".

SUMMARY OF BACKGROUND DATA: Spondylolysis of the lower lumbar vertebrae is a non-united childhood fracture of the arch of the vertebra, persisting into adult life. Symptoms of disabling low back pain appear in a minority of patients, usually for the first time in adulthood. This pain is considered to arise from several separate sources, one of which may be the spondylolysis ligament. STUDY DESIGN: The innervation of the ligament has been investigated immunohistochemically. METHODS: Specimens from eight patients were divided longitudinally for histology including hematoxylin and eosin, toluidine blue, and elastic van Gieson. Histochemistry involved immunostaining for the neuropeptides: protein gene product, calcitonin gene related peptide, substance P, vasoactive intestinal peptide, and the c-flanking peptide of neuropeptide Y. RESULTS: Immunoreactivity to calcitonin gene-related peptide, the c-peptide of neuropeptide Y, and vasoactive intestinal peptide was identified in the ligament or in the adjacent adipose tissue. CONCLUSION: The movement that the ligament allows at the fracture site may result in stimulation of the nerve endings both in the ligament and in the surrounding soft tissue.

Neuropeptides in the human intervertebral disc.

The innervation of the human intervertebral disc was investigated by immunochemical methods. Immunoreactivity to the general nerve marker protein gene product (PGP 9.5) was found in the outer annulus fibrosus of 11 of 12 discs removed during anterior arthrodesis for back pain. PGP 9.5-immunoreactive fibres ran between and across the collagenous lamellae, both in association with blood vessels and distant from them, and extended at least 3 mm into the disc. No innervation was observed in the nucleus pulposus. Fine fibres (< 1 micron in diameter) immunoreactive to calcitonin gene-related peptide and substance P (neuropeptides located in sensory and possibly nociceptive nerves) were identified in eight and four of the annuli fibrosi, respectively. Nerve fibres immunoreactive to vasoactive intestinal peptide and to the c-flanking peptide of neuropeptide Y were found in the majority of specimens of annulus fibrosus that were examined.

Localisation of vitronectin receptor immunoreactivity and tartrate resistant acid phosphatase activity in synovium from patients with inflammatory or degenerative arthritis.

The influx of cells into the synovial intima in rheumatoid joints may include osteoclasts and their precursors. The distribution of osteoclast markers--namely, tartrate resistant acid phosphatase activity and the expression of vitronectin receptor (shown with monoclonal antibodies 13C2 and 23C6)--was therefore examined in synovium obtained from patients with rheumatoid (RA) or degenerative (OA) arthritis. Tartrate resistant acid phosphatase positive cells were found in frozen sections of 60% (n = 30) of RA and 69% (n = 29) of OA synovial membranes. Whereas all synovia tested (four RA, four OA) showed diffuse staining of the lining cells with 13C2, 55% (n = 11) of RA and 57% (n = 14) of OA synovial membranes contained isolated cells stained with 23C6 scattered throughout the tissue. In cultures of synovial cells, tartrate resistant acid phosphatase positive, multinuclear, and 23C6 positive cells were found; these cells did not, however, form resorption pits on bone slices. The results show that fully differentiated osteoclasts are uncommon in synovium from patients with either degenerative or inflammatory arthropathies.

Morphological basis for back pain: the demonstration of nerve fibers and neuropeptides in the lumbar facet joint capsule but not in ligamentum flavum.

The innervation of lumbar facet capsule and ligamentum flavum was investigated using antisera to a general neuronal marker protein gene product (PGP) 9.5 and to peptide markers of sensory nerves (calcitonin gene-related peptide [CGRP] and substance P) and autonomic nerves (vasoactive intestinal polypeptide [VIP] and C-flanking peptide of neuropeptide Y [CPON]). In the facet capsule (n = 14), PGP 9.5 and CGRP-immunoreactive nerves occurred in 12 and five specimens, respectively, both around blood vessels and as free fibers in the stroma. Free fibers immunoreactive for substance P or VIP were noted in three and five specimens, whereas in nine specimens there were CPON-immunoreactive nerves located perivascularly. There was no immunoreactivity in the ligamentum flavum. This study provides further evidence that the facet capsule but not the ligamentum flavum has substantial innervation by sensory and autonomic nerve fibers and has a structural basis for pain perception.

A recombinant IGF-1 analogue stimulates [3H]thymidine incorporation in human epiphyseal growth cartilage in vitro.

The effect of a recombinant analogue of IGF-1 on human cartilage of different ages has been investigated in vitro. Addition of 100 ng/ml IGF-1 to articular and epiphyseal growth plate cartilage (9 months to 16 years) resulted in an increase in [3H]thymidine incorporation to a mean of 130 +/- 4% (SE) and 378 +/- 53% of controls, respectively. In costal and growth cartilage from normal fetuses of 15-18 weeks gestation, [3H]thymidine incorporation was increased to 119 +/- 3 and 133 +/- 11% of controls, respectively, when 100 ng/ml IGF-1 was added. The mitogenic effect of recombinant IGF-1 in vitro extends to human cartilage and is most notable in postnatal epiphyseal growth plate.

In vitro effect of multiplication stimulating activity (MSA) on human fetal and postnatal cartilage.

Although insulin-like growth factors may have a physiological role in fetal growth, little is known of their biological action on human fetal tissues. In the present study, the action of multiplication stimulating activity (MSA) on human fetal cartilage in vitro, has been examined and compared with its effect on postnatal cartilage. Addition of MSA (10-100 ng/ml) resulted in a dose dependent increase in [3H]thymidine incorporation into fetal cartilage aged between 15 and 18 weeks of gestation. The mean response with 100 ng/ml was 143 +/- 18% (n = 10) of basal levels. The increase in [35S]sulphate incorporation was variable, the mean (131 +/- 36%, n = 5) being not significantly greater than in controls. The increase in [3H]thymidine incorporation on addition of MSA was not seen in fetal cartilage of earlier (13/14 wk) or later (19 wk) gestational age. MSA-III (a highly purified component of MSA) at 100 ng/ml increased [3H]thymidine and [35S]sulphate incorporation into cartilage from a fetus of 17 weeks to 165% and 150%, respectively, but had no effect on the incorporation of either isotope into cartilage from a fetus of 19 weeks gestation. In contrast to the mitogenic effects of MSA on fetal cartilage, the same preparation had no effect on either [3H]thymidine or [35S]sulphate incorporation into postnatal cartilage. These results may reflect developmental changes in cartilage response to insulin-like growth factors similar to those reported in human brain.

Insulin-like growth factors (IGF) 1 and 2 in human foetal plasma and relationship to gestational age and foetal size during midpregnancy.

IGF-1 and IGF-2 were measured by specific radioimmunoassay after acid-ethanol extraction of plasma obtained by foetoscopy from 20 normal foetuses aged 15-23 weeks. IGF-1 and IGF-2 levels were 36 +/- 11 and 162 +/- 55 ng/ml, respectively. In comparison, levels in cord blood were 84 +/- 58 and 264 +/- 176 ng/ml, respectively, and in adult plasma were 410 +/- 106 and 818 +/- 272 ng/ml. Both IGF-1 and IGF-2 were in the normal foetal range in a further three foetuses with anencephaly and two foetuses with spina bifida. No sex difference was observed. IGF-1 was positively correlated with foetal body weight (P less than 0.001), placenta weight (P less than 0.02) and with body length measured crown-rump (P less than 0.01) or crown-heel (P less than 0.02). No correlation between IGF-2 and body weight, length, placenta weight or gestational age was found. Both IGF-1 and IGF-2 are present in the human foetal circulation earlier in gestation than has previously been demonstrated, the levels being low throughout this period of gestation in comparison with adult plasma.

Receptors for multiplication-stimulating activity (MSA) on rabbit chondrocytes.

Multiplication-stimulating activity (125I-MSA) has been shown to bind to isolated rabbit chondrocytes, the binding being dependent on time, temperature, and cell density. Nonspecific binding was approximately 15%. Unlabelled MSA at 100 ng/ml inhibited 125I-MSA binding by 50%. Porcine insulin (0.5-10 micrograms/ml) did not compete with MSA but resulted in a 10-15% increase in 125I-MSA binding. The data suggest that normal chondrocytes carry IGF2-type receptors as well as the IGF1 type previously described.

Action of multiplication-stimulating activity on [3H]thymidine incorporation in rabbit and human fetal chondrocytes in vitro.

The mitogenic action of multiplication-stimulating activity (MSA) on normal mammalian chondrocytes has been examined. Addition of MSA (NIH, PkII-MSA, 2.5-500 ng/ml or Collaborative Research, CR-MSA, 50-250 ng/ml) to primary suspensions of chondrocytes prepared by enzymic digestion of costal and articular cartilage of rabbits (356-481 g body wt) resulted in a dose-dependent increase in [3H]thymidine incorporation into the trichloroacetic acid-precipitated cell contents. CR-MSA (50-250 ng/ml) also had a significant stimulatory effect on [3H]thymidine incorporation into human fetal chondrocytes (22 weeks of gestation) prepared by enzymic digestion. When PkII-MSA was added in the presence of 1.25% of a standard adult or cord plasma to either rabbit or human fetal (18 weeks) chondrocytes, the increase in [3H]thymidine incorporation appeared to be synergistic. The mitogenic action of MSA can thus be demonstrated on primary suspensions of mammalian chondrocytes. The action of MSA on human chondrocytes has not previously been reported.

Fracture repair in the Snell dwarf mouse.

Snell dwarf mice are deficient in the somatomedin peptides which are mediators of growth hormone action on the skeleton. Tibial fractures in dwarf mice united within 6 weeks, however chondroid differentiation and osteogenesis at the fracture site were retarded in the first few weeks after fracture compared with normal mice. Administration of bovine growth hormone (5 micrograms daily) accelerated the repair process and 2 micrograms thyroxine daily resulted in rapid callus formation and ossification indistinguishable from normal controls. Normal somatomedin levels are not therefore essential for adequate fracture healing in Snell dwarf mice. The acceleration resulting from growth hormone and thyroxine administration may be due to an increased production of somatomedins locally or systemically or by direct action on connective tissue.

Plasma somatomedin activity and diabetic retinopathy.

Somatomedin activity was measured by a rabbit chondrocyte bioassay in plasma of adult patients with insulin-dependent diabetes of 11-28 years duration. Mean somatomedin activity in patients with background or proliferative retinopathy, assessed by ophthalmoscopy or fluorescein angiography, was 1.42 +/- 0.65 U/ml (n = 20) which was significantly greater than in age- and weight-matched control subjects (1.05 +/- 0.22 U/ml, n = 9). In contrast, somatomedin activity was not raised in patients who had no retinopathy (1.16 +/- 0.33 U/ml, n = 10). Diabetic patients with retinopathy also showed the greatest differences between repeat samples suggesting wider fluctuations in plasma somatomedin levels. Plasma growth hormone and glucose measured either simultaneously with somatomedin or during a 12-24 h profile were not different when diabetics with or without retinopathy were compared. The somatomedins have mitogenic actions in vivo on a variety of connective tissues. The possibility that this may extend to retinal vessels should be further evaluated.

Effect of partially purified human somatomedin on human fetal and postnatal cartilage in vitro.

Partly purified somatomedin prepared from adult human plasma stimulated [3H]thymidine and [35S]sulphate uptake into human cartilage. At a final concentration of 0.1 U/ml somatomedin the uptake of [3H]thymidine into fetal cartilage at 18 weeks of gestation was 162 +/- 23% compared with controls. Somatomedin also increased the incorporation of [35S]sulphate into fetal cartilage to 137 +/- 22% at 18 weeks compared to controls. The same concentration of somatomedin (0.1 U/ml) also increased the incorporation of [3H]thymidine into postnatal articular cartilage to 239 +/- 69% and [35S]sulphate to 133 +/- 28% compared to controls. Human somatomedin prepared from adult plasma thus has a mitogenic effect on both fetal and postnatal cartilage. The smaller effect on fetal cartilage may reflect different forms of somatomedin and/or differences in receptors for somatomedins on adult and fetal chondrocytes.

Human fetal cartilage response to plasma somatomedin activity in relation to gestational age.

The response of human fetal costal cartilage (gestational age 12--25 weeks) to plasma containing somatomedin activity has been examined in vitro. Uptake of both 3H-thymidine and 35S-sulfate into the cartilage was increased; the increase in 35S-sulfate uptake was relatively constant (mean = 198% of basal, N = 25) throughout the period studied; however, the increase in 3H-thymidine uptake varied with gestational age, reaching a maximum of about 200% at 15--17 weeks and decreasing thereafter. Such a change in cartilage sensitivity to somatomedins could be an important feature in the pattern of skeletal growth of the human fetus during gestation. The data do not confirm that human cartilage is most sensitive to somatomedins during intrauterine life.

Change in response with age of human articular cartilage to plasma somatomedin activity.

Normal male articular cartilage (34 specimens, age range 1--30 years) has been examined in vitro for response to somatomedin (SM) activity. Basal 3H-thymidine and 35S-sulfate incorporation both decreased with increasing age of the cartilage donor. However, enhancement of isotope incorporation which was attained on addition of 10% normal plasma (containing IU SM/ml) was greatest in cartilage from adolescents in the age range 12--17 years. The mean enhancement of 3H-thymidine incorporation (expressed as % basal) was as follows: age 1--10 years = 184 +/- 28 (SE), N = 9; 12--17 years = 436 +/- 101 (11); 18--30 years = 231 +/- 49 (8); and for 35S-sulfate incorporation was 1--10 years = 389 +/- 100 (8); 12--17 years = 824 +/- 273 (11); and 18--30 years = 572 +/- 56 (8). The increased response of cartilage in the 12--17 year group suggests that a greater sensitivity to the somatomedins may contribute to the increased skeletal growth during adolesence.