Specific antisera to collagen Types I, II and III and proteoglycan were used to investigate the distributions of these molecules in normal human intervertebral discs. Immunofluorescent staining indicated the presence of small amounts of Type III collagen located pericellularly in normal adult intervertebral discs. This finding had not been demonstrated previously by other methods. Similar specimens of intervertebral discs from 17 patients with scoliosis of varying aetiologies were examined, but no evidence was obtained for primary connective tissue defects. Secondary changes, especially marked vascularisation of the inner annulus, were apparent in a number of scoliotic discs, and some of these showed enhanced staining for collagen Type I and proteoglycan, and intercellular matrix staining for Type III collagen

Lesions within the articular cartilage layer of synovial joints do not heal spontaneously. Some repair cells may appear, but their failure to become established may be related to problems of adhesion to proteoglycan-rich surfaces. We therefore investigated whether controlled enzymatic degradation of surface proteoglycan molecules to a depth of about 1 μm, using chondroitinase ABC, would improve coverage by repair cells. We created superficial lesions (1.0 × 0.2 × 5 mm) in the articular cartilage of mature rabbit knees and treated the surfaces with 1 U/ml of chondroitinase ABC for four minutes. The defects were studied by histomorphometry and electron microscopy at one, three and six months. At one month, untreated lesions were covered to a mean extent of 28% by repair cells; this was enhanced to a mean of 53% after enzyme treatment. By three months, the mean coverage of both control and chondroitinase-ABC-treated defects had diminished dramatically to 0.2% and 13%, respectively, but at six months both untreated and treated lesions had a similar coverage of about 30%, not significantly different from that achieved in untreated knees at one month. These findings suggest that, with time, chondrocytes near the surface of the defect may compensate for the loss of proteoglycans produced by enzyme treatment, thereby restoring the inhibitory properties of the matrix as regards cell adhesion. This supposition was confirmed by electron microscopy. Our results have an important bearing on attempts made to induce healing responses by transplanting chondrogenic cells or by applying growth factors

The belief that an intervertebral disc must degenerate before it can herniate has clinical and medicolegal significance, but lacks scientific validity. We hypothesised that tissue changes in herniated discs differ from those in discs that degenerate without herniation. Tissues were obtained at surgery from 21 herniated discs and 11 non-herniated discs of similar degeneration as assessed by the Pfirrmann grade. Thin sections were graded histologically, and certain features were quantified using immunofluorescence combined with confocal microscopy and image analysis. Herniated and degenerated tissues were compared separately for each tissue type: nucleus, inner annulus and outer annulus. Herniated tissues showed significantly greater proteoglycan loss (outer annulus), neovascularisation (annulus), innervation (annulus), cellularity/inflammation (annulus) and expression of matrix-degrading enzymes (inner annulus) than degenerated discs. No significant differences were seen in the nucleus tissue from herniated and degenerated discs. Degenerative changes start in the nucleus, so it seems unlikely that advanced degeneration caused herniation in 21 of these 32 discs. On the contrary, specific changes in the annulus can be interpreted as the consequences of herniation, when disruption allows local swelling, proteoglycan loss, and the ingrowth of blood vessels, nerves and inflammatory cells. In conclusion, it should not be assumed that degenerative changes always precede disc herniation. Cite this article: Bone Joint J 2013;95-B:1127–33

Biochemical changes in the articular cartilage of the knees of mature dogs, one with natural and four with surgically induced osteoarthritis, have been investigated. The four dogs were killed three, six, nine and forty-eight weeks after division of the right anterior cruciate ligament, the left knees serving as controls. The cartilage of the joints operated on was thicker and more hydrated than the control cartilage; the proteoglycans were more easily extracted and had higher galactosamine/glucosamine molar ratios. The proportion of proteoglycans firmly associated with collagen, and hence not extractable, diminished before fibrillation was demonstrable by indian ink staining of the surface. These biochemical changes were present throughout the entire cartilage of the joints operated on of the dogs killed more than three weeks later, and of the dog with natural osteoarthritis. The results suggest that in response to altered mechanical stresses the chondrocytes synthesise proteoglycans that contain more chondroitin sulphate relative to keratin sulphate than normally, as in immature articular cartilage

We studied the mechanical and biochemical properties of articular cartilage from 22 osteoarthritic femoral heads obtained at operation and 97 femoral heads obtained at autopsy. Cartilage from the zenith and from the antero-inferior aspect of each head was tested both in tension and in compression. Water content, swelling ability and proteoglycan content were measured, the cartilage was examined histologically and the density of the underlying bone was assessed. Fifty-five of the autopsy specimens were defined as macroscopically normal because they exhibited no progressive fibrillation patterns on staining with Indian ink; but significant changes in water content, bone density and tensile strength related to age were seen in this group. In 20 pairs of femoral heads which were both macroscopically normal, we found, surprisingly, that cartilage from the left and right sides of the same patient was sometimes very different. Compared with the normal autopsy specimens the osteoarthritic specimens had a significantly increased swelling ability, a lower proteoglycan content and impaired mechanical properties, being both weaker in tension and softer in compression. Abnormal autopsy specimens had values intermediate between those of osteoarthritic and normal groups. Results from this abnormal group suggest that there is no primary loss of proteoglycan in early osteoarthritis

We stably transfected early passage chondrocytes with an anti-apoptotic Bcl-2 gene in vitro using a retrovirus vector. Samples of articular cartilage were obtained from 11 patients with a mean age of 69 years (61 to 75) who were undergoing total knee replacement for osteoarthritis. The Bcl-2-gene-transfected chondrocytes were compared with non-transfected and lac-Z-gene-transfected chondrocytes, both of which were used as controls. All three groups of cultured chondrocytes were incubated with nitric oxide (NO) for ten days. Using the Trypan Blue exclusion assay, an enzyme-linked immunosorbent assay and flow cytometric analysis, we found that the number of apoptotic chondrocytes was significantly higher in the non-transfected and lac-Z-transfected groups than in the Bcl-2-transfected group (p < 0.05). The Bcl-2-transfected chondrocytes were protected from NO-induced impairment of proteoglycan synthesis. We conclude that NO-induced chondrocyte death involves a mechanism which appears to be subject to regulation by an anti-apoptotic Bcl-2 gene. Therefore, Bcl-2 gene therapy may prove to be of therapeutic value in protecting human articular chondrocytes

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 × 10. 7. AdIGF-1 modified chondrocytes and the contralateral joint received 2 × 10. 7. naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months. Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model. The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation

We studied the early cartilage changes in osteoarthritis, examining the most normal appearing articular cartilage from the hips of 17 patients. Normal appearing cartilage from five patients treated for fractures was used as control material. Two different types of clone were found. The first had increased staining for proteoglycan and was thought to have been engaged in the synthesis of matrix. The other type was associated with a severe deficiency of proteoglycan, matrix streaks and evidence of degradation and phagocytosis of matrix components. Immunohistochemistry demonstrated large amounts of chondroitin 4 and 6 sulphate about the synthetic-type clones, and little or no reactivity about the degenerative clones which lay more superficially and were associated with matrix destruction. Clones appeared to be engaged in either matrix synthesis or its destruction. The disease process of osteoarthritis appeared to begin at the surface of the articular cartilage

Articular cartilage from the femoral heads of 27 patients having an arthroplasty for subcapital fracture was studied, and its mechanical and chemical properties compared to those of a group of 33 age-matched macroscopically normal autopsy specimens. Water and proteoglycan contents were measured, as were swelling ability, compressive and tensile strength of the cartilage, and the density of the underlying bone. Cartilage from the fracture specimens had a significantly reduced proteoglycan content, as measured by fixed charge density, and increased swelling ability. These results indicate that this group differs from the "normal" population and care should be taken before they are accepted as control material for studies on osteoarthritic cartilage. Another finding was that bone density was much the same in the fracture and the normal group. This casts some doubt upon the concept that patients who sustain subcapital fractures are more osteoporotic than the average for the same age range

Bone morphogenetic protein (BMP) has a crucial role in osteochondrogenesis of bone formation as well as in the repair of fractures. The interaction between hedgehog protein and BMPs is inferred from recent molecular studies. Hedgehog genes encode secreted proteins which mediate patterning and growth during skeletal development. We have shown that Indian hedgehog gene (Ihh) is expressed in cartilage anlage and later in mature and hypertrophic chondrocytes. This finding suggests that Ihh may regulate the development of chondrocytes. Our results in this study have shown that Ihh transcripts were expressed in hypertrophic chondrocytes in mice at three days but not at three weeks, although a similar expression pattern of α1 (X) collagen could be observed in both types of cartilage. To investigate the possibility that there are direct and age-dependent functions of Ihh in chondrocytes, cultured chondrocytes were treated with the amino-terminal fragment of Sonic hedgehog protein (Shh-N) which can functionally substitute for Ihh protein. Shh-N did not affect the proliferation and differentiation of chondrocytes from three-week-old mice but had a significant effect on three-day-old mice. It enhanced proliferation up to 128% of the control culture in a dose-dependent manner. Although there was no effect in Shh-N-treated cultures, Shh-N enhanced the stimulatory effect of parathyroid hormone (PTH) on the synthesis of proteoglycans. Because the effects of Shh-N on chondrocyte differentiation in this culture system differed from those of bone morphogenetic protein-2 (BMP2) and PTH, in terms of proteoglycan synthesis and ALPase activity, it is unlikely that BMP2 or PTH/PTH-related protein mediates the direct effects of Ihh in chondrocytes. Our study shows that Ihh can function in chondrocytes in a direct and age-dependent fashion

The discrepancy between successful experimental studies of cartilage repair and the clinical results is unexplained. We have evaluated the effect of metabolic alterations in joint homeostasis owing to an articular defect on the outcome of cartilage repair using tissue engineering methods. We used 21 adolescent Dutch goats divided into three groups. The control knees were left untreated while the contralateral knee was randomised to receive either no treatment (N), early treatment (E) or late treatment (L). The metabolism of proteoglycans in the surrounding joint surface was determined and correlated with the O’Driscoll score used to quantify the histological aspect of the repair of the defect. Synthesis of proteoglycan (PG) was increased in all groups. The release of glucosaminoglycan (GAG) was significantly higher in the untreated but not after early transplantation (1.3 v 1.8 NS). The cartilage repair scores in the early treatment group were not as good as those of the normal control group, but were significantly better when compared with both the untreated defects and the late treated defects. Defects which had been treated late showed a significantly decreased score when compared with those which had had early treatment or the normal control group and did not differ (p = 0.12) from those with no treatment. The histological and biochemical scores closely resembled the macroscopic and functional parameters which showed a significant deterioration for the late treated group and those without treatment compared with animals treated early. Thus, tissue-engineered cartilage repair is negatively influenced by altered matrix metabolism. Early treatment showed significantly better results for repair of cartilage than late or no treatment, with a concurrent decrease in the detrimental disturbance of cartilage metabolism which constituted a protective effect on the articulation

Aims

Platelet-rich plasma (PRP) intra-articular injections may provide a simple and minimally invasive treatment for early-stage knee osteoarthritis (OA). This has led to an increase in its adoption as a treatment for knee OA, although there is uncertainty about its efficacy and benefit. We hypothesized that patients with early-stage symptomatic knee OA who receive multiple PRP injections will have better clinical outcomes than those receiving single PRP or placebo injections.

Hemiarthroplasty of the hip and some other joints has been used for many years with satisfactory results, but the fate of articular cartilage when weight-bearing against metal has not been reported. Replacement of the head of the femur was carried out in one hip of each of 26 dogs, and the changes in acetabular cartilage studied at intervals of up to 24 weeks. There was early loss of proteoglycan, followed by surface damage to the cartilage, progressive degenerative changes, and growth of pannus from the articular margins. At 24 weeks after operation there was little remaining articular cartilage, while intense subchondral activity suggested that the bony skeleton was being remodelled to conform to the shape of the prosthesis. This study is not intended to suggest that hemiarthroplasty does not help patients

Rat patellae were preincubated with culture medium M199 for one hour and then with either fresh culture medium or Ringer's solution, Ringer lactate, Ringer glucose, normal saline or Betadine for another hour. The rate of proteoglycan synthesis in the articular cartilage was then measured by uptake of 35SO4 for the next 16 hours. Cartilage metabolism was inhibited by all of the solutions even after a recovery time of 16 hours. The inhibition was by 5% for Ringer's solution, 10% for Ringer glucose (p < 0.01), 20% for saline and Ringer lactate (p < 0.001) and 55% for Betadine (p < 0.001). Ringer's solution is therefore the best choice for joint irrigation during arthroscopy or other procedures

Our study establishes a rabbit model of disc degeneration which requires neither a chemical nor physical injury to the disc. Disc degeneration similar to that seen in man was created at levels proximal (L4-L5) and caudal (L7-S1) to a simulated lumbar fusion and was studied for up to nine months after arthrodesis. Loss of the normal parallel arrangement of collagen bundles within the annular lamellae was observed in intervertebral discs adjacent to the fusion at three months. By six months there was further disorganisation as well as loss of distinction between the lamellae themselves. By nine months the structure of the disc had been replaced by disorganised fibrous tissue, and annular tears were seen. There was an initial cellular proliferative response followed by loss of chondrocytes and notochordal cells in the nucleus pulposus. Degeneration was accompanied by a decrease in the monomer size of proteoglycans. Narrowing of the disc space, endplate sclerosis and the formation of osteophytes at adjacent disc spaces were observed radiologically

Quantitative polarised light microscopy was applied to sections of unfixed, undecalcified bone taken at operation from patients with two types of proximal femoral fracture, subcapital and trochanteric. Specimens were also taken from the equivalent sites in otherwise normal subjects at autopsy, and from various other sites of traumatic fractures; these two latter groups acted as controls. Analysis of the 57 specimens disclosed changes in the nature of the bone at the site of subcapital fractures, namely the presence of relatively large crystals of hydroxyapatite and a change in the molecular orientation, but not total content, of the acidic proteoglycans of the bone matrix. Our results have confirmed and extended the findings of others on subcapital fractures, and have also shown very similar changes in the trochanteric fractures. It thus appears that the bony changes in the two types of proximal femoral fracture are not as different as has been suggested

We studied the presence of anabolic growth factors in human herniated intervertebral discs (IVD) using a reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Messenger RNA (mRNA) was isolated from the nucleus pulposus using oligo (dT). 25. superparamagnetic beads and probing with gene-specific primers in RT-PCR. mRNA coding for TGF-α (3/10), EGF (0/10), TGF-β1 (0/10) and TGF-β3 (2/10) or the EGF receptor (EGF-R; 0/10) and TGF-β type-II receptor (0/10) was found only occasionally. Beta-actin was always present and positive sample controls confirmed the validity of the RT-PCR assay. These RT-PCR findings were confirmed using immunohistochemical staining of EGF and TFG-β, whereas TGF-α protein was always found associated with discocytes. We conclude that the nucleus pulposus of the herniated IVD is vulnerable to proteolytic degradation and depletion of proteoglycans due to the lack and/or low production of anabolic growth factors/receptors which could increase the local synthesis of the extracellular matrix

Composites of chondrocytes and polymerised fibrin were supplemented with insulin-like growth factor-I (IGF-I) during the arthroscopic repair of full-thickness cartilage defects in a model of extensive loss of cartilage in horses. Repairs facilitated with IGF-I and chondrocyte-fibrin composites, or control defects treated with chondrocyte-fibrin composites alone, were compared before death by the clinical appearance and repeated analysis of synovial fluid, and at termination eight months after surgery by tissue morphology, collagen typing, and biochemical assays. The structure of cartilage was evaluated histologically by Toluidine Blue reaction and collagen type-I and type-II in situ hybridisation and immunohistochemistry. Repair tissue was biochemically evaluated by DNA assay, proteoglycan quantitation and characterisation, assessment of collagen by reverse-phase high-performance liquid chromatography, and collagen typing using cyanogen bromide digestion and peptide separation by polyacrylamide gel electrophoresis. The results at eight months showed that the addition of IGF-I to chondrocyte grafts enhanced chondrogenesis in cartilage defects, including incorporation into surrounding cartilage. Gross filling of defects was improved, and the tissue contained a higher proportion of cells producing type-II collagen. Measurements of collagen type II showed improved levels in IGF-I-treated defects, supporting in situ hybridisation and immunohistochemical assessments of the defects. IGF-I improves the repair capabilities of chondrocyte-fibrin grafts in large full-thickness repair models

Dynamic muscle-tendon substitution for acute anterior cruciate deficiency in the dog was studied using the semimembranosus muscle-tendon. Nineteen mongrel dogs each had a semimembranosus transfer in one knee; as a control, the anterior cruciate ligament and the semimembranosus were released in the opposite knee. No postoperative immobilisation was used. The anterior drawer sign was assessed before and after operation and when the dogs were killed five months later. Dogs were excluded from the study if they developed infections or contractures of the hind legs. At five months, 11 dogs were available for study. The operated knees were examined histologically and evaluated using a reproducible index of arthritis based on: the macroscopic discoloration of the articular cartilage, the cellularity of the cartilage, the microscopic appearance of the articular surface, the loss of proteoglycans, the formation of osteophytes and the degree of subchondral osteosclerosis. There was no significant difference in the anterior drawer sign or the degree of arthritic changes between knees with a semimembranous transfer and the controls. Examination showed that a muscle-tendon transfer into the tibia was equivalent to transferring the muscle into the posterior capsule--the intra-articular tendon being weak but histologically viable. The transfer did not prevent the anterior drawer sign becoming positive nor the development of osteoarthritis. A second control group, in which three dogs had an arthrotomy and semimembranosus release in both their hind legs, showed that a semimembranosus release alone did not cause osteoarthritis

Abstract

Nerve transfer has become a common and often effective reconstructive strategy for proximal and complex peripheral nerve injuries of the upper limb. This case-based discussion explores the principles and potential benefits of nerve transfer surgery and offers in-depth discussion of several established and valuable techniques including: motor transfer for elbow flexion after musculocutaneous nerve injury, deltoid reanimation for axillary nerve palsy, intrinsic re-innervation following proximal ulnar nerve repair, and critical sensory recovery despite non-reconstructable median nerve lesions.