Aims

The aim of this study was to determine whether there is an increased prevalence of scoliosis in patients who have suffered from a haematopoietic malignancy in childhood.

Aims

The intra-articular administration of tranexamic acid (TXA) has been shown to be effective in reducing blood loss in unicompartmental knee arthroplasty and anterior cruciate reconstruction. The effects on human articular cartilage, however, remains unknown. Our aim, in this study, was to investigate any detrimental effect of TXA on chondrocytes, and to establish if there was a safe dose for its use in clinical practice. The hypothesis was that TXA would cause a dose-dependent damage to human articular cartilage.

Ketamine has been used in combination with a variety of other agents for intra-articular analgesia, with promising results. However, although it has been shown to be toxic to various types of cell, there is no available information on the effects of ketamine on chondrocytes.

We conducted a prospective randomised controlled study to evaluate the effects of ketamine on cultured chondrocytes isolated from rat articular cartilage. The cultured cells were treated with 0.125 mM, 0.250 mM, 0.5 mM, 1 mM and 2 mM of ketamine respectively for 6 h, 24 hours and 48 hours, and compared with controls. Changes of apoptosis were evaluated using fluorescence microscopy with a 490 nm excitation wavelength. Apoptosis and eventual necrosis were seen at each concentration. The percentage viability of the cells was inversely proportional to both the duration and dose of treatment (p = 0.002 and p = 0.009). Doses of 0.5 mM, 1 mM and 2mM were absolutely toxic.

We concluded that in the absence of solid data to support the efficacy of intra-articular ketamine for the control of pain, and the toxic effects of ketamine on cultured chondrocytes shown by this study, intra-articular ketamine, either alone or in combination with other agents, should not be used to control pain.

Cite this article: Bone Joint J 2014; 96-B:989–94.

We investigated whether strontium-enriched calcium phosphate cement (Sr-CPC)-treated soft-tissue tendon graft results in accelerated healing within the bone tunnel in reconstruction of the anterior cruciate ligament (ACL). A total of 30 single-bundle ACL reconstructions using tendo Achillis allograft were performed in 15 rabbits. The graft on the tested limb was treated with Sr-CPC, whereas that on the contralateral limb was untreated and served as a control. At timepoints three, six, nine, 12 and 24 weeks after surgery, three animals were killed for histological examination. At six weeks, the graft–bone interface in the control group was filled in with fibrovascular tissue. However, the gap in the Sr-CPC group had already been completely filled in with new bone, and there was evidence of the early formation of Sharpey fibres. At 24 weeks, remodelling into a normal ACL–bone-like insertion was found in the Sr-CPC group. Coating of Sr-CPC on soft tissue tendon allograft leads to accelerated graft healing within the bone tunnel in a rabbit model of ACL reconstruction using Achilles tendon allograft.

Cite this article: Bone Joint J 2013;95-B:923–8.

Hyaline articular cartilage has been known to be a troublesome tissue to repair once damaged. Since the introduction of autologous chondrocyte implantation (ACI) in 1994, a renewed interest in the field of cartilage repair with new repair techniques and the hope for products that are regenerative have blossomed. This article reviews the basic science structure and function of articular cartilage, and techniques that are presently available to effect repair and their expected outcomes.

We attempted to characterise the biological quality and regenerative potential of chondrocytes in osteochondritis dissecans (OCD). Dissected fragments from ten patients with OCD of the knee (mean age 27.8 years (16 to 49)) were harvested at arthroscopy. A sample of cartilage from the intercondylar notch was taken from the same joint and from the notch of ten patients with a traumatic cartilage defect (mean age 31.6 years (19 to 52)). Chondrocytes were extracted and subsequently cultured. Collagen types 1, 2, and 10 mRNA were quantified by polymerase chain reaction. Compared with the notch chondrocytes, cells from the dissecate expressed similar levels of collagen types 1 and 2 mRNA. The level of collagen type 10 message was 50 times lower after cell culture, indicating a loss of hypertrophic cells or genes. The high viability, retained capacity to differentiate and metabolic activity of the extracted cells suggests preservation of the intrinsic repair capability of these dissecates. Molecular analysis indicated a phenotypic modulation of the expanded dissecate chondrocytes towards a normal phenotype. Our findings suggest that cartilage taken from the dissecate can be reasonably used as a cell source for chondrocyte implantation procedures.

The aim of this study was to determine whether exposure of human articular cartilage to hyperosmotic saline (0.9%, 600 mOsm) reduces in situ chondrocyte death following a standardised mechanical injury produced by a scalpel cut compared with the same assault and exposure to normal saline (0.9%, 285 mOsm). Human cartilage explants were exposed to normal (control) and hyperosmotic 0.9% saline solutions for five minutes before the mechanical injury to allow in situ chondrocytes to respond to the altered osmotic environment, and incubated for a further 2.5 hours in the same solutions following the mechanical injury.

Using confocal laser scanning microscopy, we identified a sixfold (p = 0.04) decrease in chondrocyte death following mechanical injury in the superficial zone of human articular cartilage exposed to hyperosmotic saline compared with normal saline.

These data suggest that increasing the osmolarity of joint irrigation solutions used during open and arthroscopic articular surgery may reduce chondrocyte death from surgical injury and could promote integrative cartilage repair.

The aim of this study was to investigate the occurrence of tissue hypoxia and apoptosis at different stages of tendinopathy and tears of the rotator cuff.

We studied tissue from 24 patients with eight graded stages of either impingement (mild, moderate and severe) or tears of the rotator cuff (partial, small, medium, large and massive) and three controls. Biopsies were analysed using three immunohistochemical techniques, namely antibodies against HIF-1α (a transcription factor produced in a hypoxic environment), BNip3 (a HIF-1α regulated pro-apoptotic protein) and TUNEL (detecting DNA fragmentation in apoptosis).

The HIF-1α expression was greatest in mild impingement and in partial, small, medium and large tears. BNip3 expression increased significantly in partial, small, medium and large tears but was reduced in massive tears. Apoptosis was increased in small, medium, large and massive tears but not in partial tears.

These findings reveal evidence of hypoxic damage throughout the spectrum of pathology of the rotator cuff which may contribute to loss of cells by apoptosis. This provides a novel insight into the causes of degeneration of the rotator cuff and highlights possible options for treatment.

Chondral damage to the knee is common and, if left untreated, can proceed to degenerative osteoarthritis. In symptomatic patients established methods of management rely on the formation of fibrocartilage which has poor resistance to shear forces. The formation of hyaline or hyaline-like cartilage may be induced by implanting autologous, cultured chondrocytes into the chondral or osteochondral defect.

Autologous chondrocyte implantation may be used for full-thickness chondral or osteochondral injuries which are painful and debilitating with the aim of replacing damaged cartilage with hyaline or hyaline-like cartilage, leading to improved function. The intermediate and long-term functional and clinical results are promising.

We provide a review of autologous chondrocyte implantation and describe our experience with the technique at our institution with a mean follow-up of 32 months (1 to 9 years).

The procedure is shown to offer statistically significant improvement with advantages over other methods of management of chondral defects.

Articular cartilage repair remains a challenge to surgeons and basic scientists. The field of tissue engineering allows the simultaneous use of material scaffolds, cells and signalling molecules to attempt to modulate the regenerative tissue. This review summarises the research that has been undertaken to date using this approach, with a particular emphasis on those techniques that have been introduced into clinical practice, via in vitro and preclinical studies.

The gelatin-based haemostyptic compound Spongostan was tested as a three-dimensional (3D) chondrocyte matrix in an in vitro model for autologous chondrocyte transplantation using cells harvested from bovine knees. In a control experiment of monolayer cultures, the proliferation or de-differentiation of bovine chondrocytes was either not or only marginally influenced by the presence of Spongostan (0.3 mg/ml).

In monolayers and 3-D Minusheet culture chambers, the cartilage-specific differentiation markers aggrecan and type-II collagen were ubiquitously present in a cell-associated fashion and in the pericellular matrix. The Minusheet cultures usually showed a markedly higher mRNA expression than monolayer cultures irrespective of whether Spongostan had been present or not during culture. Although the de-differentiation marker type-I collagen was also present, the ratio of type-I to type-II collagen or aggrecan to type-I collagen remained higher in Minusheet 3-D cultures than in monolayer cultures irrespective of whether Spongostan had been included in or excluded from the monolayer cultures. The concentration of GAG in Minusheet cultures reached its maximum after 14 days with a mean of 0.83 ± 0.8 μg/10⁶ cells; mean ±, sem, but remained considerably lower than in monolayer cultures with/without Spongostan.

Our results suggest that Spongostan is in principle suitable as a 3-D chondrocyte matrix, as demonstrated in Minusheet chambers, in particular for a culture period of 14 days. Clinically, differentiating effects on chondrocytes, simple handling and optimal formability may render Spongostan an attractive 3-D scaffold for autologous chondrocyte transplantation.

The pathophysiology of intervertebral disc degeneration has been extensively studied. Various factors have been suggested as influencing its aetiology, including mechanical factors, such as compressive loading, shear stress and vibration, as well as ageing, genetic, systemic and toxic factors, which can lead to degeneration of the disc through biochemical reactions. How are these factors linked? What is their individual importance? There is no clear evidence indicating whether ageing in the presence of repetitive injury or repetitive injury in the absence of ageing plays a greater role in the degenerative process. Mechanical factors can trigger biochemical reactions which, in turn, may promote the normal biological changes of ageing, which can also be accelerated by genetic factors. Degradation of the molecular structure of the disc during ageing renders it more susceptible to superimposed mechanical injuries.

This review supports the theory that degeneration of the disc has a complex multifactorial aetiology. Which factors initiate the events in the degenerative cascade is a question that remains unanswered, but most evidence points to an age-related process influenced primarily by mechanical and genetic factors.

Human articular cartilage samples were retrieved from the resected material of patients undergoing total knee replacement. Samples underwent automated controlled freezing at various stages of preparation: as intact articular cartilage discs, as minced articular cartilage, and as chondrocytes immediately after enzymatic isolation from fresh articular cartilage. Cell viability was examined using a LIVE/DEAD assay which provided fluorescent staining. Isolated chondrocytes were then cultured and Alamar blue assay was used for estimation of cell proliferation at days zero, four, seven, 14, 21 and 28 after seeding. The mean percentage viabilities of chondrocytes isolated from group A (fresh, intact articular cartilage disc samples), group B (following cryopreservation and then thawing, after initial isolation from articular cartilage), group C (from minced cryopreserved articular cartilage samples), and group D (from cryopreserved intact articular cartilage disc samples) were 74.7% (95% confidence interval (CI) 73.1 to 76.3), 47.0% (95% CI 43 to 51), 32.0% (95% CI 30.3 to 33.7) and 23.3% (95% CI 22.1 to 24.5), respectively. Isolated chondrocytes from all groups were expanded by the following mean proportions after 28 days of culturing: group A ten times, group B 18 times, group C 106 times, and group D 154 times.

This experiment demonstrated that it is possible to isolate viable chondrocytes from cryopreserved intact human articular cartilage which can then be successfully cultured.

We used an in vivo model to assess the use of an autogenous cancellous bone block and marrow graft for augmenting tendon reattachment to metallic implants. We hypothesised that augmentation of the tendon-implant interface with a bone block would enable retention of the graft on the implant surface, enhance biological integration, and result in more consistent functional outcomes compared with previously reported morcellised graft augmentation techniques.

A significant improvement in functional weight-bearing was observed between six and 12 weeks. The significant increase in ground reaction force through the operated limb between six and 12 weeks was greater than that reported previously with morcellised graft augmented reconstructions. Histological appearance and collagen fibre orientation with bone block augmentation more closely resembled that of an intact enthesis compared with the morcellised grafting technique. Bone block augmentation of tendon-implant interfaces results in more reliable functional and histological outcomes, with a return to pre-operative levels of weight-bearing by 24 weeks.

Implantation of autologous chondrocytes and matrix autologous chondrocytes are techniques of cartilage repair used in the young adult knee which require harvesting of healthy cartilage and which may cause iatrogenic damage to the joint. This study explores alternative sources of autologous cells. Chondrocytes obtained from autologous bone-marrow-derived cells and those from the damaged cartilage within the lesion itself are shown to be viable alternatives to harvest-derived cells. A sufficient number and quality of cells were obtained by the new techniques and may be suitable for autologous chondrocyte and matrix autologous chondrocyte implantation

Ovine articular chondrocytes were isolated from cartilage biopsy and culture expanded in vitro. Approximately 30 million cells per ml of cultured chondrocytes were incorporated with autologous plasma-derived fibrin to form a three-dimensional construct. Full-thickness punch hole defects were created in the lateral and medial femoral condyles. The defects were implanted with either an autologous ‘chondrocyte-fibrin’ construct (ACFC), autologous chondrocytes (ACI) or fibrin blanks (AF) as controls. Animals were killed after 12 weeks. The gross appearance of the treated defects was inspected and photographed. The repaired tissues were studied histologically and by scanning electron microscopy analysis.

All defects were assessed using the International Cartilage Repair Society (ICRS) classification. Those treated with ACFC, ACI and AF exhibited median scores which correspond to a nearly-normal appearance. On the basis of the modified O’Driscoll histological scoring scale, ACFC implantation significantly enhanced cartilage repair compared to ACI and AF. Using scanning electron microscopy, ACFC and ACI showed characteristic organisation of chondrocytes and matrices, which were relatively similar to the surrounding adjacent cartilage.

Implantation of ACFC resulted in superior hyaline-like cartilage regeneration when compared with ACI. If this result is applicable to humans, a better outcome would be obtained than by using conventional ACI.

We attempted to repair full-thickness defects in the articular cartilage of the trochlear groove of the femur in 30 rabbit knee joints using allogenic cultured chondrocytes embedded in a collagen gel. The repaired tissues were examined at 2, 4, 8, 12 and 24 weeks after operation using histological and histochemical methods. The articular defect filling index measurement was derived from safranin-O stained sections. Apoptotic cellular fractions were derived from analysis of apoptosis in situ using TUNEL staining, and was confirmed using caspase-3 staining along with quantification of the total cellularity. The mean articular defect filling index decreased with time. After 24 weeks it was 0.7 (sd 0.10), which was significantly lower than the measurements obtained earlier (p < 0.01). The highest mean percentage of apoptotic cells were observed at 12 weeks, although the total cellularity decreased with time. Because apoptotic cell death may play a role in delamination after chondrocyte transplantation, anti-apoptotic gene therapy may protect transplanted chondrocytes from apoptosis.

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 × 10⁷ AdIGF-1 modified chondrocytes and the contralateral joint received 2 × 10⁷ 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 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.

The weight-bearing status of articular cartilage has been shown to affect its biochemical composition. We have investigated the topographical variation of sulphated glycosaminoglycan (GAG) relative to the DNA content of the chondrocyte in human distal femoral articular cartilage.

Paired specimens of distal femoral articular cartilage, from weight-bearing and non-weight-bearing regions, were obtained from 13 patients undergoing above-knee amputation. After papain enzyme digestion, spectrophotometric GAG and fluorometric DNA assays assessed the biochemical composition of the samples. The results were analysed using a paired t-test.

Although there were no significant differences in cell density between the regions, the weight-bearing areas showed a significantly higher concentration of GAG relative to DNA when compared with non-weight-bearing areas (p = 0.02).

We conclude that chondrocytes are sensitive to their mechanical environment, and that local loading conditions influence the metabolism of the cells and hence the biochemical structure of the tissue.