This retrospective matched cohort study tested the hypothesis that an incomplete periacetabular acetabuloplasty, as an added step to delayed open reduction, diminishes the risk of developing acetabular dysplasia.

29 hips from 23 patients with idiopathic DDH that underwent intentionally delayed open reduction and acetabuloplasty at our institution from 2003 to 2010 were matched for age at presentation and bilaterality to historic controls. These were 29 hips from 26 patients, treated with open reduction alone from 1989 to 2003. Residual dysplasia treated with pelvic osteotomy, AVN grade II-IV, and rate of re-intervention were the outcome measures.

The mean ages at diagnosis and at surgery were 8.62 weeks and 12.97 months, respectively. At latest follow-up, 27 hips in the acetabuloplasty group and 22 in the open reduction alone group had satisfactory radiographic outcome (Severin class Ia, Ib or II) (p=0.16). 18 of the 58 hips (31.0%) had AVN, 7 (24.14%) in the case group and 11(37.93%) in the control group. Further surgery was required in 15 of the 29 hips in the open reduction alone group. These included 2 revision of open reductions, 5 pelvic osteotomies, 3 varus derotation osteotomies, and 5 apo or epiphysiodesis whilst only one patient in the acetabuloplasty group required a medial screw epiphysiodesis for late lateral growth arrest. There is a positive association between the need for further surgery and open reductions alone: the odds ratio is 14.00 and the 95% confidence interval (1.97, 99.63), p=0.0017. The five hips in the open reduction alone group that required a pelvic osteotomy were intervened at an average of 31.45 (±9.07) months.

The addition of an incomplete periacetabular periacetabuloplasty to all hips undergoing open reduction eliminated residual acetabular dysplasia in this cohort whilst it does not appear to have deleterious effects, as evidenced by the similar Severin and McKay scores.

Aim

With the link between obesity and Slipped Upper Femoral Epiphysis (SUFE) well established and a rising number of paediatric orthopaedic patients presenting with vitamin D deficiency, the aim of our study was to establish the incidence of vitamin D deficiency in SUFE patients and whether low vitamin D levels increases the time to proximal femoral physeal fusion post surgical fixation.

Aim

Shelf acetabuloplasty is part of the armamentarium for the treatment of Legg-Perthes-Calve disease. Surgeons have used it to increase the anterolateral cover of the deformed head in advanced stages of the disease. However, others, including the senior author, advocate its use for containment of the diseased femoral head earlier in the disease, for both the prevention of further femoral head extrusion and as an aid in the remodelling process. The current study presents the results of this procedure performed from August 1999 to February 2010.

Aim

This study presents the early results of a novel procedure, both in timing and surgical technique, aimed to treat those cases of congenital hip dysplasia that present late or fail conservative treatment.

Background

Vitamin D deficiency may increase predisposition to a number of paediatric orthopaedic conditions and the prevalence of vitamin D deficiency is increasing in children in developed countries. The aim of this study was to determine the epidemiology of vitamin D deficiency and insufficiency in children presenting to a regional paediatric orthopaedic service. We also examined the relationships between vitamin D status, social deprivation and ethnicity

AIMS

We present a retrospective study of bilateral CDH. We analysed the correlation of complications to the confounding factors.

Purpose

We describe two recent cases of intraosseous (IO) access resulting in amputation in critically ill infants and make contemporary recommendations on the safe practice of this technique.

Purpose This was an observational study to determine the prevalence of 25-hydroxyvitamin D (25[OH]D deficiency in our paediatric orthopaedic patient population.

Methods We have measured serum 25(OH)D levels in 44 paediatric patients who presented with bone pain. None of these patients had a pre-existing diagnosis of 25(OH)D deficiency. The age of patients ranged from 11 months to 16.5 years. There were 23 female and 21 male patients. The range of diagnoses included hip pain/irritable hip (4), Blount's disease (4), developmental hip dysplasia (7), genu valgum (3), Legg Calve Perthes’ disease (6), slipped capital femoral epiphysis (11), knee pain (3), other (6).

Those found to be 25(OH)D deficient underwent further biochemical investigation and were referred for paediatric endocrinology review with a view to vitamin D supplementation.

Results We found 9 patients (20%) with serum 25(OH)D levels of <20ng/mL indicating 25(OH)D deficiency. 17 patients (39%) had serum 25(OH)D levels in the range 20-30ng/mL indicating possible deficiency. The remaining 18 patients (41%) had a normal level of 25(OH)D. There was no association between low serum 25(OH)D level and any specific diagnosis, nor with gender or age of patient. There was, however, a statistically significant difference between the serum 25(OH)D level in those patients with unexplained joint pain (mean 22.5ng/mL) and those with other diagnoses (mean 30.7ng/ml) (P<0.05).

Conclusion Our results are consistent with other recent prevalence studies showing a concerning level of 25(OH)D deficiency among the paediatric population, and may suggest an increasing burden of disease in the coming years arising from the problem.

Purpose of Study

To assess the radiological outcomes of medial screw epiphyseodesis of the proximal femoral physis in the management of lateral growth arrest following treatment of developmental dysplasia of the hip.

Purpose

Clinical coding is used to record information from patient admissions in the form of coded data used for monitoring the provision of health services and trends, research, audit and NHS financial planning.

Osteoarthrosis (OA) is often considered to be due to “wear and tear”, aggravated by obesity. However, if developmental dysplasia of the hip (DDH) is treated incorrectly, osteoarthrosis can also occur at a very young age. We obtained cartilage from the femoral head a 23 year-old female after arthroplasty for DDH; from a 14 year-old male, resected for paralytic dislocation, and from OA and control patients. This provided a unique opportunity to compare the cellular and epigenetic features of OA in older patients with those in a young control as well as a DDH patient. We have recently defined the cellular and epigenetic features of idiopathic OA, in particular the association between induction of proteases and loss of DNA methylation in the respective promoter regions (Arthritis Rheum2005; 52: 3110–3124). We had shown that these proteases were silenced in normal articular chondrocytes, but “unsilenced” in OA chondrocytes. The present study determined whether the phenotypic changes of idiopathic OA also take place in juvenile OA and whether loss of DNA demethylation is also associated with the abnormal expression of proteases in juvenile OA. Paraffin sections were immunostained with antibodies to MMP-3, -9, and ADAMTS-4. DNA was extracted from freezer milled cartilage. The methylation status of specific CpG sites (at which methylation occurs) was established using methylation-sensitive restriction enzymes followed by PCR. From the 23 year old female, we only obtained a 1cm thick transverse slice of femur, taken near the femoral neck. However, this contained sufficient reasonably thick cartilage in the perimeter for histology and DNA extraction.

The cartilage of the 14-year old showed high cellularity and absence of immunostaining for all proteases investigated. Apart from the higher cellularity, this was similar to the ‘control’ cartilage obtained from patients with a fracture of the femoral neck. We had previously shown that, as OA progresses, more chondrocytes become immunopositive for the degradative enzymes and these cells divide so that in the typical clones of OA all cells synthesize the proteases. The cartilage from the 23-year old DDH patient showed extensive loss of proteoglycans from the superficial zone and fibrous repair tissue covered some areas. Nearly all chondrocytes produced the proteases and clones had formed, as in idiopathic OA. Since these sample were from the base of the femoral neck, where in idiopathic OA good cartilage often remains even in severe OA, the disease process must have reached an early end-stage in this young patient. The findings indicate that severe OA, as defined by the presence of clones that produce degradative enzymes, can develop very quickly. Interestingly, the expression and synthesis of degradative enzymes by OA chondrocytes was the same in juvenile and old-age OA. and their abnormal expression was associated with “unsilencing” via DNA demethylation in both juvenile and old-age OA. The results thus suggest that age per se is not a major determinant of OA progression.

The majority of diaphyseal forearm fractures in children are treated by closed reduction and plaster immobilisation. There is a small subset of patients where operative treatment is indicated. Recent reports indicate that elastic intramedullary nailing (EIN) is gaining popularity over plate fixation. We report the results of EIN for diaphyseal fractures of the forearm in 44 children aged between 5 and 15 years during a three-year period. The indications were instability (26), redisplacement (14), and open fractures (4). Closed reduction and nailing was carried out in 18 cases. A single bone had to be opened in 16 cases and in 10 cases both bones were opened for achieving reduction. Out of the 39 both bone forearm fractures, 35 patients had stabilisation of both radius and ulna and in 4 cases only a single bone was nailed (Radius 3, Ulna 1).

Union was achieved in all the 44 cases at an average time of 7 weeks with one delayed union. All patients regained full flexion and extension of the elbow and wrist. Pronation was restricted by an average of 20° in 30% patients.

Complications were seen in 10 patients (20%). 4 patients had prominent metal work which required early removal. There was refracture in one case, which was treated by nail removal and re-fixation. Two patients developed post operative compartment syndrome requiring fasciotomy. EIN of the radius alone in a patient with fractures of both the bones of forearm, led to secondary displacement of the ulna. This resulted in ulnar malunion and a symptomatic distal radio-ulnar joint subluxation. This was successfully treated by ulnar osteotomy.

Compared to forearm plating EIN involves minimal scarring, easier removal and less risk of nerve damage. We therefore recommend EIN for the treatment of unstable middle and proximal third forearm fractures.

Idiopathic osteoarthritis (OA) is a complex, late-onset disease whose causes are still unknown. In spite of tremendous efforts, the search for the genes pre-disposing towards osteoarthritis has so far met with little success. We hypothesize that epigenetic changes play a major role in the pathology of OA. Epigenetics refers to stable, heritable, but potentially reversible modifications of gene expression that do not involve mutations in the DNA sequence, for example DNA methylation or histone modification. Epigenetic changes are gene and cell-type specific, may arise sporadically with increasing age or be provoked by environmental factors. To investigate whether epigenetic changes are significant factors in OA, we examined the DNA methylation status of the promoter regions of three genes that are expressed by OA, but not by normal, articular chondrocytes, namely MMP-3 (stromelysin-1), MMP-9 (gelatinase B) and MMP-13 (collagenase3). We hypothesized that these genes are silenced in normal chondrocytes by methylation of the cytosines of CpG dinucleotides in the respective promoter regions, but that abnormal expression is associated with a de-methylation, leading to eunsilencing f of gene expression. Cartilage was obtained from the femoral heads of 16 OA and 10 femoral neck fracture (#NOF) patients, which served as controls due to the inverse relationship between osteoporosis and OA. The cartilage was milled in a freezer mill with liquid nitrogen, DNA was extracted with a Qiagen kit, digested with methylation sensitive restriction enzymes, followed by PCR amplification. These enzymes will cut at their specific cleavage sites only if the CpGs is not methylated and thus allow us to determine methylation status of specific CpG sites.

Results. Less than 5% of the chondrocytes in superficial layer from #NOF cartilage expressed degradative enzymes, whereas all cloned chondrocytes from advanced-stage OA cartilage were immunopositive. The overall % of CpG demethylation in the promoters of control patients (whose chondrocytes did not express the enzymes) was 20.1%, whereas 48.6% of CpG sites were demethylated in degradative chondrocytes of OA patients (p< 0.001). For MMP-13, the increase in demethylation between control and OA was from 4 ^..20%; for MMP-9 from 47 ^..81% and for MMP-3 from 30 ^..57%. However, not all available CpG sites were equally demethylated. Some sites were uniformly methylated in both OA and controls, others were demethylated even in controls. However, there was at least one crucial site for each degradative enzyme, where the differences in the degree of methylation were greatest and statistically different. These sites were at −110 for MMP-13; −36 for MMP-9; −635 for MMP-3. There was no relation between the % demethylation and the patient fs age and no apparent difference between males and females.

Conclusions: We have demonstrated an association between abnormal gene expression of MMP-3, MMP-9 and MMP-13 and promoter DNA demethylation. This epigenetic dysregulation of genes appeared to be clonally inherited by daughter cells and may be typical for osteoarthritis and other complex, late-onset diseases.

Clonal chondrocytes of osteoarthritic (OA) cartilage express an aberrant set of genes. We hypothesize that this aberrant gene expression may be due to clonally inherited epigenetic changes, defined as altered gene expression without changes in genetic sequence. The major epigenetic changes are due to altered DNA methylations in crucial parts of the promoter region. If the cytosines of CpG dinucleotides are methylated, the gene will be silenced, even if the right transcription factors are present. Similarly, de-methylations may activate previously silenced genes. Our aims were to provide ‘proof-of-concept’ data by examining the methylation status of genes in OA vs non-OA chondrocytes. Articular cartilage was obtained a) from the cartilage of fracture-neck-of-femur (#NOF) patients and b) from or around the eroded regions of OA samples. The former was full thickness cartilage, the latter was partially degraded cartilage, which contained mostly clonal chondrocytes as confirmed by histology. The cartilage samples were ground in a freezer mill (Glen Creston, UK) and DNA was extracted with a Qiagen DNeasy maxi kit. To assess DNA methylation status, the genomic DNA was treated overnight with methylation-sensitive restriction enzymes. Cleavage of selected sites was detected by PCR amplifications with primer pairs designed to bracket selected promoter regions. Loss of the PCR band after digestion with the enzymes indicated absence of methylations, whereas presence of the band indicated methylated cytosine. We selected MMP-9 as one of genes that is activated in OA. Transcription of mmp-9 is regulated by a 670 bp sequence at the 5′-end flanking region, which contains 6 CpGs and a further 21 CpGs within the 1.5 kb region further upstream. A PCR primer pair was designed to bracket a 350bp sequence upstream from the transcription start site of mmp-9, which contained four of the six potential methylation sites, cleaved by the methylation-sensitive enzymes AciI and HhaI. DNA from 9 OA patients, 5 #NOF patients and 1 rheumatoid arthritic (RA) patient were digested with HhaI or AciI and examined for the presence or absence of PCR bands. In all patients, digestion with HhaI abolished the PCR band, indicating that the HhaI site was never methylated in either #NOF or OA patients. However, a remarkable difference was found after digestion with AciI: in 8/9 OA patients, the PCR band was no longer detectable, while in 4/5 #NOF patients the PCR band was still present. This suggested that all three AciI cleavage sites were methylated in the majority of chondrocytes from #NOF patients, while at least one of the three AciI cleavage sites was unmethylated in OA patients. Interestingly, the PCR band was present in the RA patient, suggesting methylation of the AciI cleavage sites. The present study provides the first ‘proof-of-concept’ data that suggest epigenetic changes may play a role in the etiology of osteoarthritis. Clearly further work is required to establish the generality of the present findings and whether de-methylations are also found in the promoter regions of other genes that are aberrantly expressed in OA.

Introduction: Chondro-epiphyseal cartilage is generally resistant to vascular invasion. At the time of formation of the secondary ossification center in skeletal ‘long’ bones, the anti-angiogenic nature of cartilage is altered in favor of angiogenesis and vascular invasion takes place. We studied the control of this angiogenic ‘switch’ by experimentally investigating two factors which might influence vascular invasion. MMP 9 is a 92Kda gelatinase which degrades collagen types IV, V and X and gelatin (denatured collagen). It has been implicated in the control of endochondral ossification at the growth plate and has been shown to modulate endothelial cell morphogenesis. Basic Fibro-blast Growth Factor (b-FGF) is a cytokine with well established angiogenic capability and has also been implicated in the development of the growth plate. We investigated whether MMP-9 caused an effect on the development of the vasculature of the chondro-epiphysis of neo-natal rabbits and compared this to the effects of b-FGF.

Materials and Methods: The CAM Culture consists of placing a small tissue explant onto the the chorioallantoic membrane of 10 day-old chick embryos and continuing culture for a further 10 days. CAM derived vessels will invade the tissue, unless anti-angiogenic factors are present. Hence, CAM culture is used as an assay system for angiogenesis and factors that will influence it. We utilized the CAM culture model to investigate vascular in-growth into explants of femoral and humeral heads from 4 day old postnatal rabbits to test the influence of MMP-9 and b-FGF. A small nylon membrane, pre-soaked in a solution containing the factor, was placed on to a tangential cut across the perichondrium. The explant was then cultured on the CAM for 3–10 days.

Results: In control epiphyses, the in-growth of CAM derived blood vessels was rare and invasion of cartilage canals through the perichondrium seldom occurred, thus confirming the anti-angiogenic nature of epiphyseal cartilage. The initial presence of MMP 9 caused a tremendous increase in the de novo vascular invasion. MMP 9 treated epiphyses contained numerous large cartilage canals. In b-FGF treated epiphyses, a greater level of vascular in-growth was seen compared with controls, but this was not as marked as with MMP 9.

Our findings indicate that b-FGF and perhaps, more interestingly, MMP-9 are implicated in the activation of the angiogenic ‘switch’ at the chondroepiphysis leading to vascular invasion. The fact that MMP-9 can act as a stimulator to angiogenesis is a novel finding. The mechanism of action remains unclear although it is possible that it is involved in the deactivation of inhibitors of vasculogenesis or the activation of angiogenic factors, or both.

A 12 year old girl presented with a history of intermittent pain in her left knee since she started walking. She was seen in the vascular clinic due to engorged veins in her left leg and was diagnosis of Klippel-Trenaunay syndrome was made. Her knee pain worsened and an orthopaedic opinion was obtained. A history of repeated knee effusion and swelling was noted.

Examination revealed partial gigantism of the left leg and reduced range of motion of the knee. There was soft tissue swelling of the knee with no effusion. Blood investigations were normal. X-rays showed an arthritic joint. MRI scans revealed synovial thickening and a vascular malformation suggesting a synovial haemangioma. She underwent Radical Open Synovectomy and excision of the haemangioma. Blood loss was minimal. Extensive haemosiderin deposition was noted along with Grade IV arthritic changes. Postoperative recovery was uneventful. Surgeons have been reluctant to excise synovial hemangiomas due to the risk of haemorrhage.

A recent paper from Switzerland suggested excision was possible with minimal blood loss. Haemosiderin deposition due to recurrent haemarthrosis may predispose to articular damage. We recommend early excision of synovial haemangiomas to minimise articular damage.

The use of designer scaffolds to deliver biologically active osteogenic growth factors such as recombinant human bone morphogenetic protein-2 (rhBMP-2) to the sites of tissue regeneration in for example orthopaedics, has tremendous therapeutic implications. The aims of this study were to generate biomimetic biodegradable porous osteogenic scaffolds using a supercritical fluid process to encapsulate rhBMP-2, and to examine the ability of the scaffolds to promote human osteoprogenitor differentiation and bone formation in vitro and in vivo.

The rhBMP-2 encapsulated in Poly(-lactic acid) (PLA) scaffolds (100ng/mg PLA) were generated using an innovative supercritical fluid mixing method. The bioactivity of rhBMP-2 encapsulated PLA scaffolds were confirmed by induction of the C2C12 promyoblast cell line into the osteogenic lineage as detected by alkaline phosphatase expression. No induction of alkaline phosphatase-positive cells was observed using blank scaffolds. BMP-2 released from encapsulated constructs promoted adhesion, migration, expansion and differentiation of human osteoprogenitor cells on 3-D scaffolds. Enhanced matrix synthesis and cell differentiation on growth factor encapsulated scaffolds was observed following culture of human osteoprogenitors on explants of chick femoral bone wedge defects in an ex vivo model of bone formation developed using the chick chorioallantoic membrane model. In vivo studies using diffusion chamber implantation and subcutaneous implantation of human osteoprogenitors on rhBMP-2 encapsulated scaffolds showed morphologic evidence of new bone matrix and cartilage formation in athymic mice as assessed by x-ray analysis, immunocytochemistry and birefringence. These studies provide evidence of controlled release of BMP-2 from biodegradable polymer scaffolds initiating new bone formation in vivo.

The generation of 3-D biomimetic structures incorporating osteoinductive factors such as BMP-2 indicates their potential for de novo bone formation that exploits cell-matrix interactions and, significantly, realistic delivery protocols for growth factors in musculo-skeletal tissue engineering.

Introduction: To review the medium term outcome of staged surgery for treating recalcitrant idiopathic talipes equinovarus.

Methods: Between 1988 and 1995, we studied 91 club feet from a series of 120 recalcitrant feet in 86 patients requiring surgical treatment. The initial results have been reported previously and this cohort has been subsequently followed up for between 7 and 15years. The mean age at initial operation was 8.9 months. Surgery consisted of an initial plantar medial release followed two weeks later by a posterolateral release. This strategy was used specifically to address the problems of wound healing associated with single-stage surgery and to ascertain the rate of relapse after a two-stage procedure. The feet were classified preoperatively and prospectively into four grades according to the system suggested by Dimeglio et al. Reported relapse at last review was 0.0% in grade 2, 20.4% in grade 3 and 65.4% in grade 4 feet. The rate of overall relapse was 30.8%. At 7 to 15 year review an additional 9.1% in grade 2, 7.4% in grade 3, 11.5% in grade 4 had relapsed. Overall a further 8.8% had relapsed and were treated with further surgery. Functional outcome of the group remains good with 95.6% overall finding no restrictions to activities.

Conclusion: This review confirms that the strategy of staged surgery is supported in the medium term when considering rates of relapse and functional outcome.

Aims: There are no large published studies examining the complication rates associated with use of Kirschner wires in þxation of a wide variety of paediatric fractures. The aim of this study is to analyse the outcomes of fracture þxation using K-wire in upper limb fractures in children and to critically assess the incidence and type of complications. Methods: This study is a retrospective review of a consecutive series of 107 fractures in 105 paediatric trauma cases treated with K-wire in between 01.09.99 to 10.09.01. Results: The fractures were fractures around Wrist (47%) and around elbow (45%). 66 (61.68%) were performed by closed percutaneous technique, 27 (25.23%) by open method and in 14 (13.08%) combined approach was used. Around there were 13 cases with over-granulation at wound site, 6 cases of Soft tissue infection, 2 cases with tendinitis, 1 case of Osteo-myelitis and 1 case with hyper-sensitive scar. 3 cases found to have postoperative neurapraxia and 1 case with axonotmesis. Metal migration was detected in 4 cases and 14 cases found to have shown wire loosening. 10 fractures have lost position in postoperative period out of which 2 cases were reoperated for Re K-wire, 1 had undergone Re-MUA and 7 left for remodelling. Conclusions: K-wires are versatile but are not inherently benign. We conclude that best results could be achieved if total life of K-wire can be restricted to 3–4 weeks. We recommend one should explain all these risks and complications during consenting for K-wiring procedures.

Background: The necessity for radiographic follow up of infants with hip clicks and normal ultrasound is not clear.

Materials and methods: Infants referred to a paediatric hip clinic whose sole risk factor for DDH was a soft tissue hip click who had a normal ultrasound scan on initial assessment were identified. A follow up six month AP pelvis radiograph was assessed and acetabular index(A.I), position of femoral ossific nucleus and Shen-ton’s line measured. Infants with rotated pelvis Xrays were excluded. Inter-observer variability for acetabular index was measured and dysplasia defined according to Tonnis.

Results: 171 infants (193 clicking hips) met the criteria for inclusion. 48 male and 109 female with unilateral clicks (57 right, 64 left) and 36 bilateral clicks. 10 were excluded due to rotation of the AP pelvis Xray. Inter-observer error for A.I. was 4°. All A.I. were within normal ranges. Shenton’s line was unbroken and all hips were located.

Conclusion: In this study infants with soft tissue hip clicks and a normal ultrasound scan on initial assessment had a normal Xray at six months.

Clinical screening aims to identify and treat infants with neonatal hip instability in order to reduce the risk of subsequent hip displacement but risks failures of diagnosis and treatment (abduction splinting) and potential iatrogenic effects. The Hip Trial aims to assess the clinical effectiveness of ultrasound (US) imaging compared to clinical assessment alone to guide the further management of infants with clinical hip instability.

Infants with clinical hip instability confirmed by a second senior doctor were recruited from 33 UK centres and randomised to standardised US hip examination at age 2–8 weeks [US group: n=314] or clinical assessment alone [no ultrasound (NU) group: n=315. ] Primary outcomes by two years were hip X-ray appearances, operative treatment, abduction, splinting and walking. Analysis was ‘intention to treat’.

Key prognostic factors were similar between the randomised groups. Protocol compliance was high (90% US; 92% NU). X-ray information was available for 91% by 12–14 months and 85% by two years. Fewer children in the US group had abduction splinting in the first two years (RR 0. 78; 95% CI 0. 65–0. 94; p=0. 01). Operative treatment was required by 21 US (6. 7%) and 25 NU (7. 9%) infants (RR 0. 84; 95% CI 0. 48–1. 47. ) By two years, subluxation, dislocation, acetabular dysplasia or avascular necrosis were identified on X-ray on one or both hips of 21 US and 21 NU children (RR 1. 00; 95% CI 0. 56 – 1. 80. ) One US and 4 NU children were not walking by two years (RR 0. 25; exact 95% CI 0. 03–2. 53; p=0. 37)

The use of US imaging in infants with screen-detected clinical hip instability allows abduction splinting rates to be reduced, and is not associated with an increase in abnormal hip development or higher rates of operative treatment by two years of age.

Programmed cell death (PCD) contributes to the pathogenesis of many diseases including osteoarthritis. The principal method is apoptosis that has a well-defined and very characteristic morphology and biochemistry.

The aim of the present study was examine whether the mechanism of cell death in OA chondrocytes was classical apoptosis.

Rat thymocytes were used as controls since these cells are known to undergo classical apoptosis. Human OA cartilage was obtained from femoral head of patients (50 – 80 years) who were undergoing joint replacement surgery. Pieces of OA samples were processed into paraffin and sections incubated with the following antibodies: M3O, an antibody that recognizes the cleavage of cytokeratin 18 by caspases; annexin V, which recognizes phosphatidylserine “flip-flop” that occurs early in the apoptotic process; bcl-2, a protein whose presence protects apoptosis and c-myc, a transcription factor thought to be associated with apoptosis. To induce apoptosis, some samples were incubated with etoposide and staurosporine.

In sections of thymus we noticed the presence of numerous apoptotic bodies. The number increased when the tissue was treated with etoposide and staurosporine. Some thymocytes were immunopositive for M3O and annexin V, and the number of positive cells increased when treated for 2h with etoposide. Chondrocytes of the articular cartilage showed chromatin condensation and many vacuoles but no fragmentation into apoptotic bodies, even when treated with etoposide or staurosporine. The OA chondrocytes were immunonegative for M3O and annexin-V, even after incubation with etoposide and staurosporine. With respect to c-myc and bcl-2, both non-weight bearing and weight-bearing areas in OA sample showed more positive cells then the thymus. More chondrocytes stained for c-myc in the superficial zone of the articular cartilage in the non-weight bearing, while in the weight-bearing areas it was more in the intermediate zone. On the other hand, there were no differences in the distribution of the cells stained for bcl-2 in the articular cartilage. It is known that some events like the phosphatidylserine flip, caspase activation and apoptotic bodies fragmentation occur quickly during apoptosis, so may be difficult to detect.

The results suggest that some features of classical apoptosis, such as phosphatidylserine flip,caspase activation and apoptotic bodies formation did not take place in OA cartilage. It is known that the molecular machinery for apoptosis is not always present in tissues that are undergoing programmed cell death, which seems to be case for OA chondrocytes.

The formation of biomimetic environments using scaffolds containing cell recognition sequence and osteo-inductive factors in combination with bone cells offers tremendous potential for bone and cartilage regeneration. In tissues, collagen forms the scaffold by mediating the flux of chemical and mechanical stimuli. Recently, a synthetic 15-residue peptide P-15, related biologically to the active domain of type I collagen, has been found to promote attachment and the osteoblast phenotype of human dermal fibroblasts and periodontal ligament fibroblasts on particulate anorganic bone mineral (ABM). The aim of this study was to exam the ability of the collagen peptide, P-15, to promote human osteoprogenitor attachment, proliferation and differentiation on cell culture surfaces and 3-D scaffolds.

Selected human bone marrow cells were cultured on particulate microporous anorganic bone mineral (‘pure ‘ hydroxyapatite based on x-ray diffraction standard JCPDS9-432) phase and polygalactin vicryl mesh adsorbed with or without P-15 in basal or osteogenic conditions. Cell adhesion, spreading and patterning were examined by light and confocal microscopy following incorporation of cell tracker green and ethidium homodimer fluorescent labels. Osteoprogenitor proliferation and differentiation was assessed by DNA content and alkaline phosphatase specific activity. Growth and differentiation on 3-D ABM structures were examined by confocal and scanning electron microscopy (SEM).

P-15 promoted human osteoprogenitor cell attachment and patterning on particulate bovine anorganic bone mineral phase and polygalactin vicryl mesh over 5–24 hours compared to culture on ABM and vicryl mesh alone as observed by photomicroscopy. Increased alkaline phosphatase specific activity was enhanced following culture on P-15 adsorbed matrices as recognized by enhanced expression of alkaline phosphatase, type I collagen, osteocalcin and cfba-1. The presence of mineralised bone matrix and extensive cell ingrowth and cellular bridging between 3-D ABM matrices and polygalactin vicryl mesh adsorbed with P-15 was observed by confocal microscopy and alizarin red staining. SEM confirmed the 3-D structure of newly formed cell constructs and cellular ingrowth on and between the P-15 modified inorganic bone mineral materials. Negligible cell growth was observed on ABM alone or polygalactin vicryl mesh alone.

These observations demonstrate that the synthetic 15-residue collagen peptide, P-15, when adsorbed to ABM or polygalactin vicryl mesh, can stimulate human osteoprogenitor attachment and spreading. They also demonstrated that P-15 coupled 3-D matrices stimulate human osteoprogenitor differentiation and materialisation. The studies indicate that a synthetic analogue of collagen provides a biomimetic environment supportive for cell differentiation and tissue regeneration and indicate a potential for the use of extracellular matrix cue in the development of biomimetic environments for bone tissue engineering.

The growth plates of rapidly growing animals have been studied extensively. Nevertheless, several questions remain unanswered, partly because many events happen simultaneously, especially at the vascular front. Terminal chondrocytes are thought to undergo programmed cell death, but the fate of the cell remnants remains unclear. Are the dying cells released into the vascular space and phagocytosed by macrophages, as one would expect for apoptosis? Or are the cells eliminated prior to opening of the lacunae, leaving empty lacunae? Do all terminal chondrocytes die or do some become bone-forming cells? Rodents maintain a growth plate into old age, long after longitudinal growth has ceased. These stationary growth plates have several features not found in the growth plates of rapidly growing animals and closer study of these features may provide answers to the above questions. Femurs and tibiae from 4–16 week-old and 62–80 week-old rats were decalcified, processed into paraffin, and the morphological changes were documented.

Between 4–16 weeks, the heights of the growth plates decreased due to loss of the large hypertrophic chondrocytes, but the various zones were still present. In the aged rats, the growth plates were identifiable as a narrow cartilaginous band with some short columns of inactive cells. The vascular front was irregular, the narrow spicules of primary spongiosa were absent and the much thicker spicules, which are normally seen in secondary spongiosa, directly abutted to the cartilage. Horizontal apposition of bone matrix onto the cartilage edge was frequently present. In addition, the following features were noted. 1) Acellular areas: Nearly all growth plates contained regions of cartilage from which all cells and their lacunae had disappeared. In some cases, these acellular regions stretched from the reserve zone to the vascular front and even persisted as a relatively wide core within the spicules of spongiosa, indicating increased resistance of acellular cartilage to resorption. The absence of cells or cell debris was consistent with an autophagic mode of cell death and subsequent collapse of the lacunae. 2) Remodelling within the growth plate; in some growth plates, large regions of growth plate cartilage had been resorbed and new bone had been laid down in a pattern similar to the remodelling of cortical bone. This suggested that the normal resistance of cartilage to vascular invasion had been lost locally, but was maintained in adjacent non-remodelled regions. 3) Trans-differentiation of chondrocytes to bone-forming cells; extensive new medullary bone formation was noted in the diaphysis of approximately 30% of the aged rats, suggesting that they had received an (unknown) osteogenic stimulus. In these rats, bone matrix was identifiable inside chondrocytic lacunae, and spreading beyond the confines of the lacunae, thus directly replacing growth plate cartilage with bone matrix.

The results suggest that i) chondrocytes are capable of self-elimination, perhaps by a mechanism similar to the autophagic cell death that occurs during insect metamorphosis; ii) resorption of cartilage and vascular invasion requires the presence of the viable chondrocytes; and iii) chondrocytes have the capacity to transdifferentiate to bone-forming cells, but only do so when receiving an increased osteogenic stimulus.

Ex vivo gene transfer of osteogenic factors into multipotential stem cells offers potentially important therapeutic implications in a variety of musculoskeletal diseases. One possible approach is the development of a cellular vehicle, namely bone morphogenetic protein (BMP)-producing bone marrow cells, created using adenoviral gene transfer. These transduced cells provide local delivery of BMP for bone formation. The aims of this study were to study the feasibility of gene transfer to human bone osteoprogenitor cells, using adenoviral vectors. Specifically, the aims were to study the efficacy of transduction with an adenoviral vector expressing BMP-2 and then to determine the ability of the transduced cells to produce active BMP-2 and to generate bone ex vivo.

Primary human bone marrow osteoprogenitor cells were expanded in culture and infected with AxCALacZ, a replication-deficient adenoviral vector carrying the E. coli lacZ gene, with a range of multiplicity of infection (MOI) of 6.25 to100. Transduced cells showed positive staining for β-galactosidase using X-Gal with an efficiency close to 100%. Uninfected cells showed no β-galactosidase activity. Efficiency was independent from MOI, however cells infected at the lower MOIs expressed lower levels of β-galactosidase. Following confirmation that primary bone marrow cells could be infected by adenoviral constructs, additional osteoprogenitors were infected with AxCAOBMP-2, a vector carrying the human BMP-2 gene, at a multiplicity of infection of 10–20. In order to determine BMP-2 activity, conditioned media from bone marrow cells expressing BMP-2 was added to promyoblast C₂C₁₂ cells. The promyoblast C₂C₁₂ cells are exquisitely sensitive to BMP-2 with induction of alkaline phosphatase activity (ED₅₀ 20 nM) in a dose-dependant manner. Alkaline phosphatase activity was induced following culture with conditioned media from BMP-2 expressing cells, in a dose dependant manner, confirming successful secretion of active BMP-2. Immunohistochemical staining for alka- line phosphatase in C₂C₁₂ cells also confirmed the bio-chemical observations. Media from uninfected control human bone marrow cells failed to produce a similar effect. The concentration of BMP-2 in the media was estimated to be 5–10 nM/10⁷ cells.

To examine whether adenoviral transfection affected the osteoblast phenotype and their ability to mineralise in vitro, adenovirally-transduced bone marrow cells expressing BMP-2 were seeded onto poly(-lactic acid co÷glycolic acid) (75:25) porous scaffolds (provided by K. Shakesheff and S. Howdle; Nottingham University) and cultured for up to 6 weeks. Expression of alkaline phosphatase activity, type I collagen formation, as well as the synthesis of osteoblast stimulating factor-1 confirmed bone cell differentiation and maintenance of the osteoblast phenotype in extended culture for up to 6 weeks.

These results indicate the ability to deliver active BMP-2 using human bone marrow osteoprogenitor cells following adenoviral infection. The maintenance of osteoblast phenotype in extended culture and generation of mineralised 3-D scaffolds containing such constructs offers a realistic approach to tissue engineer bone for orthopaedic applications.

The ossific nucleus in Developmental Dysplasia of the Hip. A study of relative ossific nuceus size in hips treated in the Pavlik harness and its predictive value in treatment outcome.

Purpose

To assess the value of measuring relative ossific nucleus (ON) size difference in Developmental Dysplasia of the hip (DDH) as a potential predictor of outcome of hips treated in the Pavlik Harness.

Study Design

Prospective study of all unilateral cases (n=68) of DDH identified in Southampton by dynamic ultrasound and treated in a Pavlik harness studying changes in relative ON size and acetabular indices over a mean follow up period of 3.6 years.

Results

All cases responding to the Pavlik harness showed a progressive correction of ON size difference. Initial ON size difference was not associated with any difference in acetabular index at the date of last follow-up. Ultrasound grading of dysplasia did not affect the rate of normalisation of ON size difference.

Conclusion and clinical relevance

In patients responding to treatment of DDH in a Pavlik harness, ON size difference was not found to be a useful prognostic indicator of outcome.

From a cohort of 110 idiopathic clubfeet, 26 feet in 18 children requiring surgery for severe relapse have been studied. Surgery was comprised of a lateral column shortening procedure (Lichtblau) plus or minus a plantarmedial release. Surgery was staged to avoid wound complications.

Pre-operatively, feet were prospectively categorised into one of four grades according to a system reported by Dimeglio. Children were reviewed on two subsequent occasions. At review, feet were again graded. In addition, appearance and functional outcome was analysed and included an assessment of gait, activity and functional limitation.

Three children were lost to follow-up, leaving 22 feet in seven male and eight female patients available for review. The mean age at surgery was 43 months (23–82). The mean time from surgery to first and second reviews was 35 and 56 months, respectively.

There was a significant improvement in grading at first review compared to pre-operative grading (Wilcoxon signed ranks test). Although there remained a significant improvement in grading at second review compared to the preoperative grading, there was a significant reduction in the number of feet in which grading had improved when compared to first review.

There was no significant change in function between the two post-operative reviews (Chi-square tests), with the majority of children experiencing little functional limitation. There were no wound complications.

Relapse surgery, involving a lateral column shortening procedure for severe clubfoot, results in a significant initial improvement when assessed using a grading system. This improvement in grading subsequently decreases over time. However, the functional outcome in such cases remains favourable.

We report 22 patients (19 women and three men) of mean age 20.8 years who had painful snapping sensations in the groin. Most were able to reproduce the click by extending the affected hip from a flexed, abducted and externally rotated position and most were tender in the adductor triangle. Plain radiographs and an arthrogram were normal. A clinical diagnosis of subluxation of the iliopsoas tendon was made. Conservative management failed in 14 patients, two of whom had bilateral pain. All 14 had surgical release of the iliopsoas tendon through a medial approach. At follow-up (mean 17 months) the click had resolved in ten hips, was occasional but painless in five and unchanged in one. The syndrome of a painful 'snapping' psoas may result in disproportionately disabling symptoms. It may be diagnosed on clinical grounds and effectively treated, when severe, by release of the iliopsoas tendon.

Between 1989 and 1992 we admitted 426 children with an irritable hip, 363 (85.2%) once and 63 (14.8%) on 143 occasions. We assessed the records retrospectively to determine whether the groups differed and in particular whether recurrence was followed by pathological sequelae. We identified no feature which distinguished between them at either presentation. The use of bone isotope scans was greatly increased in recurrent cases, without clinical benefit. No relationship between recurrence and subsequent abnormality was identified, with 22 (42%) of the recurrences taking place in the opposite hip. The only difference was a higher incidence of 'psychosocial factors' recorded in the notes of children who presented on more than two occasions. The incidence of recurrent irritable hip is larger than previously indicated and in the presence of normal radiographs and low-grade clinical signs, more detailed investigation on subsequent admission is unlikely to be helpful.

We report the preliminary results of a continuing prospective evaluation of a screening programme for congenital dislocation of the hip (CDH) which uses ultrasound imaging to provide delayed selective screening to complement neonatal clinical screening. Of 26,952 births in the Southampton district, 1894 infants were referred for secondary screening because of a clinical abnormality or the presence of a predetermined risk category for CDH. Pavlik harness treatment was required for only 118 infants, giving a treatment rate of 4.4 per 1000 births. Of those referred with clinical instability, 35% did not require treatment. Dislocation or subluxation was detected in 17 of 643 infants referred only because they fell within one of three risk categories: breech presentation, foot deformity and family history. All 17 had normal clinical examinations and cases were discovered in each category. Six children presented with CDH after 12 weeks of age, giving a late presentation rate of 0.22 per 1000 births. All had normal clinical examinations within 24 hours of birth and none was in a risk category. Surgery has been required in ten children, giving a surgical treatment rate of 0.37 per 1000 births. We conclude that, in Southampton, delayed selective secondary screening with ultrasound is more effective than clinical screening alone. It targets treatment to those infants who need it, and reveals a number of dislocated and subluxed hips that would otherwise be missed.

Of 4,617 babies born in Coventry in 1986, a total of 448 (9.7%) had either clinical abnormality of the hip or at risk factors for CDH. All were examined by ultrasound, but only 17 required treatment (3.7 per 1,000); in five of these no clinical abnormality had been detected. An additional 81 babies had ultrasound abnormalities but did not require treatment, despite the fact that ultrasound at first showed major hip displacement in 17 of them. Three late cases of CDH have presented among the babies born in 1986, but not examined by ultrasound. This incidence of late CDH is unchanged compared with the previous nine years, although ultrasound had detected covert displacement in a number of hips.

Evidence is presented to support the contention that after slipping of the upper femoral epiphysis there is a potential for the bony epiphysis to grow back to its pre-slipped position. A suggestion is made as to how this recovery may occur.

A technique of examining the infant hip joint with real-time ultrasound is described. Since the cartilaginous femoral head is clearly imaged by ultrasound, anatomical structures and their relationships can be accurately determined. Dislocated hips are easily detected and subluxations also can be visualized. We report our experience with 131 examinations in 104 patients, comprising 259 single hip studies. Of 83 patients who were previously untreated, there were 178 hip studies with three false-negative and four false-positive ultrasound results. No dislocations were missed. Twenty-seven patients who were already being treated were examined to assess hip location, comprising a total of 81 hip studies. In some cases the patients were examined while in an abduction device, cast, or Pavlik harness. In one case a dislocation was not detected. The method of examination using real-time ultrasound is considered to be reliable, accurate, and a useful adjunct to radiography. The advantages are that it is non-invasive, portable, and involves no exposure to radiation.

Certain features of the sagging rope sign recently analysed by Apley and Weintroub (1981) are examined in detail. Evidence is presented to show that the line is a radiological shadow cast by the lateral edge of a severely deformed femoral head rather than a condensation of the spongiosa within the neck. An explanation is offered to explain the common association of the presence of this radiological sign with premature epiphysial fusion.