International consensus guideline for reporting transmission electron microscopy results in the diagnosis of primary ciliary dyskinesia (BEAT PCD TEM Criteria).

Primary ciliary dyskinesia (PCD) is a heterogeneous genetic condition. European and North American diagnostic guidelines recommend transmission electron microscopy (TEM) as one of a combination of tests to confirm a diagnosis. However, there is no definition of what constitutes a defect or consensus on reporting terminology. The aim of this project was to provide an internationally agreed ultrastructural classification for PCD diagnosis by TEM.A consensus guideline was developed by PCD electron microscopy experts representing 18 centres in 14 countries. An initial meeting and discussion were followed by a Delphi consensus process. The agreed guideline was then tested, modified and retested through exchange of samples and electron micrographs between the 18 diagnostic centres.The final guideline a) provides agreed terminology and a definition of Class 1 defects which are diagnostic for PCD; b) identifies Class 2 defects which can indicate a diagnosis of PCD in combination with other supporting evidence; c) describes features which should be included in a ciliary ultrastructure report to assist multidisciplinary diagnosis of PCD; and d) defines adequacy of a diagnostic sample.This tested and externally validated statement provides a clear guideline for the diagnosis of PCD by TEM which can be used to standardise diagnosis internationally.

Effects of marine n-3 fatty acid supplementation in renal transplantation: A randomized controlled trial.

Marine n-3 fatty acids (FAs) may exert beneficial effects on inflammation, fibrosis, and endothelial function, which could preserve renal graft function. In this randomized controlled trial, 132 Norwegian renal transplant recipients received either 2.6 g of marine n-3 FAs or olive oil (control) daily for 44 weeks, in addition to standard care. Thirty patients did not complete the trial. The primary endpoint was change (Δ) in measured glomerular filtration rate (mGFR) during follow-up. We found no significant difference in Δ mGFR between the marine n-3 FA group and controls (6.7 vs 3.8 mL/min per 1.73 m2 , P = .15). Significant beneficial effects from marine n-3 FA supplementation were, however, seen in secondary endpoints plasma triglycerides, plasma high-sensitivity C-reactive protein, and brachial artery flow-mediated dilation. In the per-protocol population, the renal graft indices percent interstitial fibrosis and Chronic Allograft Damage Index also were significantly lower in the marine n-3 FA group. The cumulative incidence of adverse events did not differ between the marine n-3 FA group (n = 218) and controls (n = 240). In conclusion, marine FA supplementation did not improve renal function compared with controls, but was safe, lowered plasma triglyceride and high-sensitivity C-reactive protein levels, and improved endothelial function (Clinical.Trials.gov identifier NCT01744067).

Kidney allograft subclinical rejection modulates systemic inflammation measured by C-reactive protein at 1 year after transplantation.

Kidney allograft inflammation is associated with proinflammatory modifications of peripheral blood mononuclear cells, suggesting that renal inflammation contributes to systemic inflammation. Thus, the aim of this study was to evaluate the relationship between subclinical inflammation in surveillance biopsies performed at 1 year and systemic inflammation assessed by C-reactive protein (CRP) levels at the time of biopsy. We analyzed 544 surveillance biopsies performed at 1 year that were classified as normal (n = 368), borderline (n = 148), or subclinical rejection (SCR) (n = 28). CRP levels were divided into quartiles. Patients in 1st, 2nd, and 3rd quartile were classified as low CRP (n = 408) and patients in the 4th quartile as high CRP (n = 136). Univariate analysis showed that the proportion of patients with SCR was higher in the high CRP group (10.3% vs 3.4%, P = 0.0067). Multivariate analysis showed that independent predictors of high CRP were body mass index (odds ratio [OR] 1.072 and 95% confidence interval [CI] 1.027-1.119), a positive urine culture at the day of the biopsy (OR 2.760 and 95% CI 1.205-6.323), and the presence of SCR at 1-year surveillance biopsy (OR 7.260 and 95% CI 3.530-14.935). In summary, we describe that subclinical acute rejection constitutes an independent predictor of systemic inflammation as measured by CRP.

Development of Kidney Transplant Fibrosis Is Inversely Associated With Plasma Marine Fatty Acid Level.

OBJECTIVE(S): We assessed associations between plasma levels of polyunsaturated fatty acids (PUFAs) and degree of inflammation and interstitial fibrosis in transplanted kidneys. DESIGN: The design of the study was single center cohort study. SUBJECTS: A study population of 156 patients who received a kidney transplant at Oslo University Hospital during 2010. MAIN OUTCOME MEASURE: Kidney transplant biopsies were obtained at 2 months and 1 year after transplantation. Degree of inflammation and interstitial fibrosis in the cortex of transplanted kidneys were estimated semi-quantitatively. Plasma phospholipid fatty acids levels were measured in a stable phase 2 months posttransplant. We used multivariate linear regression to assess associations between plasma levels of PUFAs and degree of inflammation and interstitial fibrosis at 2 months and 1 year postoperatively and change in degree of interstitial fibrosis during the first year after transplantation, adjusting for inflammation and fibrosis risk factors. RESULTS: Higher plasma marine n-3 PUFA levels were associated with less development of interstitial fibrosis in the kidney transplant (unstandardized ß-coefficient -1.12, standardized ß-coefficient -0.18, P = .03) during the first year after transplantation. Plasma levels of alpha linoleic acid, linoleic acid, and arachidonic acid were not associated with development of interstitial fibrosis. No associations were found between plasma levels of PUFAs and inflammation inside fibrotic areas or outside fibrotic areas in the kidney transplant at neither 2 months nor 1 year postoperatively. Linolenic acid levels in plasma were positively associated with change in renal function during the first year after transplantation. CONCLUSION: The inverse association between plasma marine n-3 PUFA levels and development of interstitial fibrosis during the first year after kidney transplantation suggests that marine fatty acid consumption might halt progression of fibrosis.

Negative effect of zoledronic acid on tendon-to-bone healing.

Background and purpose - Outcome after ligament reconstruction or tendon repair depends on secure tendon-to-bone healing. Increased osteoclastic activity resulting in local bone loss may contribute to delayed healing of the tendon-bone interface. The objective of this study was to evaluate the effect of the bisphosphonate zoledronic acid (ZA) on tendon-to-bone healing. Methods - Wistar rats (n = 92) had their right Achilles tendon cut proximally, pulled through a bone tunnel in the distal tibia and sutured anteriorly. After 1 week animals were randomized to receive a single dose of ZA (0.1 mg/kg IV) or control. Healing was evaluated at 3 and 6 weeks by mechanical testing, dual-energy X-ray absorptiometry and histology including immunohistochemical staining of osteoclasts. Results - ZA treatment resulted in 19% (95% CI 5-33%) lower pullout strength and 43% (95% CI 14-72%) lower stiffness of the tendon-bone interface, compared with control (2-way ANOVA; p = 0.009, p = 0.007). Administration of ZA did not affect bone mineral density (BMD) or bone mineral content (BMC). Histological analyses did not reveal differences in callus formation or osteoclasts between the study groups. Interpretation - ZA reduced pullout strength and stiffness of the tendon-bone interface. The study does not provide support for ZA as adjuvant treatment in tendon-to-bone healing.

Tacrolimus and mycophenolate regimen and subclinical tubulo-interstitial inflammation in low immunological risk renal transplants.

The aim was to evaluate the relationship between maintenance immunosuppression, subclinical tubulo-interstitial inflammation and interstitial fibrosis/tubular atrophy (IF/TA) in surveillance biopsies performed in low immunological risk renal transplants at two transplant centers. The Barcelona cohort consisted of 109 early and 66 late biopsies in patients receiving high tacrolimus (TAC-C0 target at 1-year 6-10 ng/ml) and reduced MMF dose (500 mg bid at 1-year). The Oslo cohort consisted of 262 early and 237 late biopsies performed in patients treated with low TAC-C0 (target 3-7 ng/ml) and standard MMF dose (750 mg bid). Subclinical inflammation, adjusted for confounders, was associated with low TAC-C0 in the early (OR: 0.75, 95% CI: 0.61-0.92; P = 0.006) and late biopsies (OR: 0.69, 95% CI: 0.50-0.95; P = 0.023) from Barcelona. In the Oslo cohort, it was associated with low MMF in early biopsies (OR: 0.90, 95% CI: 0.83-0.98; P = 0.0101) and with low TAC-C0 in late biopsies (OR: 0.77, 95% CI: 0.61-0.97; P = 0.0286). MMF dose was significantly reduced in Oslo between early and late biopsies. IF/TA was not associated with TAC-C0 or MMF dose in the multivariate analysis. Our data suggest that in TAC- and MMF-based regimens, TAC-C0 levels are associated with subclinical inflammation in patients receiving reduced MMF dose.

Autologous cell sources in therapeutic vasculogenesis: In vitro and in vivo comparison of endothelial colony-forming cells from peripheral blood and endothelial cells isolated from adipose tissue.

BACKGROUND AIMS: Autologous endothelial cells are promising alternative angiogenic cell sources in trials of therapeutic vasculogenesis, in the treatment of vascular diseases and in the field of tissue engineering. A population of endothelial cells (ECs) with long-term proliferative capability, endothelial colony-forming cells (ECFCs), can be isolated from human peripheral blood. ECFCs are considered an endothelial precursor population. They can be expanded in cell factories in sufficient numbers for clinical applications, but because the number of isolated primary ECs is low, the culture period required may be long. Another EC population that is easily available in the autologous setting and may be expanded in vitro through several population doublings are ECs from adipose tissue (AT-ECs). METHODS: Through extensive comparisons using whole-genome microarray analysis, morphology, phenotype and functional assays, we wanted to evaluate the potential of these EC populations for use in clinical neovascularization. RESULTS: Global gene expression profiling of ECFCs, AT-ECs and the classical EC population, human umbilical vein ECs, showed that the EC populations clustered as unique populations, but very close to each other. By cell surface phenotype and vasculogenic potential in vitro and in vivo, we also found the ECFCs to be extremely similar to AT-ECs. CONCLUSIONS: These properties, together with easy access in the autologous setting, suggest that both AT-ECs and ECFCs may be useful in trials of therapeutic neovascularization. However, AT-ECs may be a more practical alternative for obtaining large quantities of autologous ECs.

Total inflammation in early protocol kidney graft biopsies does not predict progression of fibrosis at one year post-transplant.

INTRODUCTION: There is an uncertainty whether total inflammation in early protocol kidney graft biopsies is associated with fibrosis progression. We investigated whether total inflammation, both in fibrotic and non-fibrotic areas, at week 6 would predict fibrosis progression at one yr post-transplant. METHODS: We included 156 single adult ABO compatible kidney recipients with adequate week 6 and one yr transplant protocol biopsies (312 biopsies). Biopsies were scored according to the current Banff criteria. In addition, fibrosis and inflammation in fibrotic and non-fibrotic areas were scored in a 10-grade semi-quantitative eyeballing system from 0% to 100%. RESULTS: Fibrosis increased significantly from week 6 to one yr both by the 10-grade scoring system from 0.69 ± 1.07 to 1.45 ± 1.86, (mean ± SD), p < 0.001 and by Banff interstitial fibrosis (ci) scoring 0.81 ± 0.65 to 1.13 ± 0.87, p < 0.001. The 10-grade scoring system detected a larger proportion of fibrosis progressors than the Banff scoring 40.4% vs. 35.5%, p < 0.001. No significant positive association was found between inflammation at week 6 and progression of fibrosis in either of the scoring systems. CONCLUSIONS: Total inflammation in kidney transplant biopsies at week 6 did not predict progression of fibrosis at one yr post-transplant.

In patients with type 1 diabetes simultaneous pancreas and kidney transplantation preserves long-term kidney graft ultrastructure and function better than transplantation of kidney alone.

AIMS/HYPOTHESIS: In patients with type 1 diabetes and end-stage renal disease (ESRD) we aimed to determine whether long-term normoglycaemia, as achieved by successful simultaneous pancreas and kidney (SPK) transplantation, would preserve kidney graft structure and function better than live donor kidney (LDK) transplantation alone. METHODS: Estimated GFR (eGFR) was calculated in SPK (n = 25) and LDK (n = 17) recipients in a stable phase 3 months after transplantation and annually during follow-up. Kidney graft biopsies were obtained at follow-up for measurement of glomerular volume (light microscopy), glomerular basement membrane (GBM) and podocyte foot process widths and mesangial volume fraction (electron microscopy). RESULTS: SPK and LDK recipients were similar in age and diabetes duration at engraftment. Donor age was higher in the LDK group. Median follow-up time was 10.1 years. Mean HbA1c levels during follow-up were 5.5 ± 0.4% (37 ± 5 mmol/mol) and 8.3 ± 1.5% (68 ± 16 mmol/mol) in the SPK and LDK group, respectively (p < 0.001). Compared with SPK recipients, LDK recipients had wider GBM (369 ± 109 nm vs 281 ± 57 nm; p = 0.008) and increased mesangial volume fraction (median 0.23 [range 0.13-0.59] vs 0.16 [0.10-0.41]; p = 0.007) at follow-up. Absolute eGFR change from baseline was -11 ± 21 and -23 ± 15 ml min(-1) 1.73 m(-2) (p = 0.060), whereas eGFR slope was -1.1 (95% CI -1.7, -0.5) and -2.6 (95% CI -3.1, -2.1) ml min(-1) 1.73 m(-2) per year in the SPK and LDK group, respectively (p = 0.001). CONCLUSIONS/INTERPRETATION: In patients with type 1 diabetes and long-term normoglycaemia after successful SPK transplantation, kidney graft ultrastructure and function were better preserved compared with LDK transplantation alone.

Increased tartrate-resistant Acid phosphatase expression in osteoblasts and osteocytes in experimental osteoporosis in rats.

Tartrate-resistant acid phosphatase (TRAP) is known as an osteoclast marker, but osteoblasts and osteocytes in the vicinity of bone remodeling sites also express TRAP. Cell culture studies suggest that osteoblasts endocytose osteoclastic TRAP for inactivation. To evaluate whether changes in osteoclast activity could alter TRAP expression in osteoblasts and/or osteocytes in vivo, we studied the ovariectomized and vitamin D-deficient rat (Ovx-D) and rats healing from rickets. Bone sections were analyzed for TRAP gene expression by in situ hybridization, TRAP protein by immunogold labeling, and TRAP enzyme activity using the fluorescent substrate ELF97. Osteoblasts and osteocytes close to intracortical remodeling sites and bone surfaces demonstrated TRAP, most prominently in cancellous bone and osteocytes. Intracellular TRAP was located to electron-dense vesicles with similar morphology in both cell types. Ovx-D increased osteoclast activity (p < 0.001) and ELF97⁺ osteocytes (p < 0.05) in cancellous bone, but no corresponding increase was observed in the osteocyte lacunar area. The level of TRAP⁺ vesicles in cortical osteoblasts (p < 0.01) in Ovx-D rats was also increased. Enhanced osteoclast activity was noted in healing rickets after 72 h (p < 0.05), but no differences in TRAP expression were detected in osteoblasts or osteocytes. Thus, increased osteoclast activity does not affect TRAP expression in osteoblasts and osteocytes, favoring the notion that increased TRAP in these cells is rather due to increased synthesis. Although the role of TRAP in osteoblasts and osteocytes remains elusive, we speculate that the function is related to the capability of the enzyme to regulate the phosphorylation of proteins known to be expressed by these cells.

Relationship between CTX-II and patient characteristics, patient-reported outcome, muscle strength, and rehabilitation in patients with a focal cartilage lesion of the knee: a prospective exploratory cohort study of 48 patients.

BACKGROUND: C-telopeptide fragments of type II collagen (CTX-II) are created during articular cartilage breakdown and CTX-II is considered useful as a biomarker of osteoarthritis. The primary objective of the present study was to explore the relationship between urinary CTX-II concentration and patient characteristics, patient-reported outcome, muscle strength, and rehabilitation in patients with isolated focal knee cartilage lesions. Furthermore, the secondary objective was to examine differences in urinary CTX-II concentration between patients with focal cartilage lesions and healthy controls. METHODS: 48 patients (mean age 33.4 years, standard deviation 9.0) with a focal full-thickness (International Cartilage Repair Society grade 3 or 4) cartilage lesion on the medial or lateral femoral condyle were included. After baseline assessments, the patients completed a 3-month rehabilitation program and 44 patients attended the 3 month follow-up. Baseline and follow-up assessments consisted of urinary CTX-II, the Knee Injury and Osteoarthritis Outcome Score (KOOS), and isokinetic quadriceps and hamstring muscle strength measurements. CTX-II was also analysed in urine samples from 6 healthy individuals, serving as normal controls. Correlations were classified as very weak (correlation coefficient [r] < 0.20), weak (r = 0.20 - 0.39), moderate (r = 0.40 - 0.59), strong (r = 0.60 - 0.79), and very strong (r > 0.80). RESULTS: Except for age and quadriceps strength, no significant correlations were found between CTX-II concentrations and baseline characteristics, KOOS, or muscle strength. Except for age, all correlations were considered as weak or very weak. The patients with a focal cartilage lesion had significantly higher mean CTX-II concentration than the healthy control individuals both at baseline (p = 0.001) and at follow-up (p = 0.001). The mean CTX-II concentration tended to decrease during rehabilitation, but the reduction was not significant (p = 0.076). CONCLUSIONS: The current exploratory study demonstrated that patients with a focal cartilage lesion of the knee had higher concentrations of urinary CTX-II than healthy individuals. In addition, CTX-II concentration tended to decrease during rehabilitation. TRIAL REGISTRATION: ClinicalTrials.gov NCT00885729.

Quantitative proteomic analysis of eight cartilaginous tissues reveals characteristic differences as well as similarities between subgroups.

Human synovial joints display a characteristic anatomic distribution of arthritis, e.g. rheumatoid arthritis primarily affects the metacarpophalangeal and proximal finger joints, but rarely the distal finger joints, whereas osteoarthritis occurs in the distal and proximal finger joints. Pelvospondylitis has a selective localization to the spine and sacroiliac joints. Is this tropism due to differences between the cartilages at the molecular level? To substantiate this concept the present study provides a background detailed compositional analysis by relative quantification of extracellular matrix proteins in articular cartilages, meniscus, intervertebral disc, rib, and tracheal cartilages on samples from 5-6 different individuals using an optimized approach for proteomics. Tissue extraction followed by trypsin digestion and two-dimensional LC separations coupled to tandem mass spectrometry, relative quantification with isobaric labeling, iTRAQ(TM), was used to compare the relative abundance of about 150 proteins. There were clear differences in protein patterns between different kinds of cartilages. Matrilin-1 and epiphycan were specific for rib and trachea, whereas asporin was particularly abundant in the meniscus. Interestingly, lubricin was prominent in the intervertebral disc, especially in the nucleus pulposus. Fibromodulin and lumican showed distributions that were mirror images of one other. Analyses of the insoluble residues from guanidine extraction revealed that a fraction of several proteins remained unextracted, e.g. asporin, CILP, and COMP, indicating cross-linking. Distinct differences in protein patterns may relate to different tissue mechanical properties, and to the intriguing tropism in different patterns of joint pathology.

Cartilage repair in the rabbit knee: mosaic plasty resulted in higher degree of tissue filling but affected subchondral bone more than microfracture technique: a blinded, randomized, controlled, long-term follow-up trial in 88 knees.

PURPOSE: Discrepancies and variances in outcome following different surgical techniques for cartilage repair are poorly understood. Successful repair relies on proper tissue filling without initiating degenerative processes in the cartilage-bone unit. Consequently, the objective of the current study was to compare two available techniques for cartilage repair, i.e., microfracture technique and mosaic plasty, regarding tissue filling and subchondral bone changes in an experimental model. METHODS: A 4-mm pure chondral defect was created in the medial femoral condyle of both knees in New Zealand rabbits, aged 22 weeks. A stereomicroscope was used to optimize the preparation of the defects. In one knee (randomized), the defect was treated with microfracture technique whereas in the other with mosaic plasty. The animals were killed at 12, 24 and 36 weeks after surgery. Defect filling, new bone formation above the level of the tidemark and the density of subchondral mineralized tissue were estimated by histomorphometry. RESULTS: Mosaic plasty resulted in a significantly 34% higher degree of tissue filling than microfracture technique at 36 weeks, SD of mean difference being 34%. Mosaic plasty resulted in significantly more new bone formation and reduced subchondral mineralized tissue density compared to microfracture technique. The differences between the two techniques were apparent mainly at the long-term follow-up. CONCLUSION: Tissue filling is a limiting factor regarding microfracture technique when compared to mosaic plasty, whereas mosaic plasty resulted in more bone changes than microfracture technique-the implications of the latter remain to be settled. This study underlines the difficulty in predicting outcome in the single case with any of these two techniques, particularly in a long-term perspective.

Chronic Kidney Disease from Polyvinylpyrrolidone Deposition in Persons with Intravenous Drug Use.

BACKGROUND AND OBJECTIVES: Persons with intravenous drug use have a higher risk of developing CKD compared with the general population. In Norway, deposits of polyvinylpyrrolidone have been observed in kidney biopsies taken from persons with opioid addiction and intravenous drug use since 2009. Polyvinylpyrrolidone is an excipient commonly used in pharmaceuticals, and the polyvinylpyrrolidone deposits observed in these patients were caused by intravenous injection of a specific oral methadone syrup containing very high molecular weight polyvinylpyrrolidone. Here, we present the clinicopathologic findings from 28 patients with CKD associated with polyvinylpyrrolidone deposition in the kidney. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The 28 patients and their kidney biopsies were included when polyvinylpyrrolidone deposition was recognized, either retrospectively or at the time of diagnostic evaluation. Biopsies were taken between 2009 and 2016. We collected laboratory parameters and clinical data from digital patient charts. For each kidney biopsy, the glomerular volume, extent of polyvinylpyrrolidone deposition, and tubulointerstitial area with tubular atrophy were assessed quantitatively. RESULTS: All patients (mean age: 37 years) had CKD (mean eGFR: 33 ml/min per 1.73 m2) and normal urine protein or non-nephrotic-range proteinuria. Biopsies showed moderate to severe tubular atrophy (mean extent: 65%) and interstitial infiltrates of vacuolated macrophages containing polyvinylpyrrolidone (mean share of biopsy area: 1.5%). Underperfused and ischemic glomeruli were common findings. In 22 samples, ultrastructural investigation revealed polyvinylpyrrolidone-containing vacuoles in the mesangial or endothelial cells of glomeruli. At the last follow-up, most patients had stable or improved eGFR. Two patients had developed kidney failure and underwent hemodialysis. CONCLUSIONS: Intravenous injection of a specific oral methadone syrup caused polyvinylpyrrolidone deposition in the kidney in persons with opioid addiction and intravenous drug use. Kidney biopsy findings suggested an association between polyvinylpyrrolidone deposition and tubular atrophy.

Healing of long-term frozen orthotopic bone allografts is not affected by MHC differences between donor and recipient.

BACKGROUND: The use of bone grafting in orthopaedic surgery has increased dramatically in recent years. However, the degree to which immune responses are important for the survival of the allograft is not fully understood. In particular it remains unclear whether differences in the major histocompatibility complex (MHC) influence incorporation of bone allografts and their subsequent biologic performance. QUESTIONS/PURPOSES: Therefore, we asked whether isolated mismatch for MHC antigens of deep frozen bone allografts in the long-term causes (1) immune reactions, and whether these reactions have any effect on (2) morphologic features of the graft, (3) radiographic graft healing, and (4) graft strength. METHODS: We used an established orthotopic tibial segment transplantation technique that allows determination of mechanical strength, histologic evaluation, and immune responses. Tibial segments that had been deep-frozen at -80°C for 1 year were transplanted into 24 PVG (RT1 (c)) rats from either 12 syngeneic donors or 12 MHC congenic donors PVG.1U (RT1 (u)). We determined immune responses using an indirect Coombs reaction and determined graft healing radiographically and mechanically after 6 months. RESULTS: We detected no alloantibody production to graft MHC-I antigens, and found no differences between syngeneic and MHC mismatched grafts in terms of remodeling with host bone, graft healing, and mechanical strength. CONCLUSIONS: Mismatches for MHC antigens do not seem to play a decisive role in healing of long-term, deep-frozen bone allografts.

Chondrogenesis in a hyaluronic acid scaffold: comparison between chondrocytes and MSC from bone marrow and adipose tissue.

Treatment of focal lesions of the articular cartilage of the knee using chondrocytes in a hyaluronic acid (HA) scaffold is already being investigated in clinical trials. An alternative may be to use mesenchymal stem cells (MSC). We have compared articular chondrocytes with MSC from human bone marrow (BM) and adipose tissue (AT), all cultured in HA scaffolds, for their ability to express genes and synthesize proteins associated with chondrogenesis. The cells were expanded in monolayer cultures. After seeding into the scaffold, the chondrocytes were maintained in medium, while the two MSC populations were given a chondrogenic differentiation medium. Chondrogenesis was assessed by real-time RT-PCR for chondrocyte-associated genes, by immunohistochemistry and by ELISA for collagens in the supernatant. Redifferentiation of the dedifferentiated chondrocytes in the HA scaffold was shown by a modest increase in type II collagen mRNA (COL2A1) and reduction in COL1A1. BM-MSC expressed 600-fold higher levels of COL2A1 than chondrocytes after 3 weeks in the scaffold. The levels of aggrecan (AGC1) and COL1A1 were similar for chondrocyte and BM-MSC scaffold cultures, while COL10A1 was higher in the BM-MSC. AT-MSC expressed levels of COL2A1 and COL1A1 similar to chondrocytes, but less AGC1 and COL10A1. Surprisingly, little collagen II protein was observed in the scaffold. Instead, collagen II was found in the culture medium. Chondrogenesis in HA scaffolds was more efficient using BM-MSC than AT-MSC or chondrocytes. Some of the secreted collagen II escaped entrapment in the extracellular space and was detected in the culture medium.

Intraarticular location predicts cartilage filling and subchondral bone changes in a chondral defect.

BACKGROUND AND PURPOSE: The natural history of, and predictive factors for outcome of cartilage restoration in chondral defects are poorly understood. We investigated the natural history of cartilage filling subchondral bone changes, comparing defects at two locations in the rabbit knee. ANIMALS AND METHODS: In New Zealand rabbits aged 22 weeks, a 4-mm pure chondral defect (ICRS grade 3b) was created in the patella of one knee and in the medial femoral condyle of the other. A stereo microscope was used to optimize the preparation of the defects. The animals were killed 12, 24, and 36 weeks after surgery. Defect filling and the density of subchondral mineralized tissue was estimated using Analysis Pro software on micrographed histological sections. RESULTS: The mean filling of the patellar defects was more than twice that of the medial femoral condylar defects at 24 and 36 weeks of follow-up. There was a statistically significant increase in filling from 24 to 36 weeks after surgery at both locations. The density of subchondral mineralized tissue beneath the defects subsided with time in the patellas, in contrast to the density in the medial femoral condyles, which remained unchanged. INTERPRETATION: The intraarticular location is a predictive factor for spontaneous filling and subchondral bone changes of chondral defects corresponding to ICRS grade 3b. Disregarding location, the spontaneous filling increased with long-term follow-up. This should be considered when evaluating aspects of cartilage restoration.

[Osteoarthritis]. / Artrose.

BACKGROUND: Osteoarthritis is among the most common causes of functional disability and severe pain, and the prevalence of arthritic symptoms among adults is more than 50%. The article discusses epidemiology, pathology and treatment options. MATERIAL AND METHODS: The review is based on a non-systematic search in PubMed and the authors' experience with treating this patient group. RESULTS: Osteoarthritis is a degenerative disease which leads to loss of joint functioning. Symptoms usually present in the hip, hands and knees. Women are affected more often than men and the prevalence increases with increasing age. Some families have an increased prevalence of osteoarthritis, but the genetic etiology is not clear. Mechanic conditions such as overweight and heavy physical work explain some of the pathogenesis, but non-mechanical factors are probably involved as well. Loss of weight is likely to have a preventive effect, and surgical correction of mechanic conditions such as hip dysplasia and varus deformity can prevent development of osteoarthritis. Treatment of symptomatic osteoarthritis includes educating the patient and continues with stretching, physical exercise, weight reduction, technical aids (supporting braces, walking sticks) and analgesics. Subsequent options are treatment with paracetamol, NSAIDs and possibly opiates and finally insertion of an artificial joint. Many patients with disabling osteoarthritis function much better and have markedly less pain with an artificial joint. INTERPRETATION: Current treatment options alleviate but do not cure arthritic symptoms; preventive actions should be instigated when possible. Treatment of osteoarthritis involves many medical specialties and treatment modalities.

Autologous chondrocyte implantation to repair knee cartilage injury: ultrastructural evaluation at 2 years and long-term follow-up including muscle strength measurements.

Autologous chondrocyte implantation (ACI) usually results in improvement in clinical scores. However, long-term isokinetic muscle strength measurements have not been reported. Biopsies from the repair tissue have shown variable proportions of hyaline-like cartilage. In this study, 21 consecutive patients were treated with autologous cartilage implantations in the knee. Mean size of the lesions was 5.5 cm(2). Follow-up arthroscopy with biopsy was performed at 2 years in 19 patients. The biopsies were examined with both light microscopy and transmission electron microscopy (TEM) techniques including immunogold analysis of collagen type 1. Patient function was evaluated with modified 10-point scales of the Cincinnati knee rating system obtained preoperatively and at 1 and 8.1 years. Isokinetic quadriceps and hamstrings muscle strength testing was performed at 1, 2 and 7.4 years. Light microscopy and TEM both showed predominately fibrous cartilage. The immunogold analysis showed a high percentage of collagen type I. At 7.4 years, the total work deficits when compared with the contra-lateral leg for isokinetic extension were 19.1 and 11.4%, and for isokinetic flexion 11.8 and 8.5% for 60 and 240 masculine/s, respectively. Mean pain score improved from 4.3 preoperatively to 6.3 at 1 year (p = 0.031) and 6.6 at 8.1 years (p = 0.013). Overall health condition score improved from 4.1 preoperatively to 6.1 at 1 year (p = 0.004) and 6.5 at 8.1 years (p = 0.008). Three patients later went through revision surgery with other resurfacing techniques and are considered failures. In summary, the formation of fibrous cartilage following ACI was confirmed by TEM with immunogold histochemistry. Although the functional scores were generally good, strength measurements demonstrated that the surgically treated leg remained significantly weaker.

The intensity of immunogold labeling of deplasticized acrylic sections compared to deplasticized epoxy sections-Theoretical deductions and experimental data.

The purpose of this study was to compare the level of immunogold labeling of deplasticized acrylic sections and deplasticized epoxy sections. Pure protein gels of IgG, albumin and thyroglobulin were produced by glutaraldehyde fixation and embedded in non-crosslinked acrylic resin (Technovit 9100) and epoxy resin (Epon 812), respectively. Ultrathin sections of acrylic and epoxy resin were separately deplasticized in 2-methoxyethyl acetate (MEA) and sodium ethoxide. Quantitative immunogold labeling was performed with anti-IgG, anti-albumin and anti-thyroglobulin antibodies on sections of the corresponding protein gels. For all antibodies tested, the intensity of labeling for deplasticized acrylic sections was significantly higher (two to four times) than for the corresponding deplasticized epoxy sections. The results fit with a theoretically deduced relation: the quotient of the labeling of two deplasticized sections of different resins is equivalent to the square root of the quotient of the labeling of the similar sections not exposed to any kind of pre-treatment. The practical significance of the results is that immunolabeling of deplasticized non-crosslinked acrylic resin results in more intense immunogold labeling than deplasticized epoxy sections. Deplasticizing is most useful when the requirements for ultrastructural preservation according to conventional criteria are moderate. Our theoretically deduced results also indicate that deplasticized Technovit (or other non-crosslinked acrylic resins) sections will be significantly better suited for immunolabeling at the light microscopic level than deplasticized epoxy sections.